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2017-04-11T02:59:27.093Z

2015-05-26T00:00:00.000Z

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Accuracy of Predicted Genomic Breeding Values in Purebred and Crossbred Pigs Genomic selection has been widely implemented in dairy cattle breeding when the aim is to improve performance of purebred animals. In pigs, however, the final product is a crossbred animal. This may affect the efficiency of methods that are currently implemented for dairy cattle. Therefore, the objective of this study was to determine the accuracy of predicted breeding values in crossbred pigs using purebred genomic and phenotypic data. A second objective was to compare the predictive ability of SNPs when training is done in either single or multiple populations for four traits: age at first insemination (AFI); total number of piglets born (TNB); litter birth weight (LBW); and litter variation (LVR). We performed marker-based and pedigree-based predictions. Within-population predictions for the four traits ranged from 0.21 to 0.72. Multi-population prediction yielded accuracies ranging from 0.18 to 0.67. Predictions across purebred populations as well as predicting genetic merit of crossbreds from their purebred parental lines for AFI performed poorly (not significantly different from zero). In contrast, accuracies of across-population predictions and accuracies of purebred to crossbred predictions for LBW and LVR ranged from 0.08 to 0.31 and 0.11 to 0.31, respectively. Accuracy for TNB was zero for across-population prediction, whereas for purebred to crossbred prediction it ranged from 0.08 to 0.22. In general, marker-based outperformed pedigree-based prediction across populations and traits. However, in some cases pedigree-based prediction performed similarly or outperformed marker-based prediction. There was predictive ability when purebred populations were used to predict crossbred genetic merit using an additive model in the populations studied. AFI was the only exception, indicating that predictive ability depends largely on the genetic correlation between PB and CB performance, which was 0.31 for AFI. Multi-population prediction was no better than within-population prediction for the purebred validation set. Accuracy of prediction was very trait-dependent. GenPred shared data resource acrosspopulation genomic selection multi-population reproduction traits within-population Genomic selection has been widely implemented in dairy cattle breeding when the aim is to improve performance of purebred animals (Berry et al. 2009;VanRaden et al. 2009;Hayes et al. 2009b). In pigs and poultry, however, the final product is a crossbred animal. This may affect the efficiency of methods that are currently implemented for dairy cattle. In pig breeding, multiple sire and dam lines are used, with a minimum of two lines (typically for crossbred sows) and often additional sire lines to produce a three-way or four-way cross finisher pig (Merks and De Vries 2002;Lutaaya et al. 2001). Selection based on genomic estimated breeding values (GEBV) for purebreds (PB) using phenotypes on crossbreds (CB) is expected to increase the response to selection observed in CB compared to the situation in which only PB phenotypes are used. This increased response is expected when the genetic correlation between the PB and CB trait is less than 1, especially when the genetic correlation is 0.7 or less (Dekkers 2007). Genetic correlations between PB and CB performance vary and can be considerably less than 1 (Lutaaya et al. 2001;Zumbach et al. 2007;Cecchinato et al. 2010). Adding CB individuals to the training data is very expensive because, besides genotyping, it also requires additional identification and individual recording of target traits. Breeding companies are not inclined to make these investments unless there is evidence that predictions yield greater gains and higher accuracies. Simulation studies have shown that the response to selection is greater when PB animals are selected based on CB performance and that accuracy of prediction is high (Dekkers 2007;Ibánez-Escriche et al. 2009;Kinghorn et al. 2010;Toosi et al. 2010;Zeng et al. 2013). There is, however, a lack of studies using real data. The number of genotyped CB is not yet large enough to test the superiority of training on CB for PB selection. A first step toward finding the optimal genomic selection scenario for pigs is to determine predictive ability (accuracy), in real data, of GEBV for CB pigs based on PB genomic and phenotypic data. This will show how CB performance responds to the current practice of selection on GEBV in PB pigs. Recently, accuracies of within-population genomic prediction in pigs have been reported (Cleveland et al. 2010;Forni et al. 2011;Christensen et al. 2012;Tusell et al. 2013;Badke et al. 2014). These studies have shown that all traits had more than zero predictive ability within population in a variety of pig breeds using different methods. It has also been shown that using genomic information generally increased the accuracy of prediction compared to using only pedigree information (Forni et al. 2011;Christensen et al. 2012;Tusell et al. 2013). Using multi-population training might be a way to increase the accuracy of prediction further. This is especially relevant to enable genomic selection for small populations when a closely related breed, or the same breed from another country, is added to the training set (Lund et al. 2014). An unresolved question is how to obtain accurate predictions from multi-population datasets. The effectiveness of a multi-population genomic evaluation depends on many factors, e.g., differences in allele frequency and consistency of linkage disequilibrium (LD) between quantitative trait loci (QTL) and single nucleotide polymorphism (SNP), which could reduce the accuracy of prediction (Wientjes et al. 2013), whereas the larger reference population would potentially improve the accuracy. The objective of our study was to determine predictive ability (accuracy) in CB pigs using real PB genomic and phenotypic data. The outcome is a first step toward determining the optimal genomic selection scenario to select PB for CB performance. As in cattle, studying accuracy of prediction for multi-population datasets is important for species in which population size imposes upper limits to the training population size. Therefore, a second objective was to compare the predictive ability of SNPs when training is done in either single or multiple populations in pigs. Data Genotypes were available from sows with own-performance information of three pig populations born from 2005 through 2012: 1070 Dutch Landrace-based (DL) sows from 19 farms; 1389 Large Whitebased (LW) sows from 14 farms; and 287 individuals from an F1 cross between these two commercial lines (DL sire/LW dams) originating from three farms. The genotyped CB animals had no specific family structure and the majority of them were not offspring of the genotyped PB animals, i.e., a number of generations separated PB and CB. The 287 CB animals were offspring from 76 sires and 170 dams. Four female reproduction traits were analyzed: age at first in-semination (AFI); total number of piglets born (TNB); litter birth weight (LBW); and litter variation (LVR). AFI consisted of the age at the second estrus, which was the time that the first insemination was performed. TNB was the sum of all piglets born alive and stillborn. LBW was the sum of individual birth weights of all piglets born in the same litter. Finally, LVR consisted of the standard deviation (SD) of individual birth weight of the piglets from the same litter. The PB and CB sows that were selected for genotyping have phenotypic records from multiple parities on multiple traits and have a large genetic contribution to future descendants. All PB sows were breeding animals from nucleus farms, whereas the CB sows belonged to farms where combined crossbred and pure line selection (CCPS) is applied. There was no strong selection for first parity performance in the genotyped sows, reducing any possible bias in TNB and LBW due to culling after first parity. Deregressed estimated breeding values (DEBV) were used as response variable for each trait undergoing study. The estimated breeding values (EBV) were deregressed for each trait separately using the methodology proposed by Garrick et al. (2009). DEBVs, instead of EBVs, were used to compute the GEBV accuracy because this removes the influence of the parents' EBVs and rescales the EBV according to its accuracy, i.e., the DEBV of the animals reflect their genetic merit. Ostersen et al. (2011) have shown that using DEBVs rather than EBVs for genomic prediction yields higher GEBV accuracies. The number of animals and records used to estimate the EBVs are in Table 1. The EBV of each animal was obtained from the routine genetic evaluation by Topigs Norsvin using MiXBLUP (Mulder et al. 2012) in a multi-trait model (including all measured reproduction traits). The genetic evaluation was done across lines with phenotypes from the different populations treated as the same trait. A fixed line effect was included in the model for estimating EBVs. In multi-population prediction scenarios, this line effect was added back to the random additive genetic effect after estimating the EBVs, and subsequently, the line effect was again included in the genomic prediction model. Adding back the line effect allows the differences of the level of EBV between-population to be maintained in the data. Therefore, in the genomic prediction step, the mean differences between populations are still present, and this allows SNP effects (that differ in allele frequencies between lines) to explain these differences between lines. The model for obtaining the EBVs for AFI included genetic line and herd-year-season as fixed effects and an additive genetic effect (animal) as random effect. For TNB, the fixed effects were genetic line, parity, interval between weaning and pregnancy (days), whether more than one insemination procedure was performed (yes or no), and herd-year-season. The random effects consisted of service sire, a n permanent effect to account for the repeated observations of a single sow, and an additive genetic effect (animal). EBVs for LBW were obtained with a model that included genetic line, parity number, TNB, and herd-year-season as fixed effects and a permanent effect and an additive genetic effect (animal) as random effects. The model used for LVR was similar to the one used for LBW, except that TNB was removed. The reliabilities per animal, needed for deregression, were extracted from the genetic evaluation based on the methodology of Tier and Meyer (2004). The heritabilities (h 2 ) used for deregression were estimated via restricted maximum likelihood (REML) using a pedigree-based relationship matrix and were also obtained from the routine genetic evaluation. The h 2 of the traits were 0.30 for AFI, 0.11 for TNB, 0.38 for LBW, and 0.14 for LVR. The genomic h 2 of the DEBVs were estimated via REML using ASREML 3.0 (Gilmour et al. 2009). Sows were genotyped using the Illumina PorcineSNP60 BeadChip (Ramos et al. 2009). SNPs with GenCall ,0.15, unmapped SNPs, and SNPs located on either the X or the Y chromosome, according to the Sscrofa10.2 assembly of the reference genome (Groenen et al. 2012), were excluded. Quality control was performed in all populations simultaneously, which involved excluding SNPs with call rate ,0.95, minor allele frequency ,0.01, and strong deviations of Hardy-Weinberg equilibrium (x 2 . 600). After quality control, 42,139 SNPs remained out of the initial 64,232 SNPs. Individuals with missing genotype frequency .0.05 were also removed. Missing genotypes of the remaining animals were imputed using BEAGLE 3.3.2 (Browning and Browning 2007). Statistical analyses GEBVs were computed based on the genomic best linear unbiased prediction method (GBLUP). GBLUP uses a genomic relationship matrix (G) instead of the numerator relationship matrix (A). The G matrix contains genomic kinship indicating relatedness between animals and was used for prediction in all scenarios with the model: where y is the vector of DEBVs, m is the overall mean, g is the vector of random additive genetic effects assumed to be N(0, Gs 2 a ), Z is a design matrix allocating g to y, and e is a residual with heterogeneous variance due to differences in reliabilities of the DEBVs (Garrick et al. 2009). In predictions where the training set contained more than one population, the fixed line effect present in the model for estimating EBVs was also included in the GBLUP model as a fixed effect. The G matrix for within-population prediction was built according to VanRaden (2008), which was computed as G ¼ ZZ9 =2 P p i q i , where Z is a matrix of centered genotypes and p i and q i are the allelic frequencies of the i th marker based on observed genotypes. In predictions where the training set contained more than one population, the G matrix was built according to Chen et al. (2013), accounting for differences in allele frequencies between populations. We used ASREML 3.0 (Gilmour et al. 2009) to predict the GEBVs, with the G matrix entered as a user-defined matrix. Animals assigned to the prediction set had their DEBVs removed before predicting GEBV. All scenarios were also analyzed using the A matrix, which contains the average additive genetic relationships of the animals based on the pedigree (PED-BLUP). The model for these analyses was similar to the GBLUP one; however, the g vector of the random additive genetic effect was assumed to be N(0, As 2 a ). Genetic correlations between PB and CB performance were estimated for the four traits. We used records for DL, LW, and F1 animals born from 2005 through 2012 (Supporting Information, Table S1). Genetic correlations were estimated in bivariate analyses using REML in ASREML 3.0 (Gilmour et al. 2009). The effects of bivariate models were the same as those used to obtain the EBVs (see above); however, to estimate genetic correlations, PB performance and CB performance were treated as different traits (Falconer 1952), which in matrix notation is: where y i is the vector of observations with i being 1 for purebred and 2 for crossbred data, Z i is the incidence matrix for g i , which is a vector of random additive genetic effects. The additive genetic variance is expressed as: where A is the numerator relationship matrix and G 0 is a 2·2 covariance matrix with the purebred and crossbred variances in the diagonals and the covariances in the off-diagonals. Scenarios and accuracy of prediction Seventeen scenarios were investigated that can be divided into four groups according to composition of the training and validation data sets as follows: • Scenarios 1-3: Training and validation data were subsets from the same population, DL, LW, and F1, respectively, i.e., prediction was within-population. These scenarios determine how well the withinpopulation prediction performs for the different traits. • Scenarios 4-7: Same as scenarios 1-3 but the remaining PB population(s) was/were added to the training data, i.e., prediction was multi-population. These scenarios determine whether adding data from a different PB population to the training data would increase the accuracy compared to the within-population prediction. • Scenarios 8-11: One PB population was used for training to predict the other PB population. F1 data were not used in these scenarios, i.e., prediction was across breeds. These scenarios determine how well across-population predictions would perform. • Scenarios 12-17: PB populations were used for training and CB animals were used for validation. These scenarios determine how well CB genetic merit can be predicted from PB data alone, and whether inclusion of more than one parental PB population increases the accuracy. The accuracy of prediction was estimated as the correlation between the GEBV/EBV and the DEBV of the validation set animals for GBLUP/PED-BLUP. Prediction bias was calculated by regressing the validation variables (DEBV) on the prediction variables (GEBV/ EBV). Accuracies were the average of 20 random training-validation populations in scenarios 1-7, 9, 11, 13, 15, and 17. For scenarios 1-7, we randomly set aside part of the genotyped animals (N = 50) and used those in a later step to determine the accuracy of prediction. These 50 were not included in the training for those scenarios. In scenarios 9, 11, 13, 15, and 17, not all the available animals were used for training. Subsets of the training populations were sampled such that the same number of animals was used from each population per trait undergoing study. Any differences in accuracies would then be due to the different populations used, and not to differences in the number of animals. Scenarios 8, 10, 12, 14, and 16 only had one estimate of accuracy because all the animals were used in the training population to maximize prediction accuracy of animals in another population. RESULTS Estimates of genomic h 2 of the DEBVs across traits and populations ranged from 0.04 to 0.58 (Table 2). Estimates of pedigree-based h 2 of the DEBVs across traits and populations ranged from 0.03 to 0.78 (Table S2). The genomic and pedigree-based heritabilities were similar in general. Genetic correlations between PB performance and CB performance for the four traits undergoing study ranged from 0.31 for AFI to 0.90 for LBW (Table 3). Accuracies for within-population predictions for scenarios 1-3 ranged from 0.22 to 0.72 for GBLUP and from 0.21 to 0.64 for PED-BLUP across the four traits and different training sets, indicating a modest to good predictive ability (Table 4). The regression coefficient of the GEBV/EBV on the DEBV for scenarios 1-3 ranged from 1.03 to 1.70 for GBLUP and from 0.90 to 2.21 for PED-BLUP. For multi-population prediction of PB populations (scenarios 4 and 5) the accuracies ranged from 0.18 to 0.67, whereas for multipopulation prediction (two PB + one CB) of the CB population (scenarios 6 and 7) the accuracies ranged from 0.17 to 0.45 for GBLUP and from 0.32 to 0.42 for PED-BLUP. When predicting PB (scenarios 4 and 5; Table 5), the addition of the other PB population resulted in lower accuracies for all four traits in comparison to withinpopulation prediction for GBLUP. When predicting CB (scenarios 6 and 7; Table 5), the addition of PB populations resulted in lower accuracies for AFI and TNB but higher accuracies for LBW and LVR. The regression coefficient of the GEBV/EBV on the DEBV for scenarios 4 and 5 ranged from 0.86 to 1.18 for GBLUP, whereas for scenarios 6 and 7 it ranged from 0.80 to 3.11 for GBLUP and from 0.97 to 5.00 for PED-BLUP. Accuracies and regression coefficients of the EBV on the DEBV were not computed for PED-BLUP for scenarios 4 and 5 because the other PB population to be added is not related according to the pedigree. GEBV accuracy of across-breed prediction, i.e., predicting genetic merit of one PB from a different PB population, performed poorly for AFI and TNB (Table 6); accuracies were not significantly different from zero (P . 0.05). Accuracies for LBW and LVR ranged from 0.13 to 0.26 across the different training sets for GBLUP. The regression coefficient of the GEBV on the DEBV for AFI and TNB ranged from 20.71 to 1.37, whereas for LBW and LVR it ranged from 0.70 to 1.40. Accuracies and regression coefficients of the EBV on the DEBV were not computed for PED-BLUP because the two PB populations are not related according to the pedigree. Accuracy of prediction in scenarios 12-17 that predicted genetic merit of CB using PB parental populations as training data performed poorly for AFI (Table 7); accuracies were not significantly different from zero for both GBLUP and PED-BLUP (P . 0.05). For the other three traits, TNB, LBW, and LVR, however, predictive ability was observed. Accuracies ranged from 0.11 to 0.31 for GBLUP and from 0.08 to 0.22 for PED-BLUP. The regression coefficient of the GEBV/ EBV on the DEBV for AFI ranged from 21.14 to 20.15 for GBLUP and from 0.15 to 0.95 for PED-BLUP, whereas for TNB, LBW, and LVR it ranged from 0.48 to 3.82 for GBLUP and from 0.53 to 7.76 for PED-BLUP. DISCUSSION Accuracies of genomically predicted breeding values in CB and PB pigs were estimated for four female reproduction traits in 17 scenarios to optimize the use of genomic data for crossbred animals. We have used DEBVs as a response variable with a moderate to high mean reliability (ranging from 0.33 to 0.80) for the different traits and populations. The SD of the accuracies in scenarios in which we had replicates of training validation populations varied according to the type of prediction (within, multi-, across, or PB to CB). Withinpopulation and multi-population predictions showed higher SDs because the relationship between training and validation in each replicate could substantially vary due to different degrees of relationships within a population. For across-population and PB to CB predictions, the relationship between training and validation populations was naturally lower; therefore, in each replicate there was less variation. Within-population prediction LBW and LVR showed generally higher accuracies than AFI and TNB. This difference between traits may occur due to the lower reliability of the DEBV for AFI and TNB, which lowers the accuracy when the number of observations is preset. Another possibility is that there are non-additive genetic effects (e.g., dominance, epistasis) affecting AFI and TNB more, whereas LBW and LVR may be regulated mainly by an additive action of the genes. Therefore, the importance of non-additive effects needs to be further investigated. Even with the low number of genotyped CB pigs, all traits showed predictive ability within the CB. Therefore, a greater number of genotyped CB should increase these accuracies. In general, GBLUP outperformed PED-BLUP across populations and traits, which is mainly a result of a better estimation of relationships among individuals by the markers. Similar results have also been reported in other studies using pigs (Forni et al. 2011;Tusell et al. 2013). The regression coefficients of the GEBV/EBV on the DEBV for both GBLUP and PED-BLUP were, in general, close to 1, indicating that the predictions were not severely biased, except for TNB, where some of the them deviated considerably from 1. The level of accuracy found here is concordant with those found in other studies on pigs (Cleveland et al. 2010;Forni et al. 2011; n Badke et al. 2014). In these studies, as well as in ours, many traits and breeds were studied and within-population prediction always had predictive ability. One of the studies (Tusell et al. 2013) also studied TNB for two PB populations and their F1 cross and also found that prediction within the F1 cross has greater accuracy than within-PB prediction. They argued that this might be caused by the structure and effective sample size of the populations undergoing study. Accuracies found by Christensen et al. (2012) were not statistically different between single-step BLUP (SS-BLUP) and GBLUP, but both were higher than pedigree-based prediction and GBLUP was shown to be more biased. The advantage of using SS-BLUP was an increase of accuracy for non-genotyped animals. Because our aim was to predict genotyped animals, we studied accuracies of prediction using GBLUP. Multi-population prediction Adding data from a different PB population to the training data (scenarios 4 and 5) decreased the accuracy of prediction compared with within-population predictions (scenarios 1-3) for GBLUP. Adding data from the two PB populations to the CB training data (scenarios 6 and 7) had different results depending on the trait. LBW and LVR that had high genetic correlation between PB and CB performance had an increase in accuracy, whereas for AFI that had a low genetic correlation there was a decrease in accuracy. TNB had a high genetic correlation; however, the accuracy also decreased, which was unexpected. If traits are genetically very different (low genetic correlation between PB and CB), then adding more animals with the other trait to the training is not expected to increase the accuracy. When the trait is the same, however (high genetic correlation), including more animals n with the other trait (PB vs. CB) is expected to increase the accuracy. Besides having a high genetic correlation between the traits, the additional animals also need to have some (genomic) relationship to the validation animals. In addition to a low genetic correlation between PB and CB performance, the degradation of accuracy might result from differences in non-additive effects. For PED-BLUP, adding the two parental PB populations in the training also had different results depending on the trait. AFI and LBW had an increase in accuracy, whereas TNB and LVR had a slight decrease in accuracy. The regression coefficient of the GEBV/EBV on the DEBV estimated to investigate bias for scenarios 4-7 was, in general, close to 1, indicating that the predictions did not suffer from a large bias, except for AFI and TNB in scenarios 6 and 7. For these traits, whenever the PB parental populations were used as training and CB used as the validation set, the regression coefficient of the GEBV/ EBV on the DEBV indicated that the estimates were severely biased. A review regarding multi-population prediction in cattle (Lund et al. 2014) has shown that combining populations, in general, increases the accuracy of prediction when the breeds are the same but from different countries, to a lesser degree when the breeds are closely related, and has little or no benefit when the breeds are distantly related. Another study (Hayes et al. 2009a) has reported slightly higher accuracies when using multi-population prediction compared to within-population prediction in dairy cattle. Chen et al. (2013) used Angus and Charolais steers to determine the accuracy of prediction with GBLUP for within-population and multi-population predictions. In their study, accuracies did not always increase, suggesting that noise was being added to the predictions. The maximum increment in accuracy that they obtained was 0.05, whereas a decrement of 0.07 was also obtained, which is within the same range as the differences observed in the current study. These studies showed that adding another PB population to the training data in cattle did not necessarily increase the accuracy of prediction, similar to our current results in pigs. De Roos et al. (2009), using simulated data, also showed that increasing the size of the training data by adding animals from a differ-ent population does not always increase the accuracy. An increase in accuracy higher than within-population was only found when the populations were closely related, when marker density was high, or when the size of the initial within-population training data set was small. In our case, the number of markers was reasonable and in some scenarios the size of the within-population training data set was small, but we still did not have a great increase in accuracy of prediction. This suggests that the marker density might not be sufficient to have similar LD levels between QTL and markers in the different populations that are mixed. The genetic distance between the populations was probably an important factor that limited the benefit of adding training data from other populations. Across-population prediction Some predictive ability was observed when predicting across populations for LBW and LVR, whereas for AFI and TNB all the accuracies were null. Increasing the size of the training population slightly improved the accuracies of prediction, on average by 0.05. Greater accuracies were found when DL predicted LW genetic merit, rather than the other way around (scenario 9 vs. scenario 11). The regression coefficients of the GEBV on the DEBV for scenarios 8-11 were, in general, close to 1 for LBW and LVR, indicating that the predictions did not suffer from much bias. For AFI and TNB, however, regression values greatly deviated from 1, sometimes with negative values, which we attribute to the very low accuracies we found. In a study by Harris et al. (2008), the prediction across Holstein-Friesian and Jersey cattle breeds was also investigated. Predictions were not accurate, ranging from 20.1 to 0.3 for 25 traits. In another study, Hayes et al. (2009a) predicted the GEBV of Jersey animals using a Holstein population as training data and vice versa, resulting in accuracies ranging from 20.06 to 0.23 for five traits. Both studies report results that were very similar to ours that ranged from 20.05 to 0.26. The simulation study by De Roos et al. (2009) indicated that across-population prediction was substantially less accurate than within-population or multiple-population prediction. These lower n Table 6 GEBV accuracies from across-population prediction using GBLUP (scenarios 8-11) accuracies were due to differences in marker-QTL LD phase between the populations. A marker may be in LD with QTL in a given population, but it is not necessarily in LD with those QTL in the other population, resulting in poor predictions for the other population. These simulation results suggested that, for our analyses, a higher marker density would be required. However, results of Veroneze et al. (2014) show that with the same 60K porcine SNP panel, the density of SNPs is high enough to obtain reasonable levels of LD. This would predict that our SNP panel should be able to capture marker effects across breeds. Using purebred training data to predict crossbred genetic merit Using only the PB population(s) to predict the CB genetic merit with GBLUP has some predictive ability for TNB, LBW, and LVR, whereas all the accuracies for AFI were null. Increasing the size of the training data by adding another PB population increased the accuracy for TNB and LBW, whereas for AFI and LVR it did not. However, when we increased the size of the training population by adding more animals of the same PB population, the accuracies usually increased. The accuracy of prediction for predicting CB animals based on PB animals appears to depend largely on the genetic correlation between PB and CB performance. As our results demonstrate, the greater the genetic correlation, the higher the chances of having any or more predictive ability. AFI, for which the genetic correlation between PB and CB was very poor, had zero accuracy of prediction showing that selection on PB is expected to have no effect on CB genetic merit. For PED-BLUP, the accuracies were, in general, lower than for GBLUP, especially for LBW and LVR. Adding the second PB population in the training slightly increased the accuracy of prediction. The greater accuracies found for TNB, LBW, and LVR when training with DL rather than LW population can be explained by the slightly greater relationship between DL and F1 populations than between LW and F1. This higher relationship is specific for the animals included in this study. The F1 animals that were genotyped are more closely related to the DL animals that were genotyped than to the LW animals that were genotyped. To test the impact of the relationship between training and validation populations on the accuracy, we split the training data into the 50% of animals that are MOST related to the validation set and the 50% that are LEAST related to the validation set (Table S3, Table S4). For AFI, TNB, and LBW, using the 50% MOST related animals resulted in greater accuracies, whereas for LVR it did not. This indicates that if CB animals have closer genomic relationships to the PB animals used as training, then higher accuracies for scenarios 12-17 could generally be expected. In cattle, Harris et al. (2008) used PB populations (Holstein-Friesian and Jersey) to predict the genomic breeding values of a cross between these two breeds. They used data from 4500 sires genotyped for approximately 44K SNPs. Their results show that using the two breeds as the training population increased the accuracy by 5-10% compared to using only one of the breeds to predict the cross. The actual level of accuracy was not reported in their study. Our results were similar to theirs for TNB, LBW, and LVR, where the genetic n correlation between PB and CB performance is close to 1, but not for AFI. Results indicate that using a PB population to predict CB genetic merit can generate reasonable predictions. This, however, is not consistent for all traits. Although these results do not reflect the actual practice of genomic selection in pig breeding, they do provide an estimate of the accuracy of genomic prediction between CB and PB populations using real data. The results make a strong case for the genotyping and recording of CB animals, at least for a subset of traits where genetic correlations are away from 1. The low genetic correlation between PB and CB performance for AFI was also found in another study (Nagyné-Kiszlinger et al. 2013). They have reported values of 0.28 and 0.39 for Hungarian Large White and Hungarian Landrace with their reciprocal cross. Possible reasons for this low genetic correlation were reported: 1) genes that affect the trait might be different among populations; 2) this trait is affected by non-additive effects or environmental factors due to different management of PB and CB animals (Nagyné-Kiszlinger et al. 2013). One clear environmental factor that probably reduces the genetic correlation of AFI between PB and CB is the use of batch farrowing systems in the production environment of CB sows. Suppression of estrus is used to synchronize the heat of the CB gilts, which impacts the measurement of the trait and leads to these low correlations. Standardized EBVs were used; therefore, a bias would possibly be introduced during deregression due to different reliabilities between breeds (Garrick et al. 2009). Additional sources for potential bias affecting the SNP effect estimates are the differences in the population mean of the breeds. The differences in the mean between populations were remedied by reintroducing the line effect after deregression. To test the impact of deregression on bias, we investigated all 17 scenarios for the trait AFI by analyzing phenotypes, which are not standardized, instead of DEBVs. The correlation between the accuracies obtained by the two different approaches was 0.92, with a mean regression coefficient of the GEBV on the phenotype of 0.70. This correlation shows that using the phenotypes has good agreement with the accuracies calculated using DEBVs; therefore, any bias due to the process of standardization and deregression is expected to be limited. The reasonable accuracy for PB predicting CB genetic merit shows that in a current typical breeding program, selection in the PB does result in a phenotypic response in CB. AFI was an exception in our study, because the genetic correlation between PB and CB performance was very low. Further studies to compare the accuracy of genomic selection of PB for CB performance are needed. Other genomic models including breed-specific effects of SNP alleles or dominance (Ibánez-Escriche et al. 2009;Zeng et al. 2013) were described and were found to outperform an additive model only in specific cases, e.g., with high dominance levels or when the number of SNPs is small relative to the size of the training population. Using these more complex models or a multiple-trait model (Christensen et al. 2014) with real data is needed. In conclusion, there was predictive ability for purebred population (s) predicting crossbred genetic merit using an additive model in the populations studied when PB and CB traits have high genetic correlation. For practical implementation, estimation of genomic breeding values of PB animals for CB performance needs to be further studied with models that take into account the crossbred nature of training data. Multi-population prediction was no better than within-population prediction for PB populations. Accuracy of prediction was shown to be very trait-dependent; hence, for the utility of data from other breeds in the application of genomic selection, each trait needs to be studied separately and no generalizations should be made. Finally, real data accuracies were lower than what simulation studies have reported.

v3-fos

2018-04-03T00:22:50.719Z

2015-11-01T00:00:00.000Z

11466188

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Effect of milk and yogurt on streptococcus sobrinus counts and caries score in rats Background: An anti-cariogenic diet containing probiotics can be effective in caries prevention. This animal study compared the effects of milk and yogurt on Streptococcus sobrinus counts and caries score. Materials and Methods: A total of 36 male rats were infected with S. sobrinus (27,607) and divided into three groups. Group A and B received 200 mL of milk and 100 g of yogurt per day, respectively, and a control group received 2.5 mL of NCP number 2 diet twice daily for 21 days. After killing the animals, their lower left jaws were removed and sonicated to quantify the colonies of S. sobrinus. Dental caries was scored using Keyes technique. Data were analyzed using ANOVA and Kruskal-Wallis, Mann-Whitney and Wilcoxon-Signed Rank tests. Statistical significance was set at P < 0.05. Results: The mean (±standard error of the mean) of S. sobrinus colonies in the milk, yogurt and control groups were determined at 119666.67 (±20733), 46416.666 (±12846) and 163,250 (±33493), respectively. Microbial counts decreased in the yogurt group compared with the milk and control groups (P = 0.004 and P = 0.000; respectively). There were significant differences between caries scores of smooth surfaces in the milk and yogurt groups compared with the control group (P = 0.000 and P = 0.000, respectively). Both milk and yogurt significantly reduced caries score of fissured surfaces compared with controls (P = 0.004 and P = 0.000, respectively). Conclusion: Considering the limitations of this study, yogurt administration reduces S. sobrinus counts. In addition, yogurt and milk regimens reduce the caries scores of smooth and fissured surfaces. INTRODUCTION Tooth decay is the most common chronic disease of early childhood. Although it can be preventable, dental caries is considered a multi-factorial infectious and transmissible disease. [1] The most acceptable theory of caries development is the chemicoparasitic theory in which the presence of cariogenic bacteria, susceptible host and fermentable carbohydrate as a substrate for microbial action are critical; therefore, chemical and mechanical microbial plaque removal, as well as sugar discipline, are commonly advised. [2,3] Diet plays a key role in caries development. In other words, an anticariogenic diet can be effective in caries prevention. [4] The low cariogenic potential of food is attributed to high protein and mineral content as well as moderate fat content and high buffering capacity facilitating the saliva action to protect the teeth. [5] Dairy products such as milk and yogurt are excellent foods containing protein, providing essential amino acids and organic nitrogen. They also contain calcium, phosphate, casein, and lipids, which are considered factors with anticariogenic effects. These ingredients modulate the acidity of saliva and plaque, causing the remineralization of early carious lesions, in addition to having some degrees of antibacterial properties. [5] In addition, yogurt contains probiotics, living benefi cial microorganisms with an inhibitory effect on pathogenic bacteria. [6] Yogurt and other fermented milk-based products have been demonstrated to be benefi cial for general health, especially because of their probiotic content. They were proposed as an alternative to manage many disorders such as infectious diseases, cancers and gastrointestinal problems in particular. [7] Because of so many interactions known between different species of microorganisms, it has been shown that the growth of pathogenic strains can be inhibited by interfering with colonization and their interaction with nonpathogenic neighbors. [8] In this respect, probiotic therapy refers to selected removal of pathogenic bacteria by nonpathogenic competitors. [9] The most common probiotic bacteria used for caries control are some species of Bifi dobacteria and Lactobacilli, which are tested against Streptococcus mutans. [10] Mutans streptococci are the major pathogenic bacteria in the caries process. This group mainly includes S. mutans and Streptococcus sobrinus, which are responsible for caries development in both animals and humans. [5] S. sobrinus is a primary bacterial pathogen on smooth surface dental caries. It presents in 43-60% of plaque cultures of children with early childhood caries. [11] It is supposed that the inhibition of these microorganisms leads to caries prevention. This comparative study was carried out to evaluate the effects of milk and yogurt on S. sobrinus counts and caries score in rats. MATERIALS AND METHODS The present study was approved by the Ethic Committee of Babol University of Medical Sciences, and all experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23 revised in 1996). Thirty-six male Wistar rats aged 19 days and weighing 25 ± 5 g were used in this study. The S. sobrinus-free rats were screened by culturing the saliva samples on streptococcus selective agar medium. Then, they were infected with S. sobrinus PTCC 27607 (Persian Type Culture Collection, Tehran, Iran) under the cariogenic diet 2000 sterilized by deionized distilled water with 10% sucrose ad libitum to establish the infection for 6 days. [12] Plating of oral swabs showed the rats were successfully infected with S. sobrinus. All t he animals were then anesthetized with intraperitoneal injection of a mixture of ketamine (8 mg/kg) and xylazine (2.5 mg/kg) [13] and their submandibular and parotid salivary glands were surgically removed when aged 25 days. After surgery, they were divided into three experimental groups (12 in each group). Group A and B were respectively fed ad libitum with 200 mL of milk and 100 g of yogurt daily and the control group received gavage of 2.5 mL of liquid diet NCP number 2 twice daily. [14] Milk and yogurt were chosen in the same brand, with similar fat, protein and carbohydrate contents. The animals were housed one per cage (42 cm × 26 cm) and maintained on a 12-h light/12-h dark cycle (lights on at 6.00 a.m.), at a temperature of 21°C ± 1°C and relative humidity of 50-70%. They had free access to water and diet related to each group. After 21 days, the animals were sacrifi ced, and their lower left jaws were removed and suspended in 10 mL of 0.9% normal saline. [15] The samples were sonicated (BAN Delin Sonoplus, Germany) for 30 s in duty cycle 1 × 10. One loop was inoculated on streptococcus selective agar medium (Merck, Germany) and incubated at 37°C for 2 days for detecting and counting the colonies of S. sobrinus. Dental caries was detected under a stereomicroscope at magnifi cation of ×40 (Micro-Optic Industrial Group Company, China) and then scored using the Keyes technique. [16] Microbiological data and the total number of lesions on smooth and fi ssured surfaces were assessed separately. Data were analyzed using SPSS 18 (SPSS Inc., Chicago, IL, USA). Kruskal-Wallis analysis of variance was used to compare the levels of S. sobrinus. Ordinal caries scores were analyzed using Kruskal-Wallis, Mann-Whitney and Wilcoxon-Signed Rank methods. Statistical signifi cance was defi ned at P < 0.05. RESULTS The mean (±standard error of the mean) of S. sobrinus colonies in milk, yogurt and control groups were estimated at 119666.67 (±20733), 46416.666 (±12846) and 163,250 (±33493), respectively. A signifi cant reduction in S. sobrinus counts was found in the yogurt group compared with the milk and control groups (P = 0.004 and P = 0.000; respectively), but milk consumption did not signifi cantly reduce S. sobrinus counts. S. sobrinus counts are displayed in Figure 1. There were signifi cant differences between all the groups in caries scores of smooth surfaces (P = 0.000). Consumption of milk and yogurt resulted in a signifi cant decrease in dental caries scores on smooth surfaces compared with the controls (P = 0.000 and P = 0.000, respectively) but there was no signifi cant difference between yogurt and milk groups. The caries scores of smooth surfaces are displayed in Table 1. In relation to caries scores of fi ssured surfaces, a signifi cant difference was shown between all the groups (P = 0.000). Both milk and yogurt signifi cantly decreased the caries scores of fi ssured surfaces compared to the controls (P = 0.004 and P = 0.000, respectively) while there were no signifi cant differences between yogurt and milk groups. The caries scores of fi ssured surfaces are presented in Table 2. Figure 2 illustrates the rats' second molars, which were sectioned mesio-distally at the central fissure to find carious lesion based on the Keyes technique [ Figure 2]. Wilcoxon-Signed rank did not reveal any signifi cant differences between caries scores on smooth and fi ssured surfaces in all the groups. DISCUSSION This study evaluated S. sobrinus counts and caries scores by administration of milk and yogurt in rats. The results indicated that consumption of yogurt had an inhibitory effect on S. sobrinus, consistent with observations made by Caglar et al. and Petti et al. [17,18] It suggested that the yogurt affects the oral microfl ora because of its probiotic microorganisms. [19] These benefi cial bacteria are suggested as the nonpathogenic competitors against cariogenic bacteria. [9] It is supposed that interactions between microbial species prevent colonization of pathogenic bacteria by interfering with cellular adhesion and biofi lm formation. [9,20] Furthermore, some probiotic strains produce and secrete organic acids, hydrogen peroxide and bacteriocins, making the ecosystem unsafe for pathogenic neighbors. [20] Probiotic microorganisms naturally exist in some dietary products such as yogurt. In addition, they may be added to dairy products, chewing gums, food supplements, pills, etc. [21] However, probiotics-containing dairy products appear to be the most natural and the best approach for probiotic therapy. [22] Not only the probiotics content but also the other components such as high mineral and protein and moderate lipid contents in these products play a major role in caries prevention. [5] Milk consumption did not signifi cantly decrease S. sobrinus counts compared to the control group. The results of two studies conducted by Engström et al. and Lexner et al. [23,24] are consistent with the present study. This outcome can be probably explained by considering the lack of probiotics in milk, since the effi cacy of probioticscontaining milk in reduction of caries-associated bacteria has been previously demonstrated. [25,26] The anticariogenic effect of probiotic milk containing Lactobacillus rhamnosus GG was evaluated on children aged 1-6 years during 7 months. It was found that the incidence of tooth decay may decrease by a long-term daily consumption of this product. [25] Additionally, it was observed that consumption of milk and yogurt signifi cantly decreased caries scores of smooth and fi ssured surfaces compared with the control group. In this regard, Tanaka et al. [27] found that the frequent use of yogurt (more than or up to 4 times a week) might be associated with a lower incidence of dental caries in children. In addition, Levy et al. [28] showed that regular administration of milk had a protective effect on primary teeth. It is suggested that milk ingredients such as fat, casein, calcium and phosphate have a protective effect against tooth decay. [29] Aimutis [30] reported that the casein phosphopeptides (CPPs) and glycomacropeptide in dairy products prevented demineralization as well as stimulated remineralization of tooth enamel. The above-mentioned effect was also demonstrated by Ferrazzano et al. [31] about CPPs of yogurt. The CPPs stabilize high concentrations of calcium and phosphate together with fl uoride ions on the enamel surface. These ions are freely bioavailable to diffuse into early carious lesions, thereby effectively enhancing remineralization. [32] They also have a great capacity to neutralize the acid produced by cariogenic bacteria to prevent demineralization. [33] This study was designed as an animal study on rats mainly due to ethical considerations existing in human studies. In spite of natural differences between rats and humans, based on previous studies, [17,18,27,28] it seems that if this project had been performed on humans, similar fi ndings would have been achieved. However, further studies on humans, comparing the anticaries properties of dairy products on S. mutans and S. sobrinus are recommended. CONCLUSION This study showed that yog urt administration signifi cantly reduced S. sobrinus counts. Although it was not statistically signifi cant, S. sobrinus counts dropped by consumption of milk. Additionally, taking yogurt and milk reduced the caries scores of smooth and fi ssured surfaces signifi cantly. These results confi rmed the anticariogenic capacity of yogurt and milk.

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2019-03-30T13:12:02.848Z

2015-01-01T00:00:00.000Z

86201328

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Molecular Characterization of Selected Mutant Lines of Onion (Allium cepa L.) against Purple Leaf Blotch Disease Using SSR Markers Aims: The purple leaf blotch (PLB) disease, for which there is no released resistant variety in Bangladesh, causes loss in production of onion in every year. The mutants of a released variety, BARI Piaz-2 has shown to possess resistance against PLB which is yet to be characterized at molecular level. An easy and simple molecular detection technique of the gene responsible for this disease will be of great use in future. The present study was thus carried out to molecularly characterize four mutant lines of onion using Simple Sequence Repeat (SSR) marker to detect the presence or absence of PLB gene conferring resistance against purple blotch disease in onion. Place and Duration of Study: The research was carried out during the period from March, 2013 to Original Research Article Chakraborty et al.; AJEA, 8(4): 261-267, 2015; Article no.AJEA.2015.169 262 April, 2014 in the Biotechnology Laboratory of Bangladesh Institute of Nuclear Agriculture (BINA) in Mymensingh, Bangladesh. Methodology: DNA was extracted from the vigorously growing fresh leaf samples of 25 days old seedlings of four mutant lines namely, BP2-75/2, BP2 -100/1, BP2-100/2 and BP2-100/12 of onion using CTAB method. The molecular characterization was done using two sets of SSR markers, namely MatK-1RKIM-f/MatK-3FKIM-r and rbcLa-F/rbcLa-R. Results: All the four mutant lines showed clear band for the primer MatK-1RKIM-f/MatK-3FKIM-r which indicates the presence of PLB gene inferring resistance against purple blotch. Clear band was also observed with the marker rbcLa-F/rbcLa-R in all mutant lines except BP2-100/12 indicating absence of PLB gene in BP2 -100/12 which inferred susceptibility against purple blotch of onion. An unknown allele was also detected in this experiment which may be either linked with the PLB gene or a candidate gene which triggers the PLB gene responsible for purple blotch of onion. Conclusion: Both the primers seemed to be effective in detecting the presence or absence of PLB gene in the studied mutant lines of onion variety BP2. However, more number of primers should be tested for effective screening of diverse germplams that will be helpful in designing any future breeding programs. INTRODUCTION Onion (Allium cepa L. 2n=16), belonging to the family Alliaceae, is one of the most economically important and familiar vegetable and spice crops worldwide including Bangladesh [1]. The crop was originated in area, which includes Iran, Pakistan and specially their mountainous regions situated in the north of these countries [2][3][4][5][6]. Besides being used as salad and vegetable, onion is generally used as spice in most of the Asian countries. Onion has great economic importance due to it's medicinal and dietetic values [7][8][9][10]. Onion suffers from many diseases of which purple lear blotch (PLB) caused by Alternaria porri (Ellis) is a major one [11][12][13][14]. This disease caused substantial loss in both bulb and seed yield of onion in most onion growing countries including Bangladesh [15][16]. Control of this disease by using disease free seeds and fungicidal sprays are successful but have their disadvantages, such as the occurrence of fungicide resistance [17][18][19]. Due to these disadvantages, there is a constant requirement for inducing resistance to plants against the disease [20-23]. The recent development in the field of molecular markers analysis allows the rapid and accurate identification of genotypes that contain gene(s) responsible for resistance against purple blotch [7,[24][25][26]. Identification of molecular markers and marker assisted selection for purple blotch disease resistance in onion has been studied before [25][26][27]; but no report of such studies with the genotypes of Bangladesh is recorded so far. Although a good number of local varieties of onion are available in Bangladesh, no recommended or released mutant lines resistant to purple blotch disease are available till now [28][29]. However, very recently, the mutant lines of BARI Piaz-2, a biennial type summer onion variety released by Bangladesh Agricultural Research Institute (BARI), had shown to possess resistance against purple leaf blotch (PLB) disease (unpublished data). The mutant lines had been developed by Bangladesh Institute of Nuclear Agriculture (BINA) by irradiating the dry seeds of BARI Piaz-2 with 75, 100, 125 and 150 Gy doses of gamma rays using the 60 Co source of Institute of Food and Radiation Biology (IFRB), Atomic Energy Research Establishment (AERES), Bangladesh Atomic Energy Commission (BAEC). Thirteen promising mutant lines in M4 and subsequent generations had been selected based upon resistance to PLB, bulb and seed yield. The Karyological studies had revealed greater chromosomal length in these mutant lines which prompted to speculate that these modifications were taken place due to the increased gene dose effect through duplication of the chromosomal region and believed to be carrying the gene (s) responsible for resistance to PLB (unpublished data). The present piece of research program has used four of the most promising onion mutant lines to characterize the presence or absence of PLB gene conferring resistance against purple blotch using SSR markers. Genomic DNA Isolation and Polymorphism Survey for Primer Selection For the SSR analysis, young, vigorously growing fresh leaf samples were collected from 25 days old seedlings of each of the plant material which were used as the source to extract genomic DNA. The leaf samples were stored at -80ºc freezer. DNA was extracted from the leaves of each genotype using the modified Cetyl Trimethyl Ammonium Bromide (CTAB) mini-prep method [30]. The quality of the isolated DNA was sufficient for PCR analysis (data not shown). Two sets of SSR markers associated with the PLB gene conferring resistance against purple blotch were selected based on their potentiality for population discrimination which was determined by preliminary experiment with three sets of primers (data not shown). The primers are shown in Table 1. PCR Amplification Profile For MatK-1RKIM-f/MatK-3FKIM-r and rbcLa-F/rbcL-R primers set, DNA amplification was performed in an oil-free thermal cycler. The reaction mix was preheated at 94ºC for 5 minutes followed by 36 cycles of 5 minutes denaturation at 94ºC, 1 minute annealing at 55ºC and elongation or extension at 72ºC for 2 minutes. After the last cycle, a final step for 7 minutes at 72ºC to allow complete extension of all amplified fragments. After completion of cycling program, reactions were held at 4ºC. Selection of SSR Markers for Detecting the Presence of PLB Gene This study was conducted to identify the mutant lines of onions that possess the PLB gene which confers resistance against purple blotch disease using simple sequence repeats or microsatellites (SSRs) markers. The SSR markers based molecular characterization experiments on onion are limited and in In vitro condition no such reliable work is reported so far. Several codominant simple sequence repeats or microsatellites (SSRs) have been reported in onion [31], of which two sets of markers, namely rbcL and matK were used to detect the presence or absence of PLB gene present in the shorter arm of chromosome 8 in the s1/s2 locus which confers resistance to purple blotch of onion. The PCR products for rbcL and matK were obtained using modified standard CCDB protocols (http://www.ccdb.ca/bCCDB_DOCS/CCDB_Ampl ificationPlants.pdf) The 67 bp rbcL nucleotide was obtained with the primers rbcLa-F [32] and rbcLa-R [33], while the 214 bp matK nucleotide was obtained with the matK-KIM primers, MatK-1RKIM-f and MatK-3FKIM-r (http://www.ccdb.ca/CCDB_DOCSCCDB_Primer Sets-Plants.pdf) as shown in Table 1. Banding Pattern by the SSR Markers and PLB Gene Detection Using the primer MatK-1RKIM-f/MatK-3FKIM-r, clear bands were observed for all the mutant lines, BP2-100/1, BP2-100/2, BP2-75/2 and BP2-100/12 of onion variety BP2 (Fig. 1) indicating the presence of PLB gene inferring the resistance against purple blotch of onion ( Table 2). By using rbcLa-F/rbcLa-R marker clear bands were also observed in BP2variety, BP2-100/1, BP2-100/2 and BP2-75/2 mutant lines mutant lines (Fig. 2) indicating the presence of PLB gene inferring the resistance. But no band was identified in BP2-100/12 mutant line which indicates the absence of PLB gene inferring susceptibility against purple blotch ( Table 3). The reason behind the failure of rbcLa-F/rbcLa-R primer sets to identify any specific band in BP2-100/12 mutant is unclear. But a possibility behind this failure could be that the predicted high amount of gene duplications in the mutants has reduced segmental homology which restricted the primers to amplify the target genes in onion. In this experiment, another unknown allele was detected between 50-75bp by the primer set MatK-1RKIM-f/MatK-3FKIM-r in BP2, BP2-100/1 and BP2-100/2 mutant lines (Fig. 1) which may be related with the PLB gene conferring resistance against purple blotch of onion or this also may be a candidate gene which trigger the PLB gene responsible for the purple blotch of onion. However, not much is known about this allele and further investigation will be needed to detect this unknown allele. CONCLUSION The presence of the PLB gene conferring resistance to purple leaf blotch in onion was successfully detected in most of the mutant lines using two sets of SSR markers. The clear banding pattern and the easy detection of the presence of this gene shows the potentiality of using this technique for screening of wider germplasm preferably using more number of related primers that can be very helpful in selecting the parents for any breeding programs or biotechnological manipulations for improving the resistance for this disease in onion in future.

v3-fos

2016-05-04T20:20:58.661Z

2015-08-21T00:00:00.000Z

10025458

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Effects of the Sequence of Isocaloric Meals with Different Protein Contents on Plasma Biochemical Indexes in Pigs Nutrient composition and pattern of food intake may play a significant role in weight gain. The aim of this study was to document the effects of a daily 3-meal pattern with isocaloric diets containing different dietary protein contents on growth performance and different plasma biochemical indexes including amino acid plasma concentration in castrated male pigs. Then, 21 DLY (Duroc×Landrace×Yorkshire) pigs aged 60 days were assigned randomly into 3 groups: a control group (crude protein, CP 18.1%), a group receiving high then basal and then low CP meals (High-Low group) and a group receiving low then basal and then high CP meal (Low-High group) for 40 days with pigs being feed-restricted. On day 40, after 12 h fasting, blood samples were obtained for analysis. The results showed that the insulin/glucagon ratio was lower in the High-Low group (P<0.05) when compared with the control group. Compared with the control group, the average daily gain of pigs from the High-Low group increased by 14.10% (P = 0.046). Compared with the control group, serum gamma-glutamyl transferase (GGT) decreased significantly (P<0.05) in both the High-Low and Low-High groups. Plasma concentrations of branched-chain amino acids (BCAA: valine, isoleucine and leucine) increased in the Low-High group (P<0.05) when compared with the control group; and plasma methionine and serine decreased in both the two experimental groups (P<0.05). Compared with the High-Low group, all the BCAA increased significantly (P<0.05) in the Low-High group. These findings suggest that the sequence and quantity of alimentary protein intake affect the insulin/glucagon ratio, as well as amino acid concentrations including BCAA, methionine and serine. It is proposed that meal pattern with pigs receiving high then basal and then low CP meals daily may help to improve the weight gain of pigs. Introduction Pattern of nutrient consumption may reset peripheral circadian clocks and the related metabolic processes [1]. Dietary proteins are believed to participate significantly in the control of blood glucose levels. High-protein (HP) diet not only affects glucose homeostasis, but also may modify the metabolism in peripheral tissues [2]. Plasma amino acid concentrations which largely depend on the food ingested is the net result of protein digestion, amino acid and oligopeptide intestinal absorption, utilization of amino acid in anabolic pathways, release of amino acids from protein etc. [3,4]. In a recent report, it has been reported that a high protein meal given in the evening (40% of energy as protein) significantly increases the plasma free amino acids (PFAA) concentration measured on the next morning, thus even more than 12 hours after the meal [5]. All aspects of physiology, including sleep-wake cycles, cardiovascular activity, endocrine system, body temperature, renal activity, gastrointestinal tract motility, and metabolism, are influenced by the circadian clock [6]. Genome-wide circadian expression profiling studies have uncovered potential connections between circadian clocks and many aspects of metabolism, including energy, carbohydrate, amino acid, lipid, and protein metabolism [7]. Diets high in fat or sugar have been shown to alter circadian clock function [8]. Recent studies have linked energy homeostasis to the circadian clock at the behavioral, physiological, and molecular levels [9][10][11], emphasizing that certain nutrients and the timing of food intake and pattern of meals may play a significant role in weight gain [12]. This represents a paradigm shift in pig feeding, since the optimal dietary nutrient level is more and more considered as a dynamic process. Hence, we made the working hypothesis that a daily 3-meal pattern with different dietary protein contents may affect biochemical circulating parameters as well as body physiology and metabolism. Blood metabolite concentrations may help in interpretating some metabolic changes associated with different dietary modifications. Thus, the present study reports on the effects of feeding a daily 3-meal pattern with different dietary protein contents daily on several parameters including blood insulin, glucagon, and AA concentrations in the growing castrated male pigs. Animals and experimental design Twenty-one DLY (Duroc×Landrace×Yorkshire) barrows aged 60 days were obtained from Hunan New Wellful Co., Ltd. (Changsha city, 410005, China) [13]. The pigs were randomly distributed in 3 experimental groups according to the average body weight (BW = 20.80±1.74 kg, n = 21): control group, High-Low and Low-High groups, with 7 animals in each experimental groups. The pigs were housed in individual pens [14]. The barrows in the control group were fed with control diet (crude protein (CP), 18.1%) at 08:00, 13:00 and 18:00h. High-Low group of pigs was fed with high-CP diet (CP, 21.0%) at 08:00h, control diet at 13:00h and low-CP diet (CP, 15.3%) at 18:00h, while the Low-High group of pigs was fed with low-CP diet at 08:00h, control diet at 13:00h and high-CP diet at 18:00h. The pigs were feed restricted, and daily food amount was adjusted to 4.0% of BW and was given to pigs in 3 meals each day, each time with about one third of the total amount, and equal feed was given in the morning and evening for each group. The nutrients were adequate for pigs and met the NRC-recommended requirement within the weight range used in the present study (NRC, 1998). The control diet was balanced on the calculated content of digestible energy (DE) (13.72 MJ/kg), CP (181.1 g/kg), apparent ileal digestible limiting amino acids (Lys, Met+Cys, Thr, Trp), Ca and P ( Table 1). The control diet, high-protein and low-protein diets were composed as indicated in Table 1. All animals had free access to drinking water. The dietary intervention lasted 40 d. Samples collection Body weights of individual pigs were measured immediately before feeding at the beginning and end of the trial [15]. On day 40, after 12 h fasting, the pigs were immobilized and then blood samples were collected by venipuncture from the venous sinus into two tubes: heparinized tubes and non-heparinized tubes [16]. The plasma was obtained by centrifugation at 3,000×g for 10 min at 4°C and then stored immediately at -80°C until assayed [13,17]. Feed intake and average daily gain (ADG) were recorded, and feed intake/ADG (F/G) was calculated for all pigs [18]. No animals were sacrificed in this study. This study was performed in accordance with the Chinese guidelines for animal welfare and approved by the Animal Care and Use Committee of the Institute of Subtropical Agriculture, the Chinese Academy of Sciences [19]. Serum biochemical indices An Automated Biochemistry Analyzer (Synchron CX Pro, Beckman Coulter, Fullerton, CA, USA) was used to determine the concentrations of serum glucose, urea nitrogen, gamma-glutamyl transferase (GGT), alanine aminotransferase (ALT), aspartate aminotransferase (AST) activities, total protein according to the commercial kits and manufacturer's instructions [20]. All the kits were purchased from Beijing Chemlin Biotech Co., Ltd (Beijing, China). Serum hormone analyses Serum insulin and glucagon were determined by radioimmunoassay according to corresponding reagent kit manufacturer's instructions (China Institute of Atomic Energy, Beijing, China) [21]. Determination of amino acids in plasma Plasma (0.5 mL) was deproteinized with 0.5 mL of 1.5 mM HClO 4 , followed by addition of 0.25 ml of 2 M K 2 CO 3 [22]. The neutralized extract was analyzed for amino acids using highperformance liquid chromatography in Beijing Aminolabs (Beijing, China). This method involved the precolumn derivatization of amino acids with o-phthaldialdehyde and fluorescence detection [23]. Amino acids in samples were quantified on the basis of known amounts of standards (Sigma Chemicals, St. Louis, MO, USA). Statistical analysis Data are presented as the mean ± SEM obtained from triplicate experiment. Differences between mean values of multiple groups were analyzed by one-way analysis of variance (ANOVA) followed by Post-hoc Tukey tests for multiple comparisons when interactions were significant using SPSS 13.0 [24]. Differences between experimental groups were considered significant for P less than 0.05. Growth performance The effects of pattern of consumption of diets with different protein contents daily on body weigh are shown in Table 2. The results showed that, compared with the control group, the ADG of pigs from the High-Low group increased by 14.1% (P = 0.046). Compared with the control and Low-High groups, F/G in the high-low group was not significantly different but tended to modestly decrease (0.05 < P < 0.1) by 12.3% and 10.5%, respectively ( Table 2). Serum biochemical indices Serum biochemical indices are shown in Table 3. Compared with the control group, serum glucose concentration from the High-Low and Low-High groups was not significantly different but tended to modestly increased by 6.2% and 10.02%, respectively. Serum GGT decreased significantly (P < 0.05) in both High-Low and Low-High groups, compared with the control group (Table 3). Compared with the Low-High group, serum LDH in the High-Low group was lower (P < 0.05). Compared with the control group, serum urea concentration was not significantly different in the High-Low group and Low-High group but tended to increase by 20.14% and 16.67% (Table 3). Serum insulin and glucagon Serum hormone concentrations are shown in Table 4. No difference was observed for insulin, but serum glucagon was higher in the High-Low and Low-High groups, the difference being significant however only in the High-Low group. Accordingly, the insulin/glucagon ratio was lower in both the High-Low and the Low-High groups than in the control one (P < 0.05) ( Table 4). No difference was observed in the serum insulin concentrations after a High-Low or Low-High meal intake. Plasma amino acids For the essential amino acids, compared with the control group, plasma valine was higher in the Low-High group (P < 0.05), plasma leucine was lower in the High-Low group (P < 0.05) and plasma methionine was lower by 20.4% (P < 0.05) and 19.4% (P < 0.05) respectively in the High-Low and Low-High (Fig 1). For the non-essential amino acids, plasma serine and taurine were lower in the two experimental groups (P < 0.05) (Fig 2). Differences were also observed between the two experimental groups; plasma concentrations of BCAA (valine, isoleucine and leucine) were larger and plasma arginine was lower in the Low-High group than in the High-Low group. Similarly, according to the dietary pattern, diets containing an equivalent amount of nutrients had different effects on several indicators of the metabolism of amino acids (Fig 3). Indeed, plasma 3-Methyl-L-histidine (MHis) was 23.1% higher (P < 0.05) and plasma homocysteine (Hyp) was x% higher (P < 0.05) in the Low-High than in the High-Low group. Discussion Circadian clock plays a crucial role in the regulation of numerous physiological processes and the beneficial versus deleterious effects of a given diet may be partly related to the sequence of dietary intake in the daytime [12]. Circadian rhythms are generated by one of the most ubiquitous timing systems related to numerous behavioral, physiological, cellular and molecular processes that are controlled by an endogenous clock which depends on environmental factors including light, food and stress [25]. Particularly, circadian control of metabolism has been widely studied. The circadian clock controls food intake and energy homeostasis by regulating the expression and/or activity of enzymes involved in the metabolism of numerous compounds including cholesterol, amino acid, lipid, glycogen, and glucose, as well as the secretion of many hormones involved in metabolism, such as insulin, glucagon, leptin, and ghrelin hormones that exhibit circadian oscillation [26]. In addition, diet-induced thermogenesis is known to show circadian variation, with the highest response in the morning and the lowest in the evening in humans [27]. The body's nutrient digestion, absorption and utilization may differ according to the different periods of time (morning, noon and night), and according to nutritional needs. Thus, eating habits may contribute to body weight changes. The results obtained in the present study, which are primarily related to optimal pig growth in an agronomic context, may be also relevant in terms of human nutrition. Pigs, like humans, are omnivorous animal and make individual meals [28]. Besides their importance in livestock production, pigs are of particular biomedical interest because of their phylogenetic relation to humans, and sharing many physiological similarities with humans, offers several specific advantages (when compared to non-human primates). Castrated pigs can avoid unwanted or uncontrolled reproduction, and are easy for management. Interestingly, ADG in response to the High-Low feeding method was higher than that measured in to the control group and, since caloric intake was an equivalent food efficiency also tended to be increased. In a recent study, modifications of the daily-phase feeding system did not significantly modify body weight but reduced N excretion by 12% (P < 0.01) [29]. Whatever the dietary pattern of the nutrient consumption (including protein consumption) during the day, some homeostasis of the circulating and tissue nutrient concentration has to be maintained. Glucose being the primary fuel source for most cells, it is for instance important for blood glucose levels to remain relatively steady. As a matter of fact, blood glucose levels may be affected by the timing of meals and snacks. Experimental data suggest that breakfast frequency and quality may be related in a causal way to appetite and blood sugar control [30]. High-CP diets have been reported to have positive effects on glucose homoeostasis in rats [31] and humans [32]. In the present study, serum glucose concentration from the High-Low and Low-High groups was not significantly different but tended to modestly increased by 6.2% and 10.02% when compared with the control group. Transamination in cells and tissues is accomplished by enzymes called transaminases or aminotransferases. This process is an important step in the synthesis of some non-essential amino acids and urea synthesis. The activities of enzymes as well as some endocrine responses are related to these metabolic pathways and are also known to be modified by time-restricted feeding [33]. Elevated levels of serum GGT have been found to predict the risk of developmenting of type 2 diabetes in adults and children [34]. In the present study, daily 3-meal pattern with different dietary protein contents affected fasting plasma GGT significantly, suggesting that the transamination system via urea-cycle is affected in the liver at 12 hours after the ingestion of a variable nutritional levels during the day. Further experiments are needed to confirm this hypothesis. However, our results may indicate that daily 3-meal pattern with different dietary protein contents may affect amino acid requirements and metabolic availability. It has been reported recently that elevated ALT levels, within the normal physiological range, are associated with unfavorable nocturnal glucose profiles in Chinese subjects with normal glucose regulation [35]. It is noteworthy that behaviors and physiological activities in animals display circadian rhythms, allowing the organisms to anticipate and prepare for the diurnal changes in the living environment [36]. Plasma amino acid profiles also exhibit a circadian rhythm [37,38]. Amino acids, normally supplied by dietary protein or by endogenous production, are necessary for anabolic metabolism and signaling purposes including the biosynthesis of polypeptides and proteins, and the synthesis of nucleotides. In the present study, we found that among amino acids, the most significant differences between experimental groups were observed for plasma BCAA concentrations and for some urea-cycle related compounds [5]. Plasma concentrations of the BCAA (leucine, isoleucine, and valine) are more prominently affected than the concentrations of other amino acids by changes in dietary-caloric, protein, fat, and carbohydrateintake in man [39]. In our study, it is interesting to note that plasma BCAA concentrations significantly increased in pigs receiving the Low-High diet in comparison with what is observed in the High-Low group; and tended to be higher when compared with the control group. Among BCAA, leucine has been shown in animal models to promote nitrogen retention and protein synthesis as well as inhibition of protein degradation [40,41]. This finding is consistent with previous observations indicating that a high protein meal in the evening significantly increased the plasma BCAA concentration on the next morning, even more than 12 hours after the meal [5], demonstrating that varying the protein content in the meal pattern with different dietary protein contents may affect the use of some essential amino acids. These changes appear partly related to the changes of transaminating enzyme activities, even if possible causal links between these parameters remain to be demonstrated. It is well known that proteins have a notable impact on glucose homeostasis mechanisms, predominantly through their effects on insulin, incretins, gluconeogenesis, and gastric emptying [42]. Insulin and glucagon are two important hormones for blood glucose regulation. Protein, but not carbohydrate, lead to increased glucagon secretion following a meal [43,44]. According to classical studies, administration of carbohydrate has a protein-sparing effect in the fasting subject [45,46].Protein induces an increase in insulin concentrations when ingested in combination with carbohydrate. It is reported that a mixture of wheat protein hydrolysate, free leucine, phenylalanine, and carbohydrate can be applied as a nutritional supplement to strongly elevate insulin concentrations [47]. The insulin/glucagon ratio determines whether glucose uptake or output production [48]. It has been reported that the insulin/glucagon ratio is highest during daylight on a high protein diet and in late night on a control diet [49]. In the present study, it is interesting to note that in the High-Low group, we found no effect on insulin, but higher plasma glucagon than in the control group (this tendency was also observed in the Low-High group). In addition the insulin to glucagon ration was very significantly decreased in both experimental groups. This implies that the varying the protein content in the meal affected plasma glucagon and increased the drive for glucose production in the fasting state. Serum glucagon concentration has been significantly correlated with protein intake [50]. Feeding a high-protein diet resulted within 3 hours in an increase in the concentration of insulin (+100%) and glucagon (+220%) [48]. Previous studies have shown that the glucagon response to feeding with protein depends on the increase in plasma amino acid concentrations [51]. It has been reported that the insulin response is closely related to the increase in plasma amino acids, especially leucine, isoleucine, valine, phenylalanine and arginine, regardless of the rate of gastric emptying; while the glucagon response is linearly related to the increase in plasma amino acids, regardless of the rate of gastric emptying or meal composition. Among the plasma amino acids, tyrosine and methionine have been closely related to the plasma glucagon response, and it has been shown that the glucagon response to feeding with protein depends on the increase in plasma amino acid concentrations [51]. Conclusion The findings reported in the present study strongly suggest that the varying the protein content in the meal affects the insulin/glucagon ratio, and amino acids including BCAA, methionine and serine in pigs. In this study, it was found that the varying meal protein content affects circulating biochemical parameters, which may impact the growth performance of pigs, and then may be useful for pig production. In practice, this study suggests that the High-Low feeding procedure improved growth and feed efficiency. Gao, Liao Hui and Peng-fei Gao from Henan Guang'an Biology Technology Co. Ltd. provided assistance during the experiments.

v3-fos

2019-04-24T13:12:11.822Z

2015-05-01T00:00:00.000Z

128969138

s2

Temporal stability of surface soil moisture of different vegetation types in the Loess Plateau of China The temporal stability of soil moisture (TSSM) was widely applied to optimize the soil moisture sampling scheme on a catchment or even larger spatial scale over wet and dry observational periods. However, the integration of the TSSM feature with specific hydrological response at a small plot scale has not been sufficiently researched. This study analyzed the temporal stability of surface soil moisture (0-10 cm) characteristics and corresponding influencing factors of different vegetation types under two typical soil moisture changing processes including wet-to-dry (WTD) and dry-to-wet (DTW), and determined the representative points. A total of 16 microplots (60 x 60 cm each) that were composed of three vegetation types containing Andropogon, Artemisia scoparia and Spiraea pubescens and bare land cover were selected. And the soil moisture in the central point (CP) and four ambient points (APs) of each microplot were measured during the WTD and DTW processes. The results showed that, 1) from DTW to WTD processes, the distribution of the soil water content in different vegetation types indicated a significant difference. Compared with the soil moisture in the AP or CP area of other vegetation types, the soil water content in tall shrub types (S. pubescens) was the lowest. 2) The autocorrelation coefficient indicated that both in the AP and CP areas, the soil moisture of the low shrub types (A. scoparia) had a higher temporal stability than that of other vegetation types. However, the soil water content in bare land had the highest temporal fluctuation from the DTW to WTD processes. Additionally, in the CP area, the TSSM of all the vegetation types tended to decrease during the WTD process. 3) Based on the TSSM analysis system that was derived from the principle of probability and statistics, the soil moisture in the low shrub types (A. scoparia) most likely provides the best representativeness of the spatial average soil water content of heterogeneous vegetation types. The determination of the representative soil moisture point via the hydrological-trait sampling method could be supplementary and significant for a TSSM study of the available soil water resources in an arid and semi-arid ecosystem. (C) 2015 Elsevier B.V. All rights reserved. Introduction Soil moisture is an indispensable stress factor in water-controlled ecosystems (Noy-Meir, 1973). In arid and semi-arid environments, the distribution of soil moisture over a specific observational period has often been studied on many different spatial scales (Bell et al., 1980;Brocca et al., 2010;Lin et al., 2006;Qiu et al., 2001), which have a farreaching impact on the runoff and sediment generation (Calvo-Cases et al., 2003;Fitzjohn et al., 1998), the dynamic balance of water in the soil-plant-atmosphere continuum of different vegetation types , and the utilization of available water resources in arid and semi-arid ecosystems. In fact, the complex influencing factors of soil moisture response indicated that the soil water has a high variability at different spatiotemporal scales. As a result, a comprehensive analysis of the variable spatiotemporal characteristics of soil moisture is necessary to understand the behavior of soil water in arid and semi-arid environments. Under this background, the concept of the temporal stability of soil moisture (TSSM) was proposed by Vachaud et al. (1985), and was defined as the time invariant association between the spatial location and statistical parametric values based on the probability density function of the soil moisture. TSSM specifically indicates that the order of the soil moisture or of the average soil moisture arranged in the spatial pattern does not fluctuate with the observational period (Kachanoski and de Jong, 1988). The succession of the interval observation period, the homogeneity of the spatial distribution of soil moisture, and the coupling of the spatial and temporal patterns of the soil water content constituted the main elements of TSSM research. First, different spatial scales of TSSM studies were systemically investigated by many researchers. These scales ranged from multiple investigated fields scales (300 m 2 per field) (Brocca et al., 2010) to hillslope scales (approximately 900 m 2 ) (Coppola et al., 2011;Penna et al., 2013), from watershed scales (610 km 2 and 1285 km 2 respectively) to an even larger landscape scale (Martinez-Fernandez and Ceballos, 2003;Starks et al., 2006). In the Loess Plateau, many researchers (Gao and Shao, 2012;Hu et al., 2010;Jia and Shao, 2013) have also investigated the TSSM in different watersheds. Second, aiming to analyze the TSSM characteristics under extreme soil water conditions, the temporal pattern of soil moisture initially focused on two different observation periods representing the wet and dry soil moisture conditions (Grayson and Western, 1998;Grayson et al., 1997). For instance, Penna et al. (2013) systematically reported the TSSM characteristics of different soil depths under hillslope scales during the wet and dry periods in the three years. Heathman et al. (2012) divided the temporal pattern of soil moisture into three patterns-time-averaged, wettest and driest period-and mutually compared corresponding TSSM characteristics at field scale. Other researchers (Brocca et al., 2010;Gomez-Plaza et al., 2000;Williams et al., 2009) also analyzed the TSSM in the wet and dry periods on watershed and landscape scales. However, the TSSM characteristics in the two temporal patterns under different spatial scales are still debatable (Gomez-Plaza et al., 2000;Martinez-Fernandez and Ceballos, 2003;Williams et al., 2009;Zhao et al., 2010), because the main influencing factors of the TSSM-such as topography (Brocca et al., 2007(Brocca et al., , 2009, soil texture Gao and Shao, 2012;Porporato et al., 2001;Starks et al., 2006), precipitation and vegetation types (Brocca et al., 2009;Jia and Shao, 2013;Mohanty and Skaggs, 2001)-are complicated and vary with the spatial scale. Third, based on the principle of probability and statistics, Vachaud et al. (1985) introduced a series of indices into the TSSM calculation system that could quantify the TSSM characteristics. Mittelbach and Seneviratne (2012) indicated that the rank stability index could be the best way to characterize the temporal stability pattern through relative long-term measurement of soil moisture. And other TSSM evaluation indices contain the cumulative probability, autocorrelation coefficient, and mean/standard deviation of relative differences, which were derived from the probability density function, time series analysis, and statistical inference respectively. Moreover, other researchers (Jacobs et al., 2004;Penna et al., 2013;Zhao et al., 2010) further integrated these statistical methods and defined the index of temporal stability. And some new mathematical tools were also used to analyze the TSSM, such as the combination of climate simulation with TSSM characteristics (Matinez et al., 2014), geostatistical method (Brocca et al., 2009), spatial autocorrelation technique (Biswas and Si, 2011) and wavelet coherency analysis algorithm. The integration of these TSSM analysis methods and remote sensing technique could effectively promote the precision of the soil moisture estimation at large spatial scales Fig. 1. Description of research area and hydrological process-trait sampling method. (a) Study area and CP/APs' sampling sketch, (b) four types of microplot. The black square, round, triangle and diamond dispersing topographic map represent four different types of microplot including the bare (plot 1), Andropogon (plot 2), Artemisia coparia (plot 3), and Spiraea pubescens (plot 4) respectively. For each type there are four microplots whose codes were displayed in parentheses. Black circle represents the CP/APs circle area whose radius was near 5-8 cm. (Jacobs et al., 2004;Martinez-Fernandez and Ceballos, 2003;Mohanty and Skaggs, 2001). Therefore, the application of TSSM-based sampling method, the determination of the temporal pattern of soil moisture, and the construction of the TSSM calculation system were all the preparation for determination of representative soil moisture points or locations. This representativeness could effectively reflect the mean soil water content in a specific study area during the successive observational interval (Grayson and Western, 1998). Many studies (Vachaud et al., 1985;Van Pelt and Wierenga, 2001) have demonstrated that the application of the TSSM analysis system to determine representative points successfully optimized the spatial sampling points of the soil moisture and reduced the uncertainty of the soil water distribution estimation at different study regions. However, in a previous TSSM study, two types of soil moisture uniform sampling strategies including gridding and network sampling methods were used. The uniform configuration of the soil moisture monitoring points probably neglected the information about the diversity of vegetation type distribution in different land uses, as well as the corresponding impact on TSSM characteristics. Moreover, because of the temporal scale of TSSM in previous studiessuch as time-averaged, wet and dry periods of soil moisture-being relative coarse, the relationship between specific hydrological response including dry to wet (DTW) condition or wet to dry (WTD) condition processes and the corresponding TSSM characteristics has not been sufficiently analyzed. Therefore, this study combined WTD or DTW processes with corresponding TSSM characteristics, which could be beneficial to exploring and understanding the TSSM feature of heterogeneous vegetation distribution on a specific hillslope. We aimed to 1): evaluate the temporal pattern of soil moisture in different types of microplot over WTD and DTW processes affected by the integration of precipitation and radiation; 2): analyze the different soil moisture responses of vegetation types on WTD and DTW processes through using the hydrological process-trait sampling method; and finally 3): analyze the influence of different vegetation types on the corresponding TSSM during WTD and DTW processes by determining the representative points which reflect the mean soil water content in a specific hillslope. All of these contents could provide supplemental research concerning the temporal characteristics of the available soil water resources in a water-limited ecosystem. These findings could be beneficial to understanding hydrological mechanisms from the TSSM point of view and be significant for evaluating the influence of vegetation layout on the use of available water resources in water-controlled ecosystems through challenging issues of interdisciplinary research fields that are related to soil moisture. Description of study area The experiment was conducted in the Yangjuangou Catchment (36°42′N, 109°31′E, 2.02 km 2 ) site in the central part of the Loess Plateau (Fig. 1a). The elevation of this site ranges from 1050 m to 1298 m, and the slope gradient mainly fluctuate between 17.6% and 57.7%. The precipitation is influenced by North China monsoon, distributes significant inter-annual differences, occurs between June and September and totals approximately 535 mm per year . The soil type in the catchment is Loessal soil, which has a weak structure and high erosion sensitivity (Gao and Shao, 2012;Li et al., 2003;Wang et al., 2009). The main soil characteristics of the microplots are shown in Table 1. The dominant vegetation types include Stipabungeana (Andropogon), Hippophae rhamnoides, Artemisia scoparia, Spiraea pubescens and Prunus armeniaca var. ansu . We designed 16 square microplots (60 × 60 cm for each) including bare land cover (plot 1), and for another three vegetation types, the dominant plant communities are Andropogon (plot 2), A. scoparia (plot 3), and S. pubescens (plot 4). The morphological characteristics of vegetation were shown in Table 2, which describes the main statistical features of the plant. Under the background of the Grain-for-Green Project implementation in the 1980s, nearly all of the intensive-humandisturbed croplands on hillslopes, in the Yangjuangou catchment, had been returned to vegetation land which effectively prevented soil erosion. All of the vegetation in the microplots that was revegetated in the 1980s had grown for 20 years in the catchment. All microplots were randomly distributed on a 30-meter-long southwest-northeast aspect 26.8% heterogeneous vegetation hillslope (approximately 600 m 2 ). Each microplot including one single plant was fenced by four impervious PVC sheets with a thickness of 2 mm and width of 800 mm. All the sheets were perpendicularly inserted into soil layer down to a depth of approximately 50 cm. We focused on the TSSM in the surface soil layer (0-10 cm), therefore, the inserted PVC sheets could effectively prevent the soil water from generating lateral diffusion, which probably reduced the impact of the lateral effect on the accuracy of the soil moisture. Hydrological process-trait sampling method In order to compare the TSSM characteristics of the three vegetation types in different locations of microplot, we classified the soil moisture measurement points into the central point (CP), and ambient points (APs) as shown in Fig. 1b. The CP/APs' classification concerning the different influences of the vegetation morphology on WTD and DTW processes could be regarded as the hydrological process-trait sampling method. The TDR-300 Soil Moisture Meter (Spectrum Technologies, Inc, Aurora, Illinois, USA) was used to measure the soil volumetric water content in 0-10 cm depth soil layer. Two types of vegetation microplots (plot 3 and plot 4) have complex canopy structures that make the use of the CP/AP method more difficult than that of bare (plot 1) and grass (plot 2) types. Therefore, before measuring in these vegetation microplots, the first step was to determinate the CP and AP sampling areas that were characterized as circle areas of radius 5-8 cm (Fig. 1a). This step could reduce the probability of the TDR probes touching the main root or the huge soil pores that were formed by the main roots in the CP position throughout the measuring processes. It also could prevent the probes of the TDR from repeatedly disturbing the same point. The second step was to softly remove the litter layer covering over the surface of the CP and APs circle area of plot 3 and plot 4 with a brush before measurement. When the logging-data process was finished, every disturbed hole that was caused by probes was carefully filled with fine soil particles, after which the litter layer on the CP or APs circle area was recovered. Determination of the temporal pattern Temporal pattern of soil moisture consists of two typical processes. One is DTW process influenced by precipitation events, and the other process is the WTD process affected by continuous high temperature events; the average time interval of soil moisture measurement in the temporal pattern is two days. Each of the process composes an entire typical soil moisture response or pulse (Eagleson, 1978a). We selected three typical soil moisture responses to analyze the TSSM characteristics in CP and APs' sampling areas. The calculation of the TSSM by statistics The TSSM calculation system consists of four indices including mean relative difference, standard deviation, temporal stability index, and cumulative probability distribution. All the indices describe the statistical properties of TSSM. Regarding the CP area, the mean relative difference of TSSM (TSSM-MRD) was calculated as follows: where θ CP(i,j) is the soil moisture of the CP area on the ith microplot (i = 1-16) at jth observation time (j = 1-2, indicates the DTW process; j = 3-7, indicates WTD process), and θ CP ÃÁ j ð Þ represents the average soil moisture in the CP of all of the microplots at the jth time. Therefore, Δ CP(i,j) and δ CP(i,j) reflect the fluctuation of the soil moisture and relative difference in the CP at the corresponding microplot and observation time, respectively.With respect to the APs, the relative difference was calculated using Eqs. (4)-(7) as follows: where θ AP i; j ð Þ is the average soil moisture of the four APs that were located in different positions (short for p) on the ith microplot at the jth time. In all of the microplots, the average soil moisture in the APs at the jth time is displayed as θ AP ÃÁ j ð Þ . Finally, the TSSM-MRD of the CP and APs (δ CP i; j ð Þ n , δ AP i; j ð Þ n ) was calculated using Eqs. (8) and (9), respectively. n = 1-2, indicates the DTW process; n = 3~7, indicates the WTD process-the same relationship is used below Furthermore, if we synthesized the TSSM-MRD of the CP and APs, and obtained the TSSM-MRD of the specific microplot from Eqs. (10) to (14), which represents a similar meaning as the above equations, then where the TSSM-MRD indicates the fluctuation of every measuring points (Vachaud et al., 1985) compared with the average soil water content value throughout the DTW or WTD processes in the CP or APs' sampling areas. Specifically, the more closely the absolute value of TSSM-MRD approaches zero, the more likely the corresponding soil moisture represents the mean-soil-moisture in the entire spatial patterns that are formed by the CP or APs' distribution throughout the corresponding hydrological processes. Moreover, the value of TSSM-MRD indicates that whether the value of the soil moisture measurement point overestimates ( (Vachaud et al., 1985) the average value throughout the DTW or WTD period. Based on Eqs. (8), (9), and (14), the standard deviation of the MRD of TSSM (TSSM-STD) in the CP, APs and microplot The TSSM-STD reflects the fluctuation of the TSSM-MRD in a specific type of soil moisture measuring point. If the absolute value of the TSSM-STD in a given microplot or CP or APs approaches zero more closely, then this value is considered to be better representing the lower fluctuant of TSSM-MRD, and higher stability process over which the corresponding soil moisture gradually approached the entire-spatial-pattern average soil moisture. Additionally, the index of temporal stability (TSSM-ITS) in the CP, AP and microplots (ITS CPn , ITS APn and ITS Mn ) combines the TSSM-MRD and Fig. 6. TSSM-MRD/STD distribution of all microplot types over the third WTD and DTW processes. The codes in all symbols were short for the name microplot, such as P2(3) which means the plot 2(3), and the blue codes represent that the corresponding microplot has the same rank value over DTW and WTD processes. Additionally, the blue codes with star mark mean the corresponding same-rank-value microplots that have the lowest absolute value of TSSM-MRD over the DTW and WTD processes. TSSM-STD (Jacobs et al., 2004;Penna et al., 2013;Zhao et al., 2010) and uses CP as an example: The lower value of ITS in the CP or AP or some microplots indicates the higher TSSM of the corresponding soil moisture measuring point or location or vice versa. The calculation of the TSSM by a probability function The cumulative probability distribution in the TSSM (Vachaud et al., 1985) (TSSM-CumP) calculation in the CP can be used as an example in Eq. (17) as follows: where θ CP iÁÃ ð Þ is the average soil moisture of the ith microplot (i = 1-16) at CP location throughout DTW or WTD processes (j = n = 1-2, indicates the DTW process; j = n = 3-7, indicates the WTD process). The different values of θ CP iÁÃ ð Þ were ranked from lowest to highest, such as , in which the number in the square bracket indicates the order of the average soil moisture. Additionally, the average soil moisture in the CP areas of all of the microplots over n times is expressed by Eq. (18): Therefore, the probability of the lowest average soil moisture in the CP point of some microplots over n observational times p θ CP 1ÁÃ ½ À Á could be calculated by Eq. (19): Based on this equation, the TSSM-CumuP of the kth highest soil moisture in CP point of some microplot, CumuP θ CP kÁÃ ½ À Á could be expressed by Eq. (20): The TSSM-CumP reflects the rank stability of the soil moisture based on the probability density function conforming to a normal distribution. Moreover, if the TSSM-CumP of the soil moisture in the CP or AP points or a specific microplot has the same cumulative probability distribution Table 3 The characteristics of indices in TSSM analysis system over three DTW and WTD processes. Note: a means the TSSM analysis of CP area in four types of microplots over the first soil moisture pulse; b means that the TSSM-CumP of plot 1(1) was 0.49; c and d mean that based on the TSSM-MRD calculation, the soil moisture in plot 1(2), plot 1(3), plot 1(1), and plot 1(4) was on overestimation and underestimation condition respectively; e means no microplot meet the corresponding condition. in both the WTD and DTW processes, but the corresponding TSSM-CumP value is approximately 0.50, then the corresponding soil moisture in the CP or AP areas or a specific microplot could be characterized by the spatial mean soil moisture content. Results 3.1. The temporal pattern of the soil moisture-response Fig. 2 indicates the long time average soil moisture dynamics of the four microplot types which were influenced by the 10 obvious rainfall events (precipitation N 5 mm) and a total of 22 high temperature events (average temperature N 30 centigrade) during the rainy season of 2011 and 2012. There exists three typical soil moisture dynamics processes distributing throughout 2011A (June 30th, to July 14th, 2011), 2011B (from July 26 to August 15th, 2011), and 2012 (from July 18th to August 12th, 2012) respectively, which were indicated in different vegetation types and bare land on microplot (Fig. 3), AP (Fig. 4) and CP (Fig. 5) scales. Affected by the precipitation in 2012, the soil water content in the same vegetation types or bare land seemed to be higher than that of 2011A and 2011B throughout both the DTW and WTD processes. Under the same precipitation and radiation conditions, in the CP area, the fluctuations of the soil moisture in the vegetation types such as plot 3 and plot 4 ranged from 6.4% to 24.9% as well as from 6.3% to 22.0% respectively, which were more obvious than the corresponding types in the AP area at every observational interval. This diversity was more distinct between the CP and AP areas when the soil moisture changed from WTD to DTW. Meanwhile, in the same temporal pattern of soil moisture, compared to the vegetation types, the soil moisture response fluctuation of the bare land (plot 1) at CP (ranging from 6.1% to 26.7%), AP (ranging from 6.7% to 26.0%) and microplot (ranging from 6.7% to 26.4%) scales seemed to be more obvious than the corresponding positions in all of the observational intervals. Moreover, throughout the same DTW process, the average soil moisture in the bare land was larger than that of the vegetation types at the corresponding CP, AP and microplot scales. During the same WTD process, in the CP, the soil moisture in the vegetation microplots (plot 3 and plot 4) which averagely reduced 12.1% and 11.5% respectively seemed to be decreased more obviously than that of the bare land in the corresponding area. This phenomenon was more evident when the observational intervals were from August 3rd to August 12th in 2012 during the WTD processes of the third soil moisture response. TSSM-MRD/STD/ITS characteristics of different vegetation types According to the TSSM analysis calculation system, we used the third soil moisture responses as example to analyze TSSM characteristics. In Fig. 6, the TSSM-MRD and TSSM-STD distribution of all of the vegetation types and bare land under DTW and WTD processes at CP, AP and microplot scales is indicated. With respect to CP area, the rank value of plot 4(3) and plot 4(1) is 8 and 15 respectively, both of which have the same rank value over DTW and DTW processes. And other two bare microplots plot 1(4) and plot 1(2) whose rank values are 13 and 16 respectively also have the same rank values. Regarding the AP area, a grass microplot (plot 2(4)), a low shrub microplot (plot 3(2)), a tall shrub microplot (plot 4(4)) and a bare microplot (plot 1(4)) whose rank values were 8, 10, 3 and 14 respectively, have the same rank values throughout the third DTW and WTD processes. Moreover, as to the microplot scale, plot 1(2), plot 1(3) and plot 1(4) have the same rank value which were 16, 15 and 6 respectively, In addition, the samerank-value microplot whose absolute values of TSSM-MRD approaching zero indicated stronger TSSM and, better representativeness of the average soil moisture given the corresponding temporal pattern. Therefore, from the view of statistical characteristics of the TSSM, the Fig. 7. The TSSM-ITS distribution of all microplot types at AP, CP and microplot scales over three DTW and WTD processes. The code on the x-coordinate was the short for microplot name, such as P11 which means the plot 1(1). And the star mark indicates the lowest sum of TSSM-ITS at AP, CP and microplot scales over DTW or WTD process. soil moisture in plot 3(2) indicated a higher temporal persistence at the AP and microplot scales during the third soil moisture response process, and the soil water content in plot 4(3) represented stronger temporal stability in the CP areas throughout the DTW and WTD processes. And in the three soil moisture responses during the rainy seasons, the TSSM-MRD and TSSM-STD characteristics at the CP, AP and microplot scales are presented in Table 3. Additionally, the TSSM-ITS characteristics of all of the vegetation types and bare land at the CP, AP and microplot scales throughout three different soil moisture responses are shown in Fig. 7. For the first soil moisture response (2011A), throughout the DTW process, the sum the of TSSM-ITS at the CP, AP and microplot scales equaling 0.26 in plot 4(1) was the lowest in the 16 microplots, however, the lowest corresponding sum of the TSSM-ITS in all of the microplots appeared in plot 3(2) equaling 0.19 throughout the WTD process. Regarding the other two soil moisture responses in 2011B and 2012, the sum of the TSSM-ITS in plot 3(2) equaling 0.12 and 0.22 respectively in the DTW and WTD processes, and the lowest TSSM-ITS indicated that the soil moisture in plot 3(2) had more obvious temporal persistence throughout the hydrological processes. Finally, in Table 3, the TSSM analysis system including TSSM-MRD, TSSM-STD, TSSM-ITS in DTW and WTD processes indicates the difference of TSSM characteristics in the four microplot types. TSSM-CumP characteristics of different vegetation types The TSSM-CumP that was calculated by Eq. (20) was characterized as the probability-trait index to evaluate the TSSM features. The TSSM-CumP characteristics at the CP over DTW and WTD processes in 2012 are shown in Fig. 8. And values of TSSM-CumP of all vegetation types under CP, AP and microplot scales, and the determined microplots whose TSSM-CumP value approaches to 0.5 were also shown in Table 3. In the CP area, throughout the WTD and DTW processes, two high shrub microplot (plot 4(1) and plot 4(3)) and plot 1(4) had the same cumulative probability distribution respectively. In the three microplots, the TSSM-CumP of plot 4(3) equaling 0.60, was more likely to be 8. The TSSM-CumP distribution of CP in DTW and WTD processes of 2012 rainy season, the red symbols on the left side indicates the distribution of cumulative probability of soil moisture in CP of different microplots over WTD processes, and the black marks on the right side displayed the distribution of CP in different microplots over DTW processes. Additionally, the blue codes represent the corresponding microplot that has the same TSSM-CumP value over DTW and WTD processes, the blue codes with star mark means the TSSM-CumP values in corresponding microplots relative closed to 0.5. approximately 0.50. Therefore, regarding the probabilistic characteristics of the TSSM, the soil moisture in plot 4(3) indicated a greater TSSM and most likely represented the average soil moisture of all of all CP areas that were distributed on the hillslope throughout the third soil moisture pulses process. Moreover, in Table 3, regarding to the AP scales, there were more microplots with the same cumulative probability distribution throughout the two hydrological processes, although, the soil water content in plot 3(2) seemed to be more representative of higher temporal stability characteristics. At the microplot scale, compared with the other three bare lands (plot 1(2), plot 1(3) and plot 1(4)) which had the same cumulative probability distribution throughout the DTW and WTD processes, the TSSM-CumP of the low shrub microplot (plot 3(2)) seems more close to 0.5. Time series analysis (TSA) of the temporal pattern of soil moisture TSA is widely used to evaluate the temporal pattern of soil moisture, (Brocca et al., 2009;Heathman et al., 2012;Martinez-Fernandez and Ceballos, 2003;Vachaud et al., 1985) and a higher autocorrelation coefficient indicates a lower temporal fluctuation of soil moisture. In this research, the autocorrelation coefficient evaluation describes the temporal dynamic characteristics of soil moisture throughout the entire hydrological responses. A hydrological response consists of both DTW and WTD processes which were affected by the precipitation and radiation respectively. The distribution of the autocorrelation coefficient at the CP, AP and microplot scales throughout three soil moisture responses of different vegetation types is shown in Fig. 9. The autocorrelation coefficient of all vegetation types in the CP and AP areas decreased with the increasing observational duration, especially during the WTD process (the time lag from 2-3 to 6-7 in Fig. 9). TSA in CP, AP and microplot scales indicates that the consumption of water in the soil layer by evapotranspiration increased the fluctuation of the soil moisture throughout the WTD processes. The characteristics of temporal of soil moisture pattern are, to some extent, similar to those of Gomez-Plaza et al. (2000) who also reported that the heterogeneity of a plant could decrease the TSSM. However, there are also some differences in our study from the conclusion of Gomez-Plaza et al. (2000). Specifically, in the AP area, the autocorrelation coefficient, especially at the end of the WTD process (time lag from 5-6 to 6-7), stopped decreasing and even tended to increase, probably related to litter layer playing the act on restricting surface soil water from transpiring (Hartanto et al., 2003). Therefore, in the complex root-soil interface environment of soil water movement that is created by the root system extending in the soil layer of vegetation types (Caldwell et al., 1998;Dawson, 1993;Eagleson, 1978bEagleson, ,1978cHorton and Hart, 1998;Porporato et al., 2001Porporato et al., , 2002Rodriguez-Iturbe et al., 2001), the dynamic balance of water resources-such as water conservation caused by the litter layer and water consumption affected by the root system distribution in plot 3 and plot 4-could probably stabilize the temporal fluctuation of soil moisture. Furthermore, with respect to the former TSSM study related to the vegetation types, based on the TSA, Penna et al. (2013) indicated that, in the two contrasting morphology hillslopes, the characteristic of the TSSM was mainly related to the topographic properties. However, due to the homogeneous vegetation distribution on the hillslopes, these researchers neglected the influence of vegetation on the temporal pattern of soil moisture. Other researchers (Brocca et al., 2009;Jia and Shao, 2013;Mohanty and Skaggs, 2001) also considered the impact of vegetation on the TSSM, although the combination of TSSM characteristics with hydrological response that are impacted by vegetation seemed have not been sufficiently considered. Similar to the explanation of Zhao et al. (2010), the root structure distribution in the soil layer and the substances covering the soil surface caused to plant have a highly Fig. 10. Least-significant difference (LSD) analysis of soil moisture in different microplot types on CP and AP area, the same letter means no significant difference at the 0.05 level. dynamic water demand, which ultimately complicates the characteristics of the TSSM. Therefore, these authors suggested that it is necessary to detail information of vegetation dynamics, which could be beneficial to correlating the corresponding soil water content and to the TSSM study. In our study, the analysis of the TSSM characteristics of the CP/APs area in vegetation types could be regarded as the response to their suggestion. In fact, three response components-the canopy being on the ground, the litter layer covering the soil surface, and the root system dispersing in the subsurface-played different roles in the hydrological response to both the DTW and WTD processes, which finally formed the tradeoff between the water evapotranspiration and conservation processes (Zhao et al., 2010), and affected the TSSM characteristics of the CP and AP in vegetation types. Moreover, Fig. 9 also shows that, throughout the DTW process (the time lag from 1-2 to 2-3), compared with the vegetation types (plot 3 and plot 4), the temporal persistence of the soil water content in the bare land (plot 1) and grass land (plot 2) seemed to be less at the AP, CP and microplot scales, indicating that the direct disturbance of erosive rainfall on the soil moisture pattern could decrease the corresponding TSSM. Specifically, due to the lack of litter a layer and root structure to effectively resist the soil erosion (Gyssels et al., 2005) in plot 1 and plot 2, a greater probability of soil erosion could be generated, thereby increasing the temporal fluctuation of the corresponding soil moisture throughout the precipitation process. However, these complex soil water movement processes on the soil surface, the root-soil interface and the subsurface could make it difficult to determine the specific response units of the soil water dynamics and to interpret all of the TSSM characteristics in the AP and CP areas of the vegetated and bare microplots. Influential analysis of vegetation types on soil moisture response in the CP/APs Similar soil moisture response existed in different vegetation types and bare land, although, regarding the different microplot types, throughout the same DTW and WTD process, the soil moisture response in the same sampling area (CP or AP) indicates significant differences, shown in Fig. 10. These differences indicate that the diversity of the vegetation morphological characteristics lead to the more complicated hydrological response recycling in the soil-plant-atmosphere continuum (Li, 2011), and impacts the TSSM characteristics of different vegetation types (Zhao et al., 2010). First, in the CP area of the vegetation types, the soil moisture response is mainly affected by the porous structures being beneficial to infiltration or evaporation, the stem structures generating stemflow (Levia and Frost, 2003;Li et al., 2009), and the precipitation distribution. Specifically, throughout the DTW processes, due to the tall shrub microplots (plot Table 4 Determination of representative points of CP/APs and microplots over three DTW and WTD processes. (2) 4) having more obvious porous structures in the CP area, under relatively low precipitation conditions-such as the rainfall events in 2011, there existed more water infiltration to deeper soil layers, making the surface soil moisture in plot 4 lower than that of the other microplot types. However, with the increased of frequency and amount of rainfall in 2012, the higher porosity structure in the CP areas of plot 4 could retain more moisture than other microplots on the soil surface when the soil was under saturated conditions. On the other hand, throughout the WTD processes, evapotranspiration was one of the fundamental patterns of soil moisture loss on the ground. Therefore, it led the vegetation types such as plot 3 and plot 4 to have a higher rate of water loss and caused these two vegetation types to have significant lower surface soil moisture than the bare land. This study was also similar to Wang et al. (2012) who systematically analyzed the soil moisture of five different revegetation types that were impacted by the evapotranspiration in the Loess Plateau. And this huge consumption of water resources by vegetation could potentially cause the formation of a drier layer in the Loess Plateau (Chen et al., 2008a(Chen et al., , 2008b(Chen et al., , 2010Wang et al., 2012Wang et al., , 2013. Secondly, in the AP area, rather than a part of the soil crust being distributed on the surface of plot 1, indicated in Table 2, the obvious crown structure and different thickness litter layers covering the surface as well as the root system distribution of plot 3 and plot 4, could play a complicated role in intercepting precipitation retaining water resources and finally preventing soil erosion (Gyssels et al., 2005) throughout whole WTD processes. Moreover, throughout the DTW processes, due to the positive correlation between the canopy structure and the thresholds of rainfall interception , a wider average crown above the AP area of plot 4, could lead this vegetation type to have a lower probability of receiving intermittent throughfall than plot 3; meanwhile, the thicker litter layer covering on plot 4 probably has a greater ability than plot 3 to retain through fall on the soil surface and to prevent water from entering the soil medium. Therefore, during the water input processes, both of the crown and litter layer characteristics lead to the significant lower soil moisture in the AP areas than other vegetation types. In addition, Villegas et al. (2010) explored the hierarchical effects the of litter layer on restricting the evaporation of the surface soil moisture, although in our study, the litter layers' ecohydrological ability of soil evaporation control (Villegas et al., 2010) in the AP area seemed to be less than the water consumption through evapotranspiration especially triggered by high temperature weather conditions, which finally, cause the vegetation types to have a significantly lower soil water content than that of bare land in the AP area throughout the WTD processes. Representative soil moisture point distribution in the CP/APs Representative soil moisture point that represented the greatest temporal stability point of all of the moisture measuring points was derived from the consideration of the spatial optimized sampling scheme (Grayson and Western, 1998). Based on the TSSM analysis calculation system, the representative soil moisture reflects the spatial mean soil water content in the corresponding study area. Previous researchers (Martinez-Fernandez and Ceballos, 2003;Van Pelt and Wierenga, 2001) mainly focused on the representative points at the catchment scale, or even larger spatial scales. Penna et al. (2013) downscaled the spatial scale to hillslopes and determined the representative points of different soil depths. However, in our study, by further downscaling the representative point to single vegetation scale, we combined hydrological response with determination of the representative point distribution in the CP/APs' areas of different vegetation types and bare land cover, which could more effectively explore and comprehend the TSSM information as well as its influencing factors over the whole WTD and DTW processes (Lin et al., 2006). In Table 4 the representative point determination system indicated that, over DTW and WTD processes in two rainy season, TSSM characteristics in the low shrub types (plot 3) at the AP, CP and microplot scales, illuminated that the soil moisture in plot 3 could represent the spatial average soil moisture of the hillslope on which the heterogeneity vegetation types and bare land cover were distributed. In our study, the vegetation type (plot 3) characterized as the highest TSSM type could be regarded as the representative soil moisture types in all microplots, which, however, was seemed to be different from the research of Gomez-Plaza et al. (2000) who indicated that the vegetation increased the temporal fluctuation of soil moisture. And the difference was probably related to the different researching scales. Specifically, Gomez-Plaza et al. (2000) mainly focused on the soil moisture representative point at the catchment scale, but we mainly stressed the representative points at a plant scale by integrating the ecohydrological information with the corresponding TSSM characteristics under heterogeneous vegetation. Generally, Table 5 concluded all the possible influencing factors of TSSM characteristics in the four microplot types, and these factors existing in different hydrological environments and generating various hydrological behaviors also ( played the key roles in determining the representative points which reflected the mean soil water content in a specific hillslope during WTD and DTW processes. Compared with the bare land cover (plot 1), grass land (plot 2), and high shrub (plot 4), the complexity of the influencing factors-including morphological characteristics above the ground and soil surface properties affected by litter layer in the low shrub microplot (plot 3)-may be moderate and the corresponding hydrological function leads to the highest TSSM in all of the microplots, which caused plot 3 to have the best representativeness of the average soil moisture. Specifically, the hydrological response in plot 3 throughout the precipitation and radiation periods was more complex than that of plot 1 and plot 2. Because, in plot 3, the canopy structure, litter layer and the deep root system all respectively played different roles in redistributing the input and output patterns of water resources (Li et al., 2009). However, in plot 1 and plot 2, similar to some researchers' view (Gomez-Plaza et al., 2000;Jacobs et al., 2004) the soil hydrological conductivity, soil curst, probably became the main influencing factors of water resource distribution. According to some studies (Garcia-Estringana et al., 2010;Levia and Frost, 2003), the obvious stem structure in plot 4 could have high probability to generate the stemflow which would cause plot 4 to be more regarded as a precipitation collection system (Li, 2011) than plot 3 in arid and semi-arid ecosystems. Additionally, the more complex hydrological behavior in root-soil interface environment of plot 4-such as the preferential flow (Li et al., 2009), plant hydraulic lift, and the dynamics between the soil water potential and the xylem osmotic (press) potential )-could also cause the corresponding soil moisture to experience stronger temporal fluctuant process than plot 3. Furthermore, in Fig. 11, the linear regression analysis and root mean square error (RMSE) calculation was used to test the representativeness of soil moisture in plot 3 through comparing its soil moisture with the average soil moisture of all microplots. And the figure also indicated that the soil moisture in plot 3 could be the best choice of representative soil moisture point in the CP, AP as well as in microplot scales over the WTD and DTW processes. In addition, as to the influence of CP/APs sampling method on the measurement results, the disturbance of the soil layer during the soil moisture measurement processes-including designating the CP/APs' sampling circle areas, removing and recovering the litter layer condition as well as mending the disturbing hole-also inevitably brought system error into the analysis of the TSSM characteristic and the determination of the representative points. It was similar to the discussion of Zhao et al. (2010) who indicated that inaccurate measurement could cause errors in the TSSM analysis, and the fixing soil-moisturemeasuring tubes (Jost et al., 2012) into the soil layer could be an efficient method to reduce errors in future studies. Conclusions In this study, we used the hydrological-trait soil moisture sampling method to analyze the temporal persistence characteristics of the soil moisture as well as to determinate the representative soil moisture points in heterogeneous vegetation and bare microplot distribution on a hillslope of the Loess Plateau, China. And the main conclusions can be summarized as follows: precipitation and the duration of high temperature conditions could impact the soil moisture of different microplots throughout the DTW and WTD processes. The soil moisture of different microplots at the same sampling points (AP or CP) indicated significant difference under WTD or DTW processes. And the soil moisture in tall shrub microplots (plot 4) tended to be lower than other three types. The TSSM of the low shrub microplots (plot 3) at the AP, CP and microplots scales seemed to be higher than that of the other vegetation types. In the CP area, the TSSM of the vegetation types tended to decreased over whole WTD processes, although, at the end of the WTD process, the degree of the temporal fluctuation of soil moisture in the vegetation microplots in the AP area tended to decrease. Based on TSSM-MRD/STD/ITS/CumP analysis as well as the RMSE test, the plot 3 could be regarded as the representative point at the AP, CP and microplot scales, which represented the spatial mean soil water content of the heterogeneous vegetation types and bare land cover. The study on TSSM of different vegetation types supplemented the soil moisture temporal properties affected by vegetation types, and could be beneficial to understanding how to design the vegetation layout to reasonably use the available water resources in a water-limited ecosystem.

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Mentha suaveolens Ehrh. (Lamiaceae) Essential Oil and Its Main Constituent Piperitenone Oxide: Biological Activities and Chemistry † Since herbal medicines play an important role in the treatment of a wide range of diseases, there is a growing need for their quality control and standardization. Mentha suaveolens Ehrh. (MS) is an aromatic herb with fruit and a spearmint flavor, used in the Mediterranean areas as a traditional medicine. It has an extensive range of biological activities, including cytotoxic, antimicrobial, antioxidant, anti-inflammatory, hypotensive and insecticidal properties, among others. This study aims to review the scientific findings and research reported to date on MS that prove many of the remarkable various biological actions, effects and some uses of this species as a source of bioactive natural compounds. On the other hand, piperitenone oxide (PO), the major chemical constituent of the carvone pathway MS essential oil, has been reported to exhibit numerous bioactivities in cells and animals. Thus, this integrated overview also surveys and interprets the present knowledge of chemistry and analysis of this oxygenated monoterpene, as well as its beneficial bioactivities. Areas for future research are suggested. Introduction Essential oils are volatile, natural, complex compound mixtures characterized by a strong odor. They arise from the secondary metabolism of the plant, normally formed in special cells or groups of cells or in glandular hairs found on many leaves and stems. Essential oils are variable mixtures composed principally of terpenoids, including monoterpenes and sesquiterpenes (diterpenes may also be present), and their oxygenated derivatives. A variety of other molecules may also occur, such as aliphatic hydrocarbons, acids, alcohols, aldehydes, acyclic esters or lactones, and exceptionally nitrogen-and sulphur-containing compounds, coumarins and phenylpropanoid homologues. Known for their antiseptic (i.e., bactericidal, virucidal and fungicidal), medicinal properties and their fragrance, they are used in embalmment, preservation of foods and as antimicrobial, analgesic, sedative, anti-inflammatory, spasmolytic and local anesthetic remedies [1]. The spread of drug-resistant pathogens is nowadays one of the most serious threats to the successful treatment of microbial diseases. It has been well-known since ancient times that certain plants and spices have antimicrobial activity [2,3]. They produce an enormous array of secondary metabolites, and it is commonly accepted that a significant part of this chemical diversity serves to protect plants against microbial pathogens. The World Health Organization (WHO) has noted that a majority of the World's population depends on traditional medicine for its primary healthcare [4]. Many essential oils and their ingredients have been shown to exhibit a range of biological activities, including antibacterial and antifungal activity. As a result, essential oils and/or their components are becoming increasingly popular as natural antimicrobial agents used for a wide variety of purposes. Their preparations find applications as naturally occurring antimicrobial agents in pharmacology, pharmaceutical botany, phytopathology, medical and clinical microbiology and food preservation. This review focuses on the essential oil of Mentha suaveolens Ehrh. (EOMS) and one of its main chemical constituents-piperitenone oxide (PO) (Figure 1). Mentha: Species, Taxonomy, Occurrence and Uses Based on phylogenetic analysis of morphology, chromosome numbers and major essential oil constituents the genus Mentha (mint), an important member of the Lamiaceae family, is highly diverse [5,6]. It is represented by about 19 species and 13 natural hybrids, mainly perennial herbs, growing wildly in damp or wet places throughout the temperate regions of Europe, Asia, Africa, Australia and North America. Mints are fast growing, invasive and generally tolerate a wide range of agro-climatic conditions [7]. Mentha identification is difficult, since in addition to much phenotypic plasticity and genetic variability, most of the species are capable of hybridization with each other. Hybrids are frequent in Nature but can usually be recognized by their intermediate appearance and sterility, although fertile hybrid swarms occasionally occur [8]. The present literature suggests the classification of the genus Mentha into the three basic lines named: capitatae, spicatae and verticillatae, based on the characteristic inflorescence. The capitatae line includes all species with compact, head-like inflorescences; the type species is M. aquatica. The spicatae species have a spike, as shown by M. spicata, M. longifolia and MS. The third line, represented by M. arvensis, has an inflorescence vertically partitioned into whorls [9]. Mints are also classified based on the dominant monoterpene compound prevailing in the essential oil resulting from three different metabolic pathways. Thus, the production of linalool and linalyl acetate is typical for the linalool pathway; menthol, menthone and menthofuran are constituents of the menthol pathway, and carvone, dihydrocarvone and carveol characterize the carvone pathway (Scheme 1) [5,9,10]. [11]. Mentha species have been known for their medicinal and aromatherapeutic properties since ancient times. The ancient Egyptians, Romans and Greeks used peppermint as a flavoring agent for food and as a medicine, while mint essential oils have been used as perfumes, food flavors, deodorants and pharmaceuticals [7]. During the Middle Ages, powdered mint leaves were used to whiten the teeth [12]. Leaves, flowers and stems of Mentha spp. are frequently used in herbal teas or as additives in commercial spice mixtures for many foods to offer aroma and flavor. In addition, mints have been used as a folk remedy for treatment of nausea, bronchitis, flatulence, anorexia, ulcerative colitis and liver complaints, due to their anti-inflammatory, carminative, antiemetic, diaphoretic, antispasmodic, analgesic, stimulant, emmenagogue and anticatharrhal activities [13][14][15][16]. Different mint species are also used for rheumatism, dysentery, dyspepsia, skin allergies, chills, jaundice, throat infections, constipation, spasms, bladder stones, gall stone, diarrhea, toothache, stomach aches, dyspnea, gastrodynia, and as stimulant, diaphoretic, diuretic, reconstituent, stomach tonic, anti-infective, sedative, insect repellent, antimycobacterial, antifungal, antiallergic, virucidal, radioprotective, cyclooxygenase inhibitor, anti-inflammatory and hemostatic agents [17,18]. Mentha Essential Oils Recently, essential oils and various extracts of plants have provoked interest as sources of natural products. They have been screened for their potential uses as alternative remedies for the treatment of many infectious diseases and the preservation food from the toxic effects of oxidants. Research on plants from different regions has led to innovative ways to use the essential oils [19]. Particularly, the antimicrobial activities of plant oils and extracts have formed the basis of many applications, including raw and processed food preservation, pharmaceuticals, alternative medicine and natural therapies [15]. Members of the genus Mentha produce some of the most widely used essential oils. Different species vary in their essential oil content and composition. The biosynthesis and metabolism of essential oils are strongly influenced by environmental factors, such as temperature, photoperiod, nutrition and salinity [20]. Plant chemotypes, cultivation practices and method of extraction also lead to variations in oil content and composition. Other factors affecting essential oil composition relate to agronomic and genotype conditions, such as harvesting time, plant age and crop density [7]. The essential oil isolated from mint leaves has economic importance and is widely used in the food industry, cosmetics, confectionary and pharmaceutical industries [5,21] Mint is widely cultivated for this oil, produced in many countries, such as America, India, China and Canada [5]. Commercially, the most important mint species are peppermint (M. x piperita), spearmint (M. spicata) and corn (American wild) mint (M. canadensis). Many species have been studied experimentally and the efficiency of some traditional applications were confirmed in many reports. Mentha suaveolens Ehrh.: Taxonomic Characterization, Distribution and Uses MS, apple mint, woolly mint or round-leafed mint (synonyms: M. macrostachya Ten., M. insularis Req.) is an herbaceous, perennial herb with a sickly sweet scent that grows up to 100 cm in height ( Figure 1). The stem is erect, quadrangular, and sparsely hairy to densely white-tomentose. It is monopodially branched, with short internodes. The foliage is light green with opposite, wrinkled, sessile or very short petiolate leaves which are ovate-oblong to suborbicular, 3 to 4.5 cm long and 2 to 4 cm broad. Obtuse, cuspidate or rarely acute, the leaves are widest near the base, serrate, with 10-20 teeth, hairy above, usually grey or white-tomentose to lanate. A prostrate branch (creeping sucker) growing from the axil of the leaves at the base of the flowering stem propagates below the level of the ground then gives root and turns upwards to give a new shoot. Many verticillasters, usually congested, form a terminal spike 4 to 9 cm long, consisting of a number of white or pinkish flowers [8,11,22]. This species is native to Southern and Western Europe, extending northwards to The Netherlands, cultivated as a pot-herb and naturalized in Northern and Central parts of Europe. It is generally found along streams, bogs and humid places [23]. MS has been used in the traditional medicine of Mediterranean areas and has a wide range of effects: hypotensive, stimulating, stomachic, carminative, choleretic, antispasmodic, sedative, tonic, anti-convulsive, insecticidal, etc. It is also useful in cases of cough, nausea, anorexia and bronchitis [22] and finds application in digestion problems, influenza, respiratory ailments, rheumatism, skin diseases and irritation [24]. It shows depressor, analgesic, anti-inflammatory, cytotoxic, hepatoprotective and antifungal activities [10,14,25]. On reviewing the current literature on the phytochemistry of MS, flavonoids were the major constituents isolated from this species [10,26]. Concerning the biological activities, it was found that MS has antihypertensive [27], antioxidant and acetylcholinesterase inhibitory activities [28] and monoamine oxidase inhibitory activity [29]. The essential oil of MS was also found to have candidacidal activity [30,31] and a significant virucidal activity [32]. Essential Oil Composition of MS There is an ongoing effort to screen plants used therapeutically in different regions of the World. However, it is well-known that the same taxon growing in different areas may have widely differing chemical components and hence differing biological properties [23]. Ingredients of EOMS have been subjected to a number of studies which have shown a difference in its constituents depending on the region of origin [33][34][35][36][37]. In general, investigations on the chemical composition of the essential oil from different populations collected in various regions showed high percentages of oxides. These include piperitone oxide and piperitenone oxide (PO) as major components [5,10,23,37]. Other chemotypes of this species showed high percentage of alcohols such as menthol [10] or ketones such as pulegone, piperitenone and dihydrocarvone (Table 1) [23,37]. Accordingly, three profiles of EOMS have been defined previously: the first profile is rich in pulegone, the second in PO and the third one contains similar quantities of PO and piperitone oxide [23]. The populations of MS collected in various regions of Morocco showed a high percentage of oxides (PO and piperitone oxide), terpenic alcohols (fenchol, p-cymen-8-ol, geraniol, terpineol and borneol) and terpenic ketones (pulegone and piperitenone) [23]. Another investigation on Moroccan plant material from Azrou, Tetouan and Meknès confirmed the prevalence of PO (74.69%, 41.84% and 34%, respectively), while that from M'rirt is rich both in PO (81.67%) and piperitenone (10.14%) ( Table 2) [38,39]. On the other hand, the composition of the essential oils from Béni-Mellal and Boulmane is totally different, with pulegone (85.5%) and menthol (40.50%) as the major compounds, as well as oil from the Oulmès region (Rabat) where piperitenone and pulegone are the main compounds (33.03% and 17.61%, respectively) [38,40]. Dominance of menthol (48.32%) and pulegone (20.27%) is also the characteristic of the material from Córdoba, Argentina [41]. The authors explained that this oil composition was unusual and closely related to that of Mentha arvensis var. piperascens which raises the possibility that the studied sample was not really MS but rather a hybrid (M. arvensis var. piperiscens x M. suaveolens) [41]. The oil obtained from aerial parts of the wild MS ssp. timija, an endemic species of Morocco, was characterised by a very high content of oxygen-containing monoterpenes with menthone (39.4%-10.8%), pulegone (62.3%-34.3%) and isomenthone (9.3%-7.8%) as the main constituents [42]. MS is also widespread in Corsica, where it is represented by two subspecies: suaveolens and insularis. The latter species is endemic to the occidental Mediterranean Sea islands of Corsica, Sardinia, and the Balearic Islands. These two subspecies are botanically close, but various morphological characteristics allow their differentiation. Analysis of 59 oil samples isolated from MS wild plants growing in Corsica, followed by statistical analysis of the data, allowed a clear differentiation of both subspecies with respect to the composition of their essential oils: the subspecies suaveolens yielded oils dominated either by piperitenone (73.5%) or PO (72%), while all the samples of the insularis subspecies contained pulegone and cis-cis-p-menthenolide as main components [37]. Similarly, plant material of insularis subspecies from Sardinia is also characterized by prevalence of pulegone [44]. Samples originating from Uruguay and Greece have shown a preponderance of PO that reached 62.4% and 80.8% [33]. A predominance of PO (55%) was also seen in materials from the Czech Republic [45]. However, the same species in northern Algeria contained three different chemotypes: a first one characterized by the predominance of PO (29.36%) and piperitone oxide (19.72%); the second one, conversely, with piperitone oxide (31.4%) as the most abundant component, followed by PO (27.79%), and the third contains piperitenone as major constituent (54.91%) [38]. A prevalence of piperitone oxide (40.5%) followed by hydroxy-p-menth-3one (23.9%) was seen in a sample of cultivated MS in Padua (Italy) [46]. Analysis of the essential oil obtained from wild-type plants grown in the Tarquinia forests (Viterbo, Italy) showed a predominance of PO (>90%), with limonene and 1,8-cineole among minor constituents [25,30,32]. Another study on the same material was performed with the aim to analyze in details the essential oil extraction procedure in term of optimal period and extraction time. The material was submitted to hydrodistillation and the oil was collected at different times (1, 2, 3, 6, 12 and 24 h) on three different days of different months (July, August, September). The amount of the oil varied in function by both day of extraction and separation intervals. In August and September the oil yields were more than 2.5 times that of July. The maximum quantities of the oil are obtained in the first three and during the last twelve hours. In July, in the first 3 h almost 70% of the oil was extracted, while in August and September only 54% and 51%, respectively. In general, the most abundant constituent was PO, which percentage was maximum during the first three hours, disappearing in the last three extractions (after 6, 12 and 24 h). From the point of view of the extraction period, the extracted amounts were higher in the July and August samples than in the September sample. Other constituents are characteristic of the period and become important only after the fourth daily fraction. For example, in the month of July, demelverine and eucalyptol had a percentage of about 0.5%-2.0% in the first 3 h of extraction and increased to reach a maximum of about 29% after the 12-hour extraction. In August, β-caryophyllene oxide was always present and increased from about 0.4% after the 1-hour extraction to a maximum of 15.4% after the 12-hour extraction; cinerolone, that was absent in the first 3 h of extraction, showed high percentage in the next 3 h to reach a maximum of about 38.3% in the 24-hour extraction. In the September period, although PO was still the most abundant compound, the other important components seemed randomly distributed: in the first hour, contrarily from the previous period, β-caryophyllene oxide (13.5%) and cinerolone (18%) were at medium-high percentages, but then β-caryophyllene oxide decreased its percentage while cinerolone was absent but appeared again after 5 h till the last extraction (after 24 h), with a percentage varying from 2.4 to 17.5 [47]. Antimicrobial Activity of MS Essential oils and extracts have been used for thousands of years in food preservation, pharmaceuticals, alternative medicine and natural therapies [48]. They are potential sources of novel antimicrobial compounds, especially against bacterial pathogens and, in recent years, a large of number of investigations has been performed on their antimicrobial activities. Antimicrobial evaluations of essential oils are generally difficult because of their volatility, insolubility in water and complex chemistry [13,49]. Because of the mode of extraction, mostly by distillation from aromatic plants, essential oils contain a variety of volatile molecules such as terpenes and terpenoids, phenol-derived aromatic compounds and aliphatic components. The antimicrobial activity of essential oils has been extensively studied and demonstrated against a number of microorganisms, usually using direct-contact antimicrobial assays, such as different types of diffusion or dilution methods, as reviewed by many authors [50]. In these tests, essential oils are brought into direct contact with the selected microorganisms. However, due to high hydrophobicity and volatility of the essential oils, the direct-contact assays face many problems. In diffusion assays, the essential oils components are partitioned through the agar according to their affinity with water, and in dilution methods low water solubility has to be overcome by addition of emulsifiers or solvents (such as DMSO or ethanol) which may alter the activity [51]. Antimicrobial action is often determined by more than one component; each of them contributes to the beneficial or adverse effects. The major component may not be the only one responsible for the antimicrobial activity but a synergistic effect may take place with other oil components [50]. According to a literature survey, different mint species have been investigated in search of antimicrobial activities [13,15,48,[51][52][53][54], inclusing several analyses performed on MS with an aim to investigate its antibacterial, antifungal or antiviral effects. Essential oils of MS grown in several regions in Morocco were tested for their activity against 19 bacteria, including Gram-positive and Gram-negative ones, and against three fungi, using solid phase and microtitration assays [23]. The antibacterial and antifungal activities of three types of EOMS were analyzed. Essential oil rich in pulegone strongly inhibited all bacteria, while the activity of that rich in PO was weaker. The activity of the third one (rich in both PO and piperitone oxide) had a tendency to be less important. Those results indicated that the efficacy of essential oils depends on their particular chemical composition, which was confirmed with another analysis where the major aromatic components of those oils were tested against the same organisms-pulegone was the most active against all bacteria, followed by PO; the activity of piperitone oxide seems to be two-fold lower than that of PO against a number of microorganisms, in particular, yeasts. These results indicate that pulegone may be the most active aromatic component in EOMS [23]. To study the importance of the chemical structure of the major constituents of EOMS, the antimicrobial activity of a series of aromatic components was analyzed and the activities of pulegone, PO and piperitone oxide were compared with carvone, limonene and menthone [55]. These latter aromatic components are also synthesized by Mentha species and have less antimicrobial activity. Menthone, which results from the reduction of pulegone, was less active than its precursor. Limonene and carvone also had moderate antimicrobial activity compared with pulegone and PO. Similar results were obtained by other authors [55] who found that pulegone showed a more potent biocidal activity than limonene, carvone and menthone. Compared with pulegone, limonene does not possess the extra cyclic double bond between C4 and C7, which results in the loss of the antimicrobial activity of this compound. Similarly, piperitone oxide does not possess this double bond and had in general a lower activity compared with PO and pulegone. Thus, this double bond seems to be important for the antimicrobial activity of the monoterpenes, possibly by favoring an active configuration of the molecule. The presence of an epoxide between C1 and C2 in PO decreases its antimicrobial activity compared with that of pulegone [23]. Investigation of the aerial parts of MS growing in Egypt showed moderate inhibitory activity against the tested human pathogenic bacteria. In that study, the antimicrobial screening of the ethanolic extract and its subfractions were performed [10]. The oil of the fresh aerial parts showed a potent antifungal activity against Candida albicans, Saccharomyces cerevisiae and Aspergillus niger [43]. Other studies on the Egyptian plant material showed a strong antibacterial activity of the essential oil, especially against Staphylococcus aureus [6]. The oils obtained from aerial parts of the wild and cultivated MS ssp. timija, an endemic species of Morocco, have been screened for antimicrobial activity. Both oils displayed good to excellent activity against all microorganisms tested, with the oil of the cultivated form being more active [42]. Both essential oils exhibited marked antifungal activity on all the Candida species tested, especially against C. glabrata. The antimicrobial activities of timija mint essential oil can be attributed to the presence of high concentrations of pulegone and menthone, two oxygenated monoterpenes with well-documented antibacterial and antifungal potential [22,37]. However, the comparison of the antimicrobial activity between these two major compounds showed that pulegone has more potent biocidal activity than menthone, which can explain the more potent antimicrobial activity of oil obtained from the cultivated timija mint [23,42]. Antimycotic analysis on the oil from the plant material from Tarquinia (Italy) displayed high activity against all strains of Cryptococcus neoformans and different dermatophyte strains, such as Trycophyton mentagrophite, T. rubrum, T. violacee, Microsporum canis and M. gypseum, where in all cases a good antimycotic activity was observed [22]. Another study on that material assessed the in vitro and in vivo antifungal activity of essential oil in an experimental vaginal candidiasis infection model. That study showed that EOMS was both candidastatic and candidacidal in vitro, as demonstrated in an in vivo monitoring imaging system [30]. The results obtained from another study demonstrated both the effects of the essential oil on C. albicans yeast cells and biofilms, and the synergism of the oil when used in combination with conventional antifungal drugs like fluconazole (FLC) and micafungin (MCFG) [31]. The antifungal activity of this oil was investigated for differences resulting from extraction time and period of material collection. That analysis showed that the oil extracted in the first 3 h showed good antifungal activities, but decreasing activity was evident with the oils from the 6 to 24-hour extractions. The study also confirmed PO as the principal active chemical constituent responsible for the essential oil's biological activity [47]. Determination of the antibacterial activity of three essential oils was performed in vitro against strains of Pseudomonas syringe pv. actinidiae (PSA), the causal agent of bacterial kiwifruit canker. That study included oil of MS from Tarquinia, which showed significantly higher activity than other two essential oils (Rosmarinus officinalis and Melaleuca alternifolia). Also, synergistic effects of those three essential oils were analyzed. Treating PSA with the mixture caused a significant decrease in the MIC, compared to their individual values, indicating a significant synergistic effect of the three essential oils combined. Results in that study clearly demonstrated that the mixture was able to kill PSA at the concentration about 16 times lower than the MIC values of the individual oils after 1 h of exposure [56]. This oil was also investigated against Chlamydia trachomatis, the most common sexually-transmitted bacterial infection worldwide. Those results showed effectiveness towards C. trachomatis, whereby it did not only inactivate infectious elementary bodies but it also inactivated chlamydial replication. The study also revealed the efficacy of the oil in combination with erythromycin: the combination inhibited C. trachomatis replication with a considerable four-fold reduction in the minimum effective dose of antibiotic [57]. The effects of EOMS derived from Tarquinia and its active principle PO were also tested in an in vitro experimental model of infection with herpes simplex virus type 1 (HSV-1), an important human pathogen. Moreover, a synergistic action was observed in combination with acyclovir. This study demonstrated that the oil, as well as its main compound PO, exerted stronger effects when added post-infection. When the oil, or the oxide, was preincubated with the virus before infection, both showed a significant virucidal activity, thus interfering directly with the viral envelope [32]. The antimicrobial activity of the essential oil from MS growing in the north of Morocco was evaluated on Salmonella enterica, Listeria monocytogenes and Escherichia coli, as well as for antiviral activity on the cytopathogenic murine norovirus (MNV-1). The results showed weak activity against E. coli and L. monocytogenes, but moderate activity was observed against S. enterica. The oil was mainly composed of PO, which was considered to have a low antimicrobial activity due to the presence of an epoxide between C1 and C2. This study showed that the oil tested had low antiviral activity against MNV-1 [39]. The in vitro antimicrobial activity of the essential oil of the aerial parts of MS ssp. insularis grown in Sardinia was assayed against six Lactobacillus species (including four probiotic strains), Lactococcus lactis ssp. lactis and Staphylococcus xylosus. Agar diffusion test results indicated that the essential oil exhibited a low antibacterial activity potential against all tested bacteria, except to L. lactis ssp. lactis and S. xylosus upon which it exerted a slight antibacterial activity. On the other hand, all yeasts strains tested (Saccharomyces cerevisiae, Kloeckera apiculata, Candida zemplinina, Metschnikowia pulcherrima and Tetrapisispora phaffii) were inhibited, and the oil exhibited slight to strong antifungal activity [44]. The mechanisms by which essential oils inhibit microorganisms involve different modes of action, but may be due in part to their hydrophobicity. As a result, they cause lipid partitioning of bacterial cell membranes and mitochondria, disturbing the cell structures and rendering them more permeable [58,59]. Extensive leakage from bacterial cells or the exit of critical molecules and ions, will lead to death [4]. Impairment of bacterial enzyme systems may also be a potential mechanism of antimicrobial action [60]. The antimicrobial activity of the essential oils can also be explained by the lipophilic character of the monoterpenes contained. The monoterpenes act by disrupting the microbial cytoplasmic membrane, which thus loses its high impermeability for protons and bigger ions. If the membrane integrity is disrupted, then its functions are compromised not only as a barrier but also as a matrix for enzymes and as an energy transducer. However, specific mechanisms involved in the antimicrobial action of monoterpenes remain poorly characterized [31]. According to a number of authors, Gram-negative bacteria are generally less susceptible than Gram-positive bacteria to the actions of essential oils, due to their outer membrane surrounding the cell wall which restricts diffusion of hydrophobic compounds through its polysaccharide covering [61][62][63][64]. According to some authors, this effect seems to be dependent on lipid composition and net surface charge of microbial membranes [62]. This statement is not always true; indeed different authors found no differences or greater sensibility of Gram-negative bacteria than Gram-positive to essential oils [44,52]. Antioxidant Properties of EOMS Free radicals are considered to initiate oxidation reactions that lead to aging and cause diseases in human beings. Moreover, activated oxygen incorporates reactive oxygen species (ROS) which consists of free (hydroxyl radicals, superoxide anion radicals) or non-free radicals (peroxide) [65]. These ROS are liberated by virtue of stress, and thus, an imbalance is developed in the body that damages cells in it and causes health problems [18]. On the other hand, restrictions have been imposed on the use of synthetic antioxidants because of their carcinogenicity and other toxic properties, which has considerably increased interest in natural antioxidants [18,19,66]. The active ingredients of a medicinal plant are mainly its secondary metabolites which are naturally produced during the metabolic processes of the plant's growth [18]. Natural antioxidants can be phenolic compounds (tocopherols, flavonoids and phenolic acids) and carotenoids (lutein, lycopene and carotene) [19,67]. Growing evidence has shown an inverse correlation between the intake of dietary antioxidants and the risk of chronic diseases such as coronary heart disease, cancer and several other aging-related health concerns [68,69]. There are several methods to determine the antioxidant capacity of plant extracts. However, the chemical complexity of extracts could lead to scattered results obtained from different techniques, depending on the test employed. Therefore, an approach with multiple assays in the screening work is highly advisable [70]. Investigation of the antioxidant activity of MS extracts from Morocco showed that the phenol extract presented a high antioxidant activity equivalent to that of butylated hydroxytoluene (BHT), an inhibitor well-known for its antioxidant activity [70,71]. Effectively, phenolic compounds are considered a major group of compounds that contribute to the antioxidant activities of botanical materials because of their scavenging ability on free radicals due to their hydroxyl groups [72]. The antioxidant activity of phenolic compounds is described as being largely influenced by the number of hydroxyl groups on the aromatic ring [73]. This activity is also due to their ability to scavenge free radicals, donate hydrogen atoms or electrons, or chelate metal cations [74]. The highest ferrous ion chelating activity was found in phenol and methanol extracts [71]. There is a linear correlation between the content of total phenolic compounds and their antioxidant capacity [29,72,75,76]. The results of this study indicate that phenolic compounds present in MS could be the major contributors of antioxidant capacities of this species [70]. Nine mint species from Pakistan were investigated as new potential sources of natural antioxidants. The methanolic extract assays revealed that significantly higher activity (82%) was observed in MS [18], which showed appreciable antioxidant activity only in the polar fractions while its decoction was also very effective in the inhibition of AChE and as a scavenger of radicals [28]. Mentha species prevent cell damage through their strong antioxidant activity, by scavenging free radicals and neutralizing bacterial invaders. They also promote the release of superoxide dismutase, a powerful antioxidant especially potent in destroying free radicals caused by imbalanced oxidation. Radical scavenging activity was observed when discoloration occurred, and MS were observed to produce high discoloration, followed by the other investigated species [18]. Analysis of some Egyptian mint species gave different results, since the lowest antioxidant activity was found in EOMS-only 6% [6]. Other analysis of the Egyptian EOMS showed potent in vivo (96% relative to vitamin E) and moderate in vitro antioxidant activities [43]. Ethanolic extracts of aerial parts of MS cultivated in Egypt and its subfractions (n-hexane, chloroform, ethyl acetate and n-butanol) were also evaluated. The ethyl acetate fraction showed the highest antioxidant activity: in vivo (as it restored the glutathione level in diabetic rats by 98%) and in vitro as it had the highest free radical scavenging activity (IC50 = 31 μg·mL −1 ) [77]. Extracts of MS gathered from the interior of Portugal only showed appreciable antioxidant activity in the polar fractions [28]. Insecticidal Activity of EOMS Plant insecticides have been used to fight pests for centuries. For instance, the use of plant extracts and powdered plant parts as insecticides was widespread during the Roman Empire. However, after the Second World War the few plants and plant extracts that had shown promising effects and were of widespread use were replaced by synthetic chemical insecticides. Later on, the adverse effect of chemical insecticides was realized with the appearance of problems like environmental contamination, residues in food and feed and pest resistance. Since the majority of plant insecticides are biodegradable, this has led to a revival of growing interest in the use of either plant extracts or essential oils. More than 1500 plant species have been reported to have insecticidal value [7]. Many plant secondary metabolites, such as alkaloids, monoterpenoids or phenylpropanoids are toxic to insects; in addition, essential oils extracted from plants have been widely investigated for pest control properties, with some providing to be toxic [78]. Mentha has historical significance as a medicinal and insecticidal plant in the traditional knowledge system. In the last few decades, many studies have been reported on the insecticidal activity of several Mentha species, largely in terms of adulticidal activity. EOMS from Azrou (Morocco) was investigated for its insecticidal activity against adults of Sitophilus oryzae. Considerable differences in insect mortality due to essential oil fumigation were observed using different concentrations and exposure times. Results showed that the essential oil was very toxic against S. oryzae, but the degree of this toxicity was influenced by the concentration applied and the exposure time [38]. Another study of the oil from the Moroccan material was carried out on two species of devastating insects of stored foodstuffs: Sitophilus oryzae and Rizopertha dominica. Mortality was 100% for amounts of 50 µL and 12 µL of the oil, while for the amount of 3 µL an acute toxicity was observed causing the mortality of 85% on the first and 100% on the second day [40]. To assess the biological activity of EOMS (also from Morocco), four concentrations were tested as fumigants against Callosbruchus maculatus reared on chickpea seeds. Great effectiveness of the oil was noticed in that study [79]. Oil from the Czech Republic had larvicidal activity against Culex quinquefasciatus [45]. The insecticidal activity of essential oils depends closely on their chemical composition. Monoterpenes have been well-documented as active fumigants and insecticides [80] and EOMS contains up to 86.2% of monoterpenes such as PO, pulegone, limonene, piperitenone, β-pinene, α-pinene and p-cymene. Their toxicity was proved toward stored product pests [34,40,[79][80][81][82][83][84]. The fumigant toxicity of tested oil can be correlated with the abundance of PO. This oxygenated monoterpene possessed high toxicity against pests [45,80,82,84]. Differences in the chemical structures of monoterpenes are another factor influencing biological potency. Oxygenated terpenoids are more toxic than the non-oxygenated ones, and further, even among oxygenated ones, biological activity is differentiated by their other chemical groups and saturation [45]. Other components are present at low levels but may exert a synergistic effect [38]. The mode of action of essential oils and their constituents as insecticides is not known. The lipophilic nature of plant essential oils allows them to interfere with basic metabolic, biochemical, physiological and behavioral functions of insects [7]. Recent studies reported that essential oils and their constituents affect biochemical processes, which specifically disrupt the endocrine balance of insects. They may be neurotoxic or may act as insect growth regulators, disrupting the normal process of morphogenesis [85]. Further, monoterpenes have been investigated for their neurotoxicity [7]; they are typically volatile and rather lipophilic compounds that can penetrate into insects rapidly and interfere with their physiologic functions [86] by inhibiting acetylcholinesterase activity [80,87] and acting on insects' octopaminergic sites [88]. Additional Bioactivities of EOMS EOMS from Egypt was screened for certain other biological activities. It exhibited analgesic and acute anti-inflammatory activities (75% and 82% relative to indomethacin) [43]. In addition, it exerted moderate cytotoxic and hepatoprotective activities [43]. Further investigation included different fractions of the ethanolic extract of the aerial parts of MS growing in Egypt. Analgesic and acute anti-inflammatory activities of the oral administration of the ethanolic extract and its subfractions (n-hexane, chloroform, ethyl acetate and n-butanol) were evaluated, using indomethacin as a standard drug. As a result, the ethanolic extract showed the most potent analgesic activity (78.5% potency compared to indomethacin), followed by the ethyl acetate and n-butanol fractions whose potency percentages were 66.3% and 54.7%, respectively. On the other hand, the ethyl acetate fraction was the most potent anti-inflammatory (88% potency) as compared to indomethacin, followed by the ethanolic extract (82.9%) and n-butanol fraction (62.6%) [10]. It is obvious that both analgesic and anti-inflammatory activities were exerted by the ethanol extract, the ethyl acetate and n-butanol fractions. It could be concluded that these activities may be attributed to their phenolic contents. The potent anti-inflammatory activities of the ethanolic extract may be due to its content of sterols, triterpenes, phenolic acids and flavonoids which have been proved to exert anti-inflammatory activity [10]. The hepatoprotective activity of the ethanolic extract and its subfractions was also evaluated. It revealed that the ethyl acetate fraction had the highest activity, as it prevented the increase caused by CCl4 in the levels of aspartate amino transferase (AST), alanine amino transferase (ALT) and alkaline phosphatase (ALP) enzymes by 51.6%, 57.0% and 56.7%, respectively. The ethanolic extract, as well as its subfractions, were also tested for their cytotoxic activity. The results showed a significant activity of the ethanolic extract on liver carcinoma and larynx cancer cell lines (IC50 = 7.28 and 7.35 µg·mL −1 , respectively). The ethyl acetate fraction showed the highest activity against human liver carcinoma cell line (IC50 = 5.1 µg·mL −1 ), the chloroform fraction was the most potent on the colon carcinoma cell line (IC50 = 14.40 µg·mL −1 ) while n-hexane was the most active regarding the breast carcinoma cell line (IC50 = 13.5 µg·mL −1 ) [77]. Essential oils, ethanolic extracts and decoction of 10 plants from interior Portugal were analyzed for their activity towards acetylcholinesterase (AChE) enzyme. MS showed AChE inhibitory capacity higher than 50% in the essential oil fraction, while the ethanolic extract was less potent. A high value of AChE inhibitory activity was found in a decoction of MS [28]. The pharmacological activity of a methanol extract of the leaves and stems of MS was analyzed in in vivo and in vitro models. The extract exhibited a central nervous system depressant action but no anticonvulsive activity. In order to determine the type of analgesia induced, the activity was evaluated using three different pain stimuli, i.e., heat, mechanical and chemical agents such as acetic acid. The extract evaluated lacked any analgesic effect arising from CNS action since it did not increase the response time in the hot plate test. However, the extract showed a significant effect on mechanical and chemical stimuli, thus suggesting the induction of a peripheral analgesic effect. The extract also showed significant anti-inflammatory action inhibiting the rat paw oedema induced by carrageenan. Moreover, the in vitro studies showed a significant diminution in the contractile effects induced by histamine, serotonin and acetylcholine [14]. Since MS preparations have been applied in the traditional medicine of the Mediterranean areas as a hypotensive, the methanol and dichloromethanol extracts of the leaves and stems of this plant were tested for their effects on resting arterial blood pressure, heart rate and noradrenaline induced hypertension. Both extracts reduced the mean arterial blood pressure and heart rate, while only the dichloromethanol extract prevented the noradrenaline induced hypertension [27]. Mutagenicity tests revealed that the oil was not endowed with any particular toxic effect [47]. Piperitenone Oxide-the Main EOMS Chemical Components 1,2-Epoxypulegone (PO) is an important chemical constituent of the essential oils of many Mentha species, where it is formed by epoxidation of piperitenone (Scheme 2). It was firstly named rotundifolone, since it was isolated from the essential oil of M. rotundifolia cultivated in Japan [89,90]. The taxonomic status of this species is confusing. Namely, according to Flora Europea [8], this was a misapplied name for MS (Mentha rotundifolia auct., non (L.) Huds.) and some authors consider it as its synonym [91], as mentioned in some articles [33,35,92]. On the other hand, there is a hybrid of MS and M. longifolia. named Mentha × rotundifolia. This problem has been discussed and clarified by some authors [93]. (9); and the terpenoid epoxidase (10). OPP denotes the diphosphate moiety [94]. This monoterpenoid ketone (C10H14O2, molecular weight 166) is highly dextro-rotatory and has a low melting point (27.5 °C). The wavelengths of the ultraviolet absorption of PO and its semicarbazone are 260 and 273 nm, respectively, which indicates that this oxide has an α,β-unsaturated carbonyl system. PO does not give a ferric chloride reaction in methanol nor in aqueous suspension, but shows a positive Malaprade reaction. It reduces ammoniacal silver nitrate and Fehling solution. PO is soluble in ethanol, methanol, benzene and petroleum ether, but not in water and aqueous alkali [91]. PO is considered as one of the major common constituents of EOMS. This oxygenated monoterpene exhibits interesting activities, including cardiovascular, antimicrobial and insecticidal [38,79,82]. According to recent investigations, the plant species Lippia pedunculosa (Verbenaceae) may be considered as the new important source of PO since it is a predominant constituent (≈72%) in its essential oil [118]. For sure, PO is not characteristic of the genus Lippia, but some of the other species also contains it as a minor or significant constituent [119]. Thus, L. turbinate essential oil contains 30% [120], the amount in L. juneliana oil is from 22.9% to 47.7% [121], while the oil of L. alnifolia was shown to have 44.6% of PO [122]. PO is also found to be the main constituent of some Satureja oils (Lamiaceae). Thus, S. parvifolia from Argentina contains 69.8% of PO [123,124], while the endemic S. kallarica from Iran has a 71.2% content [125]. Some Calamintha species (Lamiaceae) are also recognized to be quite rich in PO. The oils of C. incana from Turkey [126] and C. nepeta ssp. glandulosa from Belgium [127,128] contain up to 66.6% and 52% of PO, respectively. Besides being available from natural sources, PO can be also produced by chemical synthesis. One of the ways is the epoxidation of piperitenone [119]. The monoterpenoid piperitenone (1) is of interest because it can be converted to a wide variety of important compounds. It has been obtained by several chemical routes [129,130]. The best yield was achieved with the process involving condensation of mesityl oxide (2) in tetrahydrofuran (THF) with methyl vinyl ketone (3) in THF and a solution of Triton-B (Scheme 3) [131]. Another synthetic procedure involves the same condensation, but uses sodium tert-butoxide as condensing agent in toluene [129]. However, this process mainly yields isoxylitone, the self-condensation product of 2 and less than 8% of 1. Subsequently, numerous investigations have attempted to attain a high yield of 1 by suppressing simultaneously the formation of isoxylitone. It had been found that when an alkali metal alkoxide was used as condensing agent, the self-condensation of 2 occurred in an aprotic solvent (e.g., n-hexane, benzene) but little or no reaction occurred if the solvent was replaced by a protic solvent (e.g., ethanol), tetrahydrofuran (THF) or a hydrous aprotic solvent. A heterogeneous system of potassium hydroxide and THF restricts the formation of isoxylitone considerably [130]. It has been reported that 1 is capable of forming a water-soluble sodium bisulfite addition compound. Treatment of the reaction mixture from the condensation reaction with bisulfite followed by ether extraction gave an aqueous solution from which 1 could be regenerated and isolated in 54% overall yield. Another condensation includes sodium hydride or a potassium 2-butoxide as catalysts [130]. Scheme 3. Synthesis of piperitenone from mesityloxide and methyl vinyl ketone. In the epoxidation step 30% aqueous hydrogen peroxide and 10% potassium hydroxide should be added to 1 in isopropyl alcohol. Two main fractions are obtained: the unreacted 1 mixed with PO, which could be separated by the column chromatography (Scheme 4) [119]. Bioactivities of PO The monoterpenic ketone PO has been evaluated in relation to the following different biological activities: cardiovascular, hypotensive, bradycardic, insecticidal, trypanocidal, schistosomicidal, antimicrobial and antinociceptive properties. It has been shown that PO exhibited central analgesic activity in mice and rats [97]. PO was analyzed for potential activity on smooth muscle. The experimental model was the guinea pig ileum, and the main conclusion of that study was that PO had a relaxant, depressant activity on intestinal smooth muscle [132]. PO exhibited strong toxic effect against the larvae of the mosquito species Aedes aegypti [103] but also against other mosquito species [82,84]. Although it has shown a good larvicidal effect against the Ae. aegypti, it was less potent than its essential oil of origin, which may be due to a synergistic interaction between PO and other compounds. Comparing PO and its analogues confirmed that the different functional groups and their positions in the p-menthane skeleton influence the larvicidal activity. In general, replacement of C=C double bonds by epoxide groups decreases the larvicidal potency. It can be concluded that with appropriate structural modification in the monoterpenes it may be possible to develop new larvicidal agents [103]. The essential oil from the leaves of Lippia pedunculosa and its main compounds, the monoterpenes PO and (R)-limonene, were evaluated for their trypanocidal activity against epimastigote and trypomastigote forms of Trypanosoma cruzi. PO was the most active compound. The effects of the oil and isolated compounds on the intracellular form of the parasite were also evaluated in cultures of macrophages infected with T. cruzi, but the treatment with (R)-limonene and PO caused a moderate reduction in the percentage of macrophages [118]. Biological effects of the M. x villosa essential oil and its main constituent PO were evaluated on adult worms of Schistosoma mansoni (Plathelminths). Worms were incubated with different concentrations of the oil and PO, which resulted in decreased worm motility continuing until 96 h of observation. At higher concentrations (100 and 70.96 µg·mL −1 , respectively), both the essential oil and PO caused mortality among adult S. mansoni worms [101]. Cardiovascular effects of intravenous treatment with the essential oil of M. x villosa were investigated. Additionally, that study examined whether the major constituent PO was the active principle mediating changes in mean aortic pressure and heart rate. The study showed that the treatment with oil in rats induced hypotensive and bradycardic effects, which appeared mostly attributed to the action of the main compound of the oil, PO [95]. The acute cardiovascular effects of PO were also investigated in rats by using a combined (in vivo and in vitro) approach. The acute administration of PO induced a short-lasting and dose-related decrease in arterial pressure, followed by a significant bradycardia, probably due to a non-specific muscarinic receptor stimulation. Furthermore, in vitro studies suggested that PO induced vasodilatation [97]. The effects of PO on vascular smooth muscle were analyzed, and the major finding was that PO-induced vasodilatation of the rat aorta was apparently mediated by an inhibitory effect on Ca 2+ influx and inhibition of intracellular Ca 2+ release from stores [96]. The authors concluded that the hypotensive effect was possibly due to a reduction in heart rate associated to a reduction of peripheral vascular resistance, both due to muscarinic activation [133]. PO induces muscle contraction in both depolarized and non-depolarized sartorius muscle of toad (Bufo paracnemis). The study demonstrated that PO induced muscle contraction by releasing calcium from the sarcoplasmic reticulum, probably by the activation of the ryanodine receptor [134]. Assessment of the antinociceptive activity of PO and the analogous compounds was performed using the acetic acid-induced writhing model in mice. All compounds showed to be more antinociceptive than PO against the pain response induced by acetic acid. It was found that the functional groups and their position on the ring of PO contributed to its antinociceptive activity [135]. All those hypotheses were subsequently strengthened by studies characterizing the molecular mechanism of action involved in relaxation produced by PO. The findings suggested that PO induced vasodilatation through two distinct but complementary mechanisms that clearly depended on the concentration used [136]. Another study was performed to evaluate the vasorelaxant effects of different monoterpenes and establish the structure-activity relationship of PO and its structural analogues. The results showed that both oxygenated and non-oxygenated monoterpenes exhibited relaxation activity. The absence of an oxygenated molecular structure was not a critical requirement for the molecule to be bioactive. It was also found that the position of ketone and epoxide groups in the monoterpene structures influenced the vasorelaxant potency and efficacy [137]. The oil of M. x villosa and its major component PO together with four similar analogues (limonene oxide, pulegone oxide, carvone epoxide and (+)-pulegone) were evaluated in relation to the antimicrobial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans and a strain of meticilin-resistant Staphylococcus aureus (MRSA). The essential oil, PO and the analogues showed antibacterial activity on S. aureus and antifungal activity on C. albicans. Limonene oxide and carvone epoxide were the substances with the lowest antimicrobial potential. None of the products showed antimicrobial activity against strains of the Gram negative bacteria E. coli and P. aeruginosa [138]. Conclusions and Future Perspectives In general, plants have provided a source of inspiration for novel drug compounds. The increased interest in alternative natural substances is driving the research community to find new uses and applications for these substances and has led to a considerable increase in the use of medicinal plants. The results of the cited studies indicate that MS and PO, as the main compound of EOMS, show a wide range of biological activities. Keeping in mind that the biological properties can be the result of synergism, investigation of the main compounds alone seems questionable. However, PO is usually the predominant compound (sometimes more than 90%) in EOMS and was found to reflect quite well the biophysical and biological features of the whole oil. PO seems to be responsible for a lot of bioactivities although it is possible that its activity is slightly modulated by the other minor molecules. Neither the essential oil nor PO have very strong antibacterial effects. Pulegone is the most important Mentha constituent responsible for antibacterial activity, but the oil of this species is usually not rich in it. The effect of the oil is significant only in cases where other compounds are present in reasonable amounts, such as pulegone, menthone or β-cymene. On the other hand, the oil and PO seem to be potent antifungal agents. In that sense, further investigations of PO can be proposed. There are some data indicating certain antiviral effects of the oil, as well as PO. Thus, that field is also interesting for the future examinations. It should be added that the further studies are needed to evaluate the in vivo potential in animal experimental models since there are little data about that aspect. The essential oil has strong antioxidant potential. In some cases, it can be compared with butylated hydroxytoluene which is a well-known synthetic antioxidant additive. However, to the best of our knowledge, investigation of PO in this field is missing. Insecticidal properties of EOMS and PO were evaluated. Since they usually exhibit strong toxic effects, this can definitely be a field for future study. The oil contains a huge amount of monoterpenes (often, even more than 85%) which have been well-documented as active fumigants and insecticides. On the other hand, oxygenated terpenoids are more toxic than the non-oxygenated ones. Thus, the fumigant toxicity of this oil can be justified by the high content of the monoterpene PO, which has already been well explored in that sense. What can be emphasized is that appropriate structural modification in the monoterpenes may lead to the development of new insecticidal agents. Additional bioactivities of EOMS, as well as PO, have been investigated, and the results showed good potential in relation to analgesic, anti-inflammatory, antihypertensive and AChE inhibitory activities. There are also a significant number of studies on the different extracts, not the essential oil, that can be continued avenues of study in the future. Future studies should further explore the possible beneficial synergistic properties of combining PO (or the whole oil) with other natural or synthetic compounds.

v3-fos

2017-07-06T02:21:44.879Z

2015-02-13T00:00:00.000Z

5045228

s2

Accumulation, Allocation, and Risk Assessment of Polycyclic Aromatic Hydrocarbons (PAHs) in Soil-Brassica chinensis System Farmland soil and leafy vegetables accumulate more polycyclic aromatic hydrocarbons (PAHs) in suburban sites. In this study, 13 sampling areas were selected from vegetable fields in the outskirts of Xi’an, the largest city in northwestern China. The similarity of PAH composition in soil and vegetation was investigated through principal components analysis and redundancy analysis (RDA), rather than discrimination of PAH congeners from various sources. The toxic equivalent quantity of PAHs in soil ranged from 7 to 202 μg/kg d.w., with an average of 41 μg/kg d.w., which exceeded the agricultural/horticultural soil acceptance criteria for New Zealand. However, the cancer risk level posed by combined direct ingestion, dermal contact, inhalation of soil particles, and inhalation of surface soil vapor met the rigorous international criteria (1×10−6). The concentration of total PAHs was (1052±73) μg/kg d.w. in vegetation (mean±standard error). The cancer risks posed by ingestion of vegetation ranged from 2×10−5 to 2×10−4 with an average of 1.66×10−4, which was higher than international excess lifetime risk limits for carcinogens (1×10−4). The geochemical indices indicated that the PAHs in soil and vegetables were mainly from vehicle and crude oil combustion. Both the total PAHs in vegetation and bioconcentration factor for total PAHs (the ratio of total PAHs in vegetation to total PAHs in soil) increased with increasing pH as well as decreasing sand in soil. The total variation in distribution of PAHs in vegetation explained by those in soil reached 98% in RDA, which was statistically significant based on Monte Carlo permutation. Common pollution source and notable effects of soil contamination on vegetation would result in highly similar distribution of PAHs in soil and vegetation. Ethics statement Vegetable fields are common resources in the outskirts of Xi'an, the capital of Shaanxi Province, China. Thus, no specific permissions were required for these locations/activities. Additionally, we confirm that the field study did not involve endangered or protected species. Sampling In late July of 2012, surface soil and vegetables samples were collected from vegetable fields in the outskirts of Xi'an, the capital of Shaanxi Province, China (108°48 0 to 109°14 0 E, 34°10 0 to 34°25 0 N). The vegetable fields in the outskirts of Xi'an have an area of 60,000 ha with a typical warm temperate monsoon climate. The annual mean temperature is 13.6°C, the annual precipitation is 595.9-732.9 mm, and the predominant direction of the wind throughout the year is northeast. The main soil type in the region is brown earth [20] and the main vegetable crop is Brassica chinensis. Seven sampling areas were selected from the villages of Dujia, Xicha, Gaomiao, Xiwang, Guanmiao, Jiangwu, and Liulin in the Weiyang region. Four sampling areas were selected from the villages of Shijia, Moling, Weijia, and Changjia in the Baqiao region. Two sampling areas were selected from the villages of Liangzhao and Xingnan in the Lintong region (S1 Table and Fig. 1). Each surface soil sample (0-20 cm depth) was homogeneously mixed with five diagonally sampled subsamples in each block (approximately 0.5 ha). Vegetation samples were handled in the same way. The blocks were randomly selected in each sampling area, with the total number of blocks varying with size of the planting area. A portion of each sample was filled into a pre-cleaned aluminum box and transported to the laboratory at a temperature of 4°C, where it was freeze-dried and ground to pass through 1 mm mesh for analysis of PAHs. The remainders of the soil sample portions were transported to the laboratory in plastic bags for analysis of basic properties. The soil and vegetable samples were labeled by S and V before the names of villages, respectively. Samples were further distinguished by the numbers after the names of villages. PAH analysis For extraction of the petroleum hydrocarbons, soil samples (approximately 30 g) or vegetation samples (approximately 10 g) were pressurized-liquid extracted with dichloromethane using an ASE-300 accelerated solvent extractor (Dionex, USA). During the extraction, the cells were pressurized to 1500 psi/1.0 × 10 7 Pa and heated to 100°C for 5 min. The static extraction was held for 5 min, after which the sample was flushed with solvent (60% of the cell volume) and purged with nitrogen for 60 s at 150 psi/1.0 × 10 6 Pa [21,22,23]. The aliphatic hydrocarbons were obtained through elution with approximately 20 ml n-hexane after purification with an alumina and silica gel chromatography column [22,24,25] and then discarded. The PAHs were obtained through elution with approximately 30 ml dichloromethane/n-hexane (2:1, v/v), after which they were concentrated to 1 ml for analysis [26]. PAHs in the extracts of all samples were analyzed by gas chromatography-mass spectrometry [Agilent, 6890N GC, 5975B mass spectrometric detector (MSD), USA] equipped with a DB-1MS capillary column (30 m, 0.25 mm inner diameter × 0.25 μm film thickness, Agilent, USA). The carrier gas was helium (high purity, 99.99%) applied at a constant flow rate of 1 ml/min. The oven temperature was initially set at 50°C, after which it was held for 1 min, then increased to 200°C at 19°C/min, where it was held for 2 min. The oven temperature was then increased to 240°C at 4.5°C/min, which was held for 2 min, and finally increased to 290°C at 2.5°C/min. MSD was operated in electron impact mode at 70 eV with an ion source temperature of 300°C. Mass Accumulation of PAHs in Soil-Plant System spectra were recorded with a selected ion mode to identify the PAHs. The concentrations of PAHs were given as dry mass of the sample and in the form of (mean±standard error). Quality control The quantification of 16 PAHs designated as priority control pollutants by USEPA (S2 Table) was conducted based on the peak area external reference method with a mixture of PAH standards (Accustandard, USA). The SPAHs value was the sum of the 16 PAHs. The analytical procedure was comprehensively evaluated against quality control acceptance criteria [27]. Calibration graphs were constructed by plotting the peak area against the reference material concentration every 2 days. A linear relationship with r 2 >0.999 was obtained. The method detection limits (MDLs) and the recoveries were evaluated using spiked soil samples. The MDLs of 2-, 3-, 4-, 5-, and 6-ring PAHs were 13, 15, 16, 14, and 14 μg/kg, respectively. The results of the blanks extracted under the same conditions were below the detection limits. The sample results without blank corrections were also presented. The recoveries were 50% to 105% (average percentage recovery: 81%) with a relative standard deviation lower than 11%. Soil properties analysis The standard methods recommended by the Chinese Society of Soil Science were used to determine basic soil properties. Soil pH was measured using a pH meter (PSH-3C, Leici, China), and the ratio of water to soil was 2.5:1 (v/w). The potassium dichromate method was used to determine the OM content, while the semimicro-Kjeldahl method and the alkali fusion-ascorbic acid-molybdate (ammonium molybdate and antimony tartrate) spectrophotometric method were used to determine total N and P in soil, respectively [28]. The particle size distribution was determined using the hydrometer method, and the cation exchange capacity (CEC) was measured using the ammonium acetate method [28]. Soil moisture was measured with an infrared moisture meter (MA30, Sartorius, Germany). Statistical analysis Non-parametric tests such as Mann-Whitney U test, Kruskal-Wallis H test, Nemenyi test, Spearman correlation, and regression analyses were conducted using SPSS Statistics 17.0 (SPSS, USA). PCA and RDA were performed with the CANOCO 4.5 software bundled with CanoDraw for Windows (Microcomputer Power, USA). Vegetation and soil samples were set as species variables in PCA, whereas vegetation samples were set as species variables and soil samples were set as environmental variables in RDA. All analyses were scaled on inter-species correlations and species-centered by dividing species scores by their standard deviation to obtain correlation matrices. The original data were ln transformed (original data + 1) so that all variables came as close to a normal distribution as possible. The average values of the species were 0 by centralization in PCA, whereas the average values of the species were 0 and their standard deviations were 1 by centralization and standardization in RDA. In PCA and RDA, the arrows representing vegetation and soil (as species or environmental variables) started from the origin point (0, 0), which pointed in the direction of increasing concentration of PAH monomer. By projecting the PAH monomer (as sample in PCA and RDA) symbol (circle in S1 Fig. but not present in Fig. 2) on the arrow line and ranking the projection points, a ranking of the concentration of PAH monomers in the vegetation or soil represented by the corresponding arrow could be obtained. Moreover, the approximate linear correlation coefficient between two species or environmental variables was equal to the cosine value of the angle between the corresponding arrows. However, the factor scores for vegetation (as species variables) were linear fitted by soil (as environmental variables) in RDA. These scores were different from those obtained in PCA. Risk assessment The cancer risks (CR) of PAHs in soil were calculated using an RBCA tool kit for chemical releases (GSI, USA) [9]. The exposure pathways of direct ingestion, dermal contact, inhalation of soil particles, and inhalation of surface soil vapor were accounted for. The pH and OM content of the local soil were used, while other parameters of exposure and soil properties were set as stated in appendix G of the guidelines for risk assessment of contaminated sites by the Ministry of Environmental Protection of the People's Republic of China (China MEP) [29]. The cancer risk values of vegetation ingestion were calculated using a modified version of equation (1) proposed by the USEPA [30] and exposure parameters stated in the guidelines for risk assessment of contaminated sites by China MEP [24]. where C PAHs is the contaminant concentration in vegetation (mg/kg) and the TEQ value of PAHs mixture was used as C PAHs in this study; SF is the slope factor for Bap = 7.3 kgÁday/mg; the cancer risk values were the cumulative effect from exposure to PAHs during two phases, child and adult; IR is the ingestion rate of vegetation [child: 0.2234 kg/day, adult: 0.3607 kg/day [31]; ED is the exposure duration (child: 6 years, adult: 24 years); BW is the body weight (child: 15.9 kg, adult: 56.8 kg); CF is a conversion factor for fresh weight vegetable to dry weight = 0.085 [32]; EF is the exposure frequency = 365 (day/year); AT is the averaging time (period over which exposure is averaged) = 26,280 days. Risk and distribution analyses of PAHs in vegetation and soil The Mann-Whitney U test results showed that all specific PAHs except Pyr, Chr, and Bbf, as well as total PAHs were significantly higher in vegetation than soil (p<0.05). The concentration of total PAHs was (207±31) μg/kg in soil, and the concentration of total PAHs was (1052±73) μg/kg in vegetation. Therefore, the bioconcentration factor (BCF) for total PAHs, defined as the ratio of total PAHs in vegetation to total PAHs in soil, was 8±1. The TEQ values for soil ranged from 7 to 202 μg/kg d.w., with an average of 41 μg/kg d.w. in the outskirts of Xi'an according to the USEPA [5]. The cancer risks of PAHs in soil ranged from 1×10 −7 to 3×10 −6 (mean = 7×10 −7 ) through exposure pathways of direct ingestion, dermal contact, inhalation of soil particles, and inhalation of surface soil vapor using RBCA and local parameters. The TEQ values for vegetation ranged from 11 to 117 μg/kg d.w. with an average of 84 μg/kg d.w., and the cancer risks posed by ingestion of vegetation ranged from 2×10 −5 to 2×10 −4 (mean = 1.66×10 −4 ). Distributions of PAHs in soil and vegetation among various sampling areas and regions are shown in Figs. 3 and 4. For PAHs in both soil and vegetation, no significant differences were observed among three regions by the Kruskal-Wallis H test (p>0.05). The average concentration of total PAHs in soil was higher in Weiyang (247±47 μg/kg) and lower in Lintong (137±37 μg/kg), whereas the average concentration of total PAHs in vegetation was higher in Lintong (1212±107 μg/kg) and lower in Weiyang (1006±110 μg/kg). For soil, the concentration of total PAHs in Jiangwu of Weiyang was highest (583±123 μg/kg), and was significantly higher than those in Xicha, Xiwang, Shijia, Changjia, and Xingnan Village (p<0.05) based on the Nemenyi test. The average concentration of total PAHs in vegetation was highest in Xiwang Village. with soil moisture (p>0.05). In addition, few PAHs were correlated with total N and total P in soil (p<0.05). However, seven high-ring PAHs (4-to 6-ring PAHs: Fla, Bbf, Bkf, Bap, Daa, Bgp, and I1p), as well as total PAHs in vegetation had significant positive correlations with soil CEC. Moreover, eleven PAHs (Any, Phe, Fla, Baa, Chr, Bbf, Bkf, Bap, Daa, Bgp, and I1p), as well as total PAHs in vegetation had significant positive correlations with soil fine particles (clay and silt), whereas these eleven PAHs (Any, Phe, Fla, Baa, Chr, Bbf, Bkf, Bap, Daa, Bgp, and I1p), as well as total PAHs in vegetation had significant negative correlations with sand. The p values resulted from Spearman correlation between PAHs and soil properties were summarized in Table 2. Both the total PAHs in vegetation and BCF for total PAHs were determined by pH and particle composition, and the regression equations are shown in Table 3. Multivariate statistical analysis of PAHs in vegetation and soil The resulting factor scores of both vegetation and soil samples as species/independent variables based on the composition of PAHs were presented in PCA. These values were obtained by an iterative ordination algorithm. The variance of the species scores on each ordination axis reflected the importance of the axis. Consequently, the species scores on the first axis would show a larger spread than those on the second axis. PCA revealed that the percentage variances of the PAHs in soil and vegetation explained by the first and second axes were 60 and 16, respectively. Linear correlation coefficients between the distributions of PAHs in the soil and those in vegetation were indicated by the cosine values of the angles between their arrows. Therefore, the distributions of PAHs in vegetation samples were positively related to those in vegetation samples (S1 Fig.). The resulting factor scores of distributions of PAHs in soil (species/independent variables) and vegetation (environmental/dependent variables) were presented in RDA by carrying out multiple regressions within the iterative algorithm. In RDA, the regression model was inserted in the ordination model. As a result, the ordination axes appeared in the order of the variance explained by linear combinations of environmental variables. The cumulative percentage variance of distributions of PAHs in vegetation samples explained by those in vegetation was as high as 98 in the RDA, which was statistically significant based on unrestricted Monte Carlo permutation under a reduced model (p<0.05). As shown in Fig. 2 Contamination and risk level for vegetation and soil The TEQ value for soil (mean = 41 μg/kg d.w.) in the outskirts of Xi'an was comparable with that in farmland of the low urbanization area (mean = 51 μg/kg d.w.) and the Hunpu wastewater irrigated region (mean = 52 μg/kg d.w.) [33,34]. These values were higher than the agricultural/horticultural soil acceptance criteria provided for surface (<1 m) contamination in the petroleum hydrocarbon guidelines of New Zealand (27 μg/kg) [7]. However, they were much lower than 600 μgÁkg -1 , which is Bap TEQ calculated by the Canadian Council of Ministers of the Environment (CCME) to protect human health for all land uses (cancer risks = 1×10 -6 ) by the exposure pathways of direct ingestion, inhalation and dermal exposures [8]. The cancer risks posed by PAHs in soil collected from all villages except Dujia, Gaomiao, and Jiangwu met the acceptable cancer risk level proposed by China MEP (1×10 −6 ) [29]. Excess lifetime risk limits for carcinogens typically range from 10 -6 to 10 -4 [9]. The cancer risks posed by PAHs in soil from all villages were lower than 1×10 −4 . The concentration of total PAHs (122-1832 μg/kg) in vegetation was comparable to that in vegetation near an e-waste recycling site in south China (199-2420 μg/kg) [35]. The TEQ values for vegetation (mean = 84 μg/kg d.w.) was comparable to that of Spinacia oleracea L. in the wastewater irrigated region of south China (mean = 70 μg/kg) [36]. was higher than international excess lifetime risk limits for carcinogens (1×10 −4 ) [9]. The cancer risk values were calculated by assuming a constant diet of vegetables contaminated with PAHs, which would overrate the risks. However, the cancer risks posed by PAHs in the outskirts of Xi'an should receive great attention and further investigation based on these findings. Jiangwu Village has a developed traffic network and a thriving tourist industry, which has led to increased petroleum hydrocarbons and PAHs in soil. High molecular weight PAHs with lower bioavailability were dominant in soil of Dujia, which are represented by the lowest LPAHs/HPAHs ratios (Table 1). Therefore, the average concentration of total PAHs in vegetation of Dujia was lowest although the average concentration of total PAHs in soil of Dujia was higher than that in soil of all villages except Jiangwu. The vegetation in Xiwang absorbed low Source apportionment of PAHs in vegetation and soil LPAHs/HPAHs <1.0, Ant/(Ant+Phe) >0.1 reflect pyrogenic sources, such as combustion-derived particles present in urban atmospheric dust. LPAHs are predominant in fuel oil or lightrefined petroleum products, which have LPAHs/HPAHs >1.0 and Ant/(Ant+Phe) <0.1 [15,37,38]. The Fla/(Fla+Pyr) ratio is below 0.50 for most petroleum samples, with values closer to 0.40 than 0.50. The ratio is between 0.40 and 0.50 for liquid fossil fuel combustion and above 0.50 in grass, most coal, and wood combustion samples [5,39,40]. Baa/(Baa+Chr) ratios <0.20 indicate petroleum, while those of 0.20 to 0.35 indicate either petroleum or combustion, and >0.35 indicate combustion [5,41]. I1p/(I1p+Bgp) ratios <0.20 likely indicate petroleum, while ratios between 0.20 and 0.50 indicate liquid fossil fuel combustion, and ratios >0.50 indicate grass, wood, and coal combustion [42,43]. According to the Ant/(Ant+Phe), Fla/(Fla+Pyr), Baa/(Baa+Chr), and I1p/(I1p+Bgp) ratios, the PAHs in vegetation and soil in the outskirts of Xi'an were mainly from pyrogenic sources. The Fla/(Fla+Pyr) ratios and the I1p/(I1p+Bgp) ratios approaching 0.5 indicated that the PAHs were mainly from liquid fossil fuel (vehicle and crude oil) combustion. The LPAHs have greater water solubility, volatility, and bioavailability, which resulted in LPAHs/HPAHs ratios higher than 1 for vegetation [44,45]. The affinity to soil organic matter (Koc partition coefficient) of PAHs with 4-6 rings is two or three times higher than that of PAHs with 2-3 rings [46]. Moreover, PAHs in aerosol samples collected from October 2005 to October 2007 mainly originated from liquid fossil fuel (vehicle and crude oil) combustion in Xi'an [10]. Based on these findings, it is important to implement vehicle exhaust controls to protect the atmosphere, soil, and vegetation. Effects of soil properties on PAHs in soil and vegetation Increases in pH resulted in higher negative charges on both OM and inorganic solid surfaces in the soil, as well as high soil CEC. The repulsion between the soil surface and OM could cause the desorption of OM from solid surfaces. Second, increases in pH increased the soil particle dispersion, which resulted in more colloid-associated OM being filtered into the operationally defined soluble portion [47]. Therefore, PAHs as hydrophobes were strongly adsorbed onto the surface of the particles associated with OM and positively correlated with OM and sand, whereas they were negatively correlated with pH, soil CEC, and fine particles (clay and silt) [15,48,49]. The finer materials only adsorb PAHs on the surface, while sand adsorbs PAHs on the surface and within larger sized particles. Therefore, PAH concentrations in soil increased with increasing amounts of sand [43,50]. Conversely, PAHs increase in vegetation with increasing pH, soil CEC, and fine particles as well as decreasing OM and sand in soil. Similarity and implication of PAHs in vegetation and soil Due to the physicochemical properties of PAHs and soil, a portion of each PAH retained in the soil and other portions migrated from soil into vegetation. A negative correlation was observed between specific PAHs or total PAHs in soil and vegetation, although this correlation was not statistically significant (p>0.05). Results from analysis of specific PAH or total PAHs values would not accurately represent the relationship between PAH distribution in soil and in vegetation; therefore, multivariate models were employed to analyze the entire dataset. In PCA, the distributions of PAHs in vegetation samples were positively related to those in vegetation samples, which indicated a similar composition and source of PAHs in soil and vegetation. Tavakoly Sany et al. [17] found that 46% of the total variances of the PAHs of sediments originate from coal combustion and vehicular emissions, while 36% of the total variances are related to petrogenic sources, biogenic sources, and unknown sources based on PCA of the PAHs congeners. Wang et al. [35] conducted PCA to evaluate PAHs congeners and found that they could be divided in to LPAH and HPAH groups based on their different physicochemical properties. Almost all of the soil and vegetation samples were close to each other in the score plot, indicating that PAHs near e-waste recycling sites may originate from the same source via dispersion of the ash or gaseous emissions from open burning activity. In our study, geochemical indices indicated that the PAHs in soil and vegetables mainly originated from vehicle and crude oil combustion. We conducted PCA and RDA to investigate soil and vegetation samples rather than PAH congeners. The similarity between distributions of PAHs in soil and those in vegetation and the effects of soil contamination on vegetation were quantified. In RDA, 98% of the total variances for PAH distributions in vegetation samples could be explained by PAH distributions in soil. The results of RDA confirmed that they had a common source and demonstrated the important effects of soil contamination on vegetation. Accumulation of PAHs in both farmland soil and vegetables through gaseous deposition, as well as migration of PAHs from the soil to vegetables through roots resulted in highly similar distribution of PAHs in soil and vegetation [11,12,13]. Conclusions In this study, the cancer risks posed by exposure pathways of direct ingestion and dermal contact PAHs in soil, inhalation of soil particles and surface soil vapor met the rigorous acceptable cancer risk level (1×10 −6 ). However, the Bap TEQ values of PAHs in soil were higher than agricultural/horticultural soil acceptance criteria provided for surface (<1 m) contamination in the petroleum hydrocarbon guidelines for New Zealand. The cancer risks posed by ingestion of vegetation ranged from 2×10 −5 to 2×10 −4 with average of 1.66×10 −4 , which was higher than international excess lifetime risk limits for carcinogens (1×10 −4 ). Both the total PAHs in vegetation and BCF for total PAHs were determined by pH and particle composition, and the PAHs were found to increase in vegetation with increasing pH and decreasing sand in soil. The results of the PCA and the RDA confirmed that PAHs in soil and vegetation had a common source (vehicle and crude oil combustion) and demonstrated the notable effects of soil contamination on vegetation. Supporting Information S1 Fig. Principal components analysis of PAH distribution in soil-vegetation. The percentage variances of the PAHs in soil and vegetation explained by the first and second axes were 60 and 16, respectively. The approximated linear correlation coefficient between two species variables (vegetation and soil samples) was equal to the cosine of the angle between the corresponding arrows. (EPS) S1

v3-fos

2016-05-17T16:14:43.309Z

2015-05-30T00:00:00.000Z

14607288

s2

Genome-wide association study on legendre random regression coefficients for the growth and feed intake trajectory on Duroc Boars Background Feed intake and growth are economically important traits in swine production. Previous genome wide association studies (GWAS) have utilized average daily gain or daily feed intake to identify regions that impact growth and feed intake across time. The use of longitudinal models in GWAS studies, such as random regression, allows for SNPs having a heterogeneous effect across the trajectory to be characterized. The objective of this study is therefore to conduct a single step GWAS (ssGWAS) on the animal polynomial coefficients for feed intake and growth. Results Corrected daily feed intake (DFIAdj) and average daily weight measurements (DBWAvg) on 8981 (n = 525,240 observations) and 5643 (n = 283,607 observations) animals were utilized in a random regression model using Legendre polynomials (order = 2) and a relationship matrix that included genotyped and un-genotyped animals. A ssGWAS was conducted on the animal polynomials coefficients (intercept, linear and quadratic) for animals with genotypes (DFIAdj: n = 855; DBWAvg: n = 590). Regions were characterized based on the variance of 10-SNP sliding windows GEBV (WGEBV). A bootstrap analysis (n =1000) was conducted to declare significance. Heritability estimates for the traits trajectory ranged from 0.34-0.52 to 0.07-0.23 for DBWAvg and DFIAdj, respectively. Genetic correlations across age classes were large and positive for both DBWAvg and DFIAdj, albeit age classes at the beginning had a small to moderate genetic correlation with age classes towards the end of the trajectory for both traits. The WGEBV variance explained by significant regions (P < 0.001) for each polynomial coefficient ranged from 0.2-0.9 to 0.3-1.01 % for DBWAvg and DFIAdj, respectively. The WGEBV variance explained by significant regions for the trajectory was 1.54 and 1.95 % for DBWAvg and DFIAdj. Both traits identified candidate genes with functions related to metabolite and energy homeostasis, glucose and insulin signaling and behavior. Conclusions We have identified regions of the genome that have an impact on the intercept, linear and quadratic terms for DBWAvg and DFIAdj. These results provide preliminary evidence that individual growth and feed intake trajectories are impacted by different regions of the genome at different times. Electronic supplementary material The online version of this article (doi:10.1186/s12863-015-0218-8) contains supplementary material, which is available to authorized users. Background The use of genomic information to infer the estimated breeding value (EBV) of an individual, referred to as genomic-EBV (GEBV), has become a routine practice in several livestock species due to the rapid expansion and cost effective nature of genotyping technology. Currently, the majority of traits utilized when estimating GEBV are measures occurring at a single time point or averaged across several time points. Alternatively, longitudinal models that describe the trajectory across time can be utilized to characterize the variation across animals across the time horizon for a specific trait. Models such as random regression or splines have been utilized in the past and are advantageous since they allow for the covariance between age classes (age (d) of an animal) to vary continuously across the trajectory [1][2][3][4]. While these models have seen widespread application with the use of pedigree data their use in conjunction with dense SNP panels, either for genomic prediction or trait architecture dissection is far less common. Previous research has utilized random regression models to characterize the effect of individual SNP across time using either simulated [5,6] or real data [7] on a small number of SNPs. Characterizing SNP effects across a trajectory when the data is derived from dense SNP arrays (i.e. 1000+ SNPs) remains computationally demanding. In spite of this a genome wide association study (GWAS) using a longitudinal model offers several advantages, the main one being the ability to account for the heterogeneity of marker effects across time. Growth and feed intake are economically important traits in swine production [8] and have been previously investigated using average daily gain and average daily feed intake, respectively [9,10]. Complex traits such as growth and feed intake are often the result of dynamic systems. It is conceivable that different genes might play different roles along the growth and feed intake trajectory [11]. Longitudinal models offer the possibility to explicitly account for this heterogeneity. A recent GWAS was conducted by Tetens et al. [12] based on degressed EBV from a random regression model using Legendre polynomials for feed intake across specific phases of the lactation curve in dairy. To the authors' knowledge no previous research has utilized the animal specific polynomial coefficients as a phenotype in a GWAS. The use of the polynomial coefficients could allow for the characterization of genes that impact specific components of the trajectory. Knowledge of these regions would in turn be advantageous to potentially identify genetic antagonisms involving the shape of the growth and feed intake trajectory. Recently, a GWAS approach, referred to as single-step GWAS (ssGWAS), that utilizes all genotypes, phenotypes, and pedigree information jointly in one step has been proposed by Wang et al. [13] and validated using field data [14][15][16]. This approach allows for complex models such as random regression and multiple traits to be efficiently implemented. Furthermore, greater power and more precise estimates of variance components can be achieved by including non-genotyped animals if the number of genotyped animals is limited. The objective of this study is to perform a ssGWAS on the animal polynomial coefficients in order to identify genomic regions that impact specific polynomial coefficients of the growth and feed intake curves in Duroc boars. Genetic parameters Corrected electronic FIRE (Feed Intake Recording Equipment, Osborne Industries, Inc., Osborne, KS, USA), daily feed intake (DFI Adj ) and average daily weight measurements (DBW Avg ) on 8981 (n = 525,240 observations) and 5643 (n = 283,607 observations) animals were utilized in a random regression analysis (order = 2). A blended relationship matrix (H) containing a SNP-derived genomic relationship matrix (G) and a pedigree numerator relationship matrix (A) was constructed to model the additive genetic relationship between animals [17]. The trajectory of each individual was split into three phases based on age classes. Phase 1, 2 and 3 included ages from 90 to 118d, 119 to 146d and 147 to 175d, respectively and the average heritability reported within each phase. Descriptive statistics and the number of observations within each class for both DFI Adj and DBW Avg are outlined in Table 1 and Fig. 1, respectively. The estimated heritability for the traits ranged from 0.34 to 0.52 and 0.07 to 0.23 for DBW Avg and DFI Adj across the trajectory. Genetic correlations across the trajectory for DBW Avg and DFI Adj are depicted in Fig. 2. Correlations across age classes were large and positive for the majority of the trajectory for DBW Avg (mean correlation: 0.75), although the correlation decreased slightly as the age classes grew further apart from each other. The mean correlation between phase 1 and 3 was 0.48. The genetic correlations across age classes for DFI Adj (mean correlation: 0.54) were large and positive for age classes that were near each other. As the age distance increased, the correlation decreased with the lowest correlation found between age classes at the beginning and end of the trajectory. The mean correlation between phase 1 and 3 was 0.01. The average heritabilities for phase 1, 2 and 3 were 0.37, 0.45 and 0.50 for DBW Avg and 0.08, 0.12 and 0.17 for DFI Adj . GEBV correlations and heritability within and across traits for each polynomial coefficient are outlined in Table 2. The correlation between the intercept and linear coefficient for DFI Adj and DBW Avg was moderate, while negligible between the intercept and quadratic coefficient. For both traits the correlation between the linear and quadratic coefficient was negative and moderate. Genome-wide association study A ssGWAS as described by Wang et al. [13] was conducted on the animal polynomial coefficients (i.e. intercept, linear and quadratic) for both DFI Adj and DBW Avg . A total of 855 and 590 animals with both phenotypes and genotypes for DFI Adj and DBW Avg were used to conduct the association analysis on 31,366 autosomal SNP. The G part of the H matrix was utilized to iteratively estimate individual SNP effects from the animal specific GEBV for each polynomial coefficient. To characterize regions of the genome that had an impact on a given coefficient and to limit statistical noise and reduce the number of false positives 10-SNP sliding windows GEBV (WGEBV) was used. This was done to account for marker effects potentially being shared by adjacent SNP in high linkage-disequilibrium (LD). For each polynomial the significance level of the putative QTL window was estimated using a bootstrap analysis with 1000 replicates. Briefly, a bootstrap sample was generated for each observation by replacing the putative QTL windows with a sample from an independent standard normal distribution that was scaled by the residual variance from the full model. For each bootstrap sample the data was reanalyzed and the WGEBV re-computed. The p-value of a window was obtained based on the number of times a bootstrap sample WGEBV from the 1000 simulated exceeded the WGEBV from the real data. An arbitrary genome-wide significance value of P < 0.001 was adopted. Based on this, gene annotations for significant windows were obtained using the Biomart platform on Ensemble [18] through the 'Biomart' R package (http://www.bioconductor.org). To characterize the genetic relationship between polynomial coefficients within and across traits, the covariance between WGEBV across the genome for each trait polynomial combination was obtained. In addition, the WGEBV correlation averaged across the genome was compared to the GEBV correlation within and across traits. Multiple regions were found to be significantly associated with specific polynomial coefficients based on the Table 3. Furthermore, the region on SSC9 was associated with both intercept terms for DFI Adj and DBW Avg . Additional file 1: Figure S1 and Additional file 2: Figure S2 display the contribution of each WGEBV to the overall WGEBV variance for a given polynomial coefficient for DFI Adj and DBW Avg , respectively. In general, the contribution of a particular region is heterogeneous across polynomial coefficients for both DFI Adj and DBW Avg . The cumulative variance explained (number of windows) by significant windows for DFI Adj was 1.0 (n = 10), 0.3 (n = 3) and 0.6 (n = 1) percent for the intercept, linear and quadratic polynomial coefficients, respectively. Similarly, cumulative variances explained (number of windows) by significant windows for DBW Avg were 0.9 (n = 6), 0.2 (n = 1) and 0.5 (n = 5) percent for the intercept, linear and quadratic part of the trajectory, respectively. The WGEBV variance explained by significant regions for the trajectory was of 1.54 and 1.95 % for DBW Avg and DFI Adj . The covariance between WGEBV polynomial coefficients across the genome is outlined in Additional file 3: Figure S3 and Additional file 4: Figure S4 for DFI Adj and DBW Avg , respectively. In addition, the WGEBV correlation averaged across the genome is outlined in Table 2. Additional file 3: Figure S3 and Additional file 4: Figure S4 show how there are regions across the genome with a large degree of covariance across polynomial coefficients. In particular, a region on SSC9 (8.4-9.5) with a large and positive covariance between the intercept and quadratic coefficient for DBW Avg was tagged as a putative QTL for both coefficients, although was not declared significant after the bootstrap analysis. The same region was declared significant for the intercept term for DFI Adj . Also, for DBW Avg a region on SSC14 (15.6-17.2) had a positive covariance between the intercept and quadratic coefficient and the region was declared significant for the intercept term. Knowledge of regions that have a covariance that deviates from the average between two polynomial coefficients allows for the potential to alter genetic antagonisms regarding the shape of the trajectory through selection. Genes within regions with a significant impact on the intercept coefficient for DFI Adj were identified, involving energy homeostasis (TBC1D1, UCP2, UCP3), anti-satiety and adipogenesis (TPP2), behavior (GLRA3), glucose homeostasis (IGFBP5), host immune response and cell-tocell interactions (SIGLEC-5), vasoconstriction and kidney function (EDN1). Furthermore, significant regions for higher order polynomial coefficients (i.e. linear and quadratic) included genes related to Cysteine homeostasis (CDO1), polyamine synthesis regulation (AZIN1) and cell signaling (GPR126). Regions that impacted the intercept coefficient for DBW Avg included genes related to insulin signaling (PHLPP1), feeding behavior and regulation of metabolism (MC4R), energy homeostasis (NDUFAF6) and cell growth and division (VRK1). Similarly for linear and quadratic coefficients for DBW Avg genes within significant regions were identified involved in the formation of skeletal elements (IMPAD1), the negative regulator of cell proliferation (CABLES1), clearing of metabolic waste (STAB2) and tryptophan metabolism (KYNU). Discussion The heritability estimates derived from our study for DBW Avg and DFI Adj are in line with previous random regression estimates although genetic correlations between age classes are lower than previous studies. Utilizing FIRE systems, Haraldsen et al. [2] and Wetten et al. [3] estimated the heritability for the growth trajectory in Norwegian Duroc and Landrace boars using pedigree information. Estimates ranged between 0.32 to 0.35 and 0.17 to 0.25, respectively while genetic correlations across test days were never below 0.80. Using three weight measurements across the growth period Huisman et al. [4] estimated the heritability to range from 0.13 to 0.20 and the genetic correlation was the lowest (0.378) for measurements at the beginning and end of the growth phase. Zumbach et al. [19] using a population related to the one in the current study obtained heritability estimates of 0.04, 0.06 and 0.09 for daily, weekly, and bi-weekly intervals, respectively, using a repeatability model. Schnyder et al. [1] estimated the heritability for weekly mean daily feed intake from castrated Large White male pigs using pedigree information to range from 0.20 to 0.38 and the genetic correlation between weekly mean daily feed intake was large and positive with the lowest (r g = 0.80) occurring for feed intake at week 1 and weeks at the end of the test period. Wetten et al. [3] estimated the heritability along the feed intake trajectory for Norwegian Duroc and Landrace boars using pedigree information to be from 0.09 to 0.11 for both breeds. The heritability for the polynomial coefficient for weekly mean feed intake was estimated by Schnyder et al. [1] and the majority of the variation was captured by the intercept (h 2 = 0.32) with a smaller proportion captured by the linear (h 2 = 0.06) and quadratic (h 2 = 0.03) regression coefficients. An alternative way to model growth curves using genomic information has been investigated by Silva et al. [20] using a nonlinear logistic regression model to estimate the regression functions for mature weight, start weight, and growth rate, and then used these as phenotypes in a GWAS. Silva et al. [20] estimated a moderately negative genetic correlation (r g = −0.69) between mature weight and growth rate. This is in line with our results with moderately negative WGEBV and GEBV correlation between the linear and quadratic coefficients for DFI Adj and DBW Avg . Although the method utilized by Silva et al. [20] for modeling growth curves does provide a way of obtaining mature weight breeding values, the ability to put different degrees of selection pressure across the trajectory and on specific polynomial coefficients is not possible. Random regression might allow to "bend the growth curve". This has been investigated for example on lactation curves in dairy cattle using a restricted selection index in order to make cattle more persistent (i.e. reduced rate of decline in milk yield after peak milk yield) [21]. A different method could involve constructing a traitspecific marker derived relationship matrix as outlined by [22] that weights the genomic relationship matrix based on specific polynomials in order to place more emphasis on certain regions of the genome. Future research would need to verify the effectiveness of either approach for growth and feed intake in pigs. A limited number of GWAS studies have investigated regions that impact feed intake and growth in pigs using average daily feed intake (ADFI) and average daily gain (ADG) as phenotypes [9,10]. A common alternative metric to determine feed efficiency has been often utilized, referred to as residual feed intake (RFI) [9,10,[23][24][25][26]. RFI is usually defined as the difference between the observed feed intake and the feed intake predicted based on production traits [27]. The limitations of using ADG, ADFI or RFI for a GWAS is that an animals feed intake and growth trajectory is not characterized and more importantly the gene effects are considered consistent across time. Due to a higher level of muscle deposition at the beginning of the trajectory compared to a higher level of fat deposition towards the end of the trajectory it is expected that different metabolic pathways are being differently regulated. A GWAS on the polynomial coefficient in a random regression model directly is advantageous because it allows for the additive genetic architecture to be understood for each polynomial coefficient. Furthermore, regions that have an effect across polynomial coefficients can be identified in order to characterize genetic antagonisms for the feed intake and growth trajectory. Longitudinal models could account for the fact that a gene effect might potentially not being consistent across time. It is expected that effects associated with the intercept coefficient would be homogenous across time while higher order polynomial coefficients, such as the linear and quadratic terms would capture transient effects across the trajectory. In the current study, a bootstrap analysis was conducted to declare significance based on WGEBV variance. A similar method has been utilized previously in GWAS studies [10,28] and provides a robust, albeit computationally intensive way to conduct significant testing, when using the ssGWAS method. In a previous study by Jiao et al. [10], a GWAS was conducted on ADG and ADFI using the same genetic line employed in the current work. A region located on SSC1 (166-170 Mb) was significantly associated with both ADG and ADFI. In the current study the same region was identified as a putative QTL for the ADFI intercept coefficient, but not ADG. The region did not pass the bootstrap significance threshold. A potential reason for the discrepancy is that the marker map we used was based on the 2 nd version of the SNP60k bead chip, whereas Jiao et al. [10] used the 1 st version. The marker map used in the current study is outlined in the supplementary attached marker map file (MarkerMap.xlsx). The linkage disequilibrium was investigated based on both genotypes used by Jiao et al. [10] and genotypes employed in the current study for SSC1 (168 -180 Mb) and is illustrated in Additional file 5: Figure S5 and Additional file 6: Figure S6, respectively. As shown, there is strong LD between the region that Jiao et al. [10] found significant for ADG and ADFI and the MC4R gene based on the genotypes used in the current study, while LD was much weaker based on the genotypes used in the previous study. This could explain why the MC4R region instead of the region found by Jiao et al. [10] was found to be associated with DBW Avg in the current study. Other reasons for the differences between the two analyses may be due to the fact that a Bayesian method was utilized in the previous study, a larger number of phenotyped and smaller number of genotyped individuals in the current dataset and different modeling techniques that allow for the covariance to change between age classes. A comparative analysis between Bayesian alphabet methods and ssGBLUP conducted by Wang et al. [15] highlighted how the strength and detection of associations depends on the methodology utilized and both have their advantages. Multiple regions identified in the current study have been found to be previously associated with metrics related to feed intake and growth in both livestock species and humans. A region on SSC6 (50.8-53.4 Mb) was found to harbor the SIGLEC-5 gene, which is contained within a large family of cell-surface transmembrane receptors that regulate host immune responses [29]. It has been found that SIGLEC-5 weakly binds to leptin and potentially regulates leptin levels [30]. The region on SS7 (8.4-9.6 Mb) is in proximity of the EDN1 (9.15 Mb) gene, a powerful endogenous vasoconstrictor peptide that is produced and released by the vascular endothelium [31]. A consistent body of literature in humans has shown how variants within this gene are associated with hypertension and obesity (see for example Tiret et al. [32]). A previous study by Onteru et al. [9] also found an association 2 Mb downstream of EDN1. The TBC1D1 gene on SSC8 has been previously found to be associated with carcass traits in pigs [33]. The TBC1D1 gene is a Rab-GTPase-activating related protein implicated in regulating the trafficking of glucose transporter 4 (GLUT4) storage vesicles to the cell surface in response to insulin and AMPK-activating stimuli in skeletal muscle [33]. A previous GWAS study for RFI by Do et al. [23] also found an association 2 Mb downstream of the TBC1D1 gene. The two genes on SS9, UCP2 and UCP3, produce carrier proteins of the inner membrane of the mitochondria that release protons in respiring mitochondria and expression of these enzymes is nutritionally and hormonally regulated and plays a role in the regulation of energy balance [34]. It has been shown in transgenic mice that overexpressing UCP2 and UCP3 result in decreased adiposity and increased hypothalamic NPY concentrations and feed intake [35]. The TPP2 gene on SSC11 has been shown to have anti-satiety roles via the degradation of the satiety peptide cholecystokinin 8 and is required for mammalian adipogenesis [36]. A previous study by Gleason et al. [37] found that the absence of IGFBP5 in mice results in an increase in size and mild glucose intolerance and is accentuated during diet-induced obesity. The region on SS1 that contained the gene (GPR126) was associated with the quadratic coefficient for DFI Adj and has been previously found to be associated with human height [38] and weight gain in German Landrace boars [39]. Furthermore, a region 5 Mb upstream of GPR126, PEX7 and MAP3K5, was found to be associated with RFI by Do et al. [26]. The GPR126 gene is involved in cell signaling and has been shown to give rise to adolescent idiopathic scoliosis in humans, which is characterized by spinal deformations [40]. The progression of idiopathic scoliosis has been shown to be related to the growth and age of the individual therefore it is perhaps not surprising that the SNP effect would change across time in a non-linear manner based on functional analysis in humans [41]. Regions associated with the intercept coefficient for DBW Avg was the gene PHLPP1 on SSC1 which encodes a phosphatase that can terminate Akt signaling which in turn is able to regulate insulin levels. Andreozzi et al. [42] found that PHLPP1 abundance is increased in adipose tissue and skeletal muscle of obese individuals, and is also significantly related to BMI and insulin resistance. A region 1.5 Mb upstream on SSC1, MC4R, has been previously found to be associated with ADFI and ADG [43]. Although the variant that has been shown to be associated with ADFI and ADG is not on the current chip, the region comprising PHLPP1 and MC4R display high levels of linkage disequilibrium, as shown in Additional file 6: Figure S6, therefore it is possible either one or both of the genes are associated with the intercept coefficient for DBW Avg . The region on SSC6, which was associated with the quadratic coefficient for DBW Avg contained the gene, CABLE1, which encodes a protein involved in cell cycle regulation by interacting with several cyclin-dependent kinases and has been previously found to be associated with height and menarche in humans [44]. The STAB2 gene functions as a scavenger receptor to clear metabolic waste products from the circulation and in mice lacking the protein have been shown to display reduced hepatic clearance of waste products in the blood [45]. The region on SSC15 harbors the KYNU gene, which is involved in the kynurenine pathway, which is a major route for the majority of ingested tryptophan [46]. Tryptophan is the precursor of a wide array of metabolites, which are involved in a variety of aspects related to nutrition and metabolism [46]. Conclusions The incorporation of genomic information into random regression models has allowed for the identification of regions that are potentially associated with the shape of the growth and feed intake curve. These results have confirmed that the polynomial coefficients describing the individual's growth and feed intake curve are impacted by different regions of the genome. Furthermore, the WGEBV covariance between growth and feed intake polynomial coefficients have been identified. Regions and genes with heterogeneous effects across time were identified by including linear and quadratic terms in the random regression model. Future research will involve using genomic information to modify the trajectory by constraining certain polynomials for both DBW Avg and DFI Adj . Data set No animal care approval was required for the present manuscript because all records came from field data. [47]. Briefly, feed intake editing techniques developed by Casey et al. [48] were used to identify and adjust for errors associated with feed intake observations. The editing procedures were: 1.) Identify and remove errors for each visit based on 16 criteria [48]; 2.) Sum errorfree feed intake within each day for each pig; 3.) Estimate the effect of error counts on error-free feed intake by fitting a linear mixed model to error-free daily feed intake observations with the 16 error counts, contemporary group (concatenation of season, pen and year of birth), body weight on that day and ADG as covariates and pig as a random effect; 4.) Adjust error-free DFI Adj for each pig and day for feed consumed during error visits based on estimates obtained from part 3. Lastly, animals with less than 20 DFI adj observations were removed. The final number of DFI Adj observations totaled 525,240 on 8981 animals. Weight editing techniques developed by Zumbach et al. [19] were utilized to identify and remove errors. Briefly, utilizing robust regression with a bisquare weight function weight was fit to a quadratic regression of ontest day and linear regression of on-test age. Each data point from a robust regression procedure is assigned a weight (from 0 to 1) and weights that were less than 0.5 were treated as outliers and removed. Lastly, on-test ADG was computed by regressing weight on age and values less than .4 kg or greater than 2.0 kg were removed and the remaining weights were averaged by day (DBW Avg ). The final number of DBW Avg observations was 283,607 on 5643 animals. Descriptive statistics for DFI Adj and DBW Avg and the number of observations for each age (age (d) of an animal) is outlined in Table 1 and Fig. 1, respectively. Genotypic data was derived from the Illumina Porci-neSNP60K Bead (Illumina Inc., San Diego, CA; n = 3699) and the GGP-Porcine containing roughly 10,000 SNP (GeneSeek Inc., a Neogen Co., Lincoln, NE; n = 3621). Prior to the imputation of missing genotypes and imputation of low-density to medium-density, multiple quality control edits were conducted including removal of animals with call rates ≤ 0.90, SNP with call rates ≤ 0.90, SNP with a minor allele frequency (MAF) ≤ 0.02, and pvalue < 0.0001 of a chi-square test for Hardy-Weinberg equilibrium. The Beagle software was used for imputation [49] and the mean (± SD) imputation accuracy (Beagle r 2 ) across all SNP was 0.85 (± 0.15). The SNP unmapped to the swine genome build 10.2 and SNP on sexual chromosomes were also excluded from the analysis. Furthermore, the map file used in the current analysis was based on version 2 of the Illumina PorcineSNP60K Bead genotype platform and any markers that were not in common were removed. Only animals with both phenotypes and genotypes were used in the analysis and totaled 858 and 590 for DFI Adj and DBW Avg , respectively. Animals were derived from both the medium-density (DFI Adj : n = 786; DBW Avg : n = 587) and the low-density panel (DFI Adj : n = 70; DBW Avg : n = 3). Prior to analysis the MAF for the genotyped animals used in the analysis was checked and SNP with a MAF < 0.002 were removed, resulting in 31,366 SNP utilized in the analysis. Statistical analysis Legendre polynomials (order = 2) were used to model the trajectory of DFI Adj and DBW Avg . Prior to the analysis, age was standardized to have values from −1 to 1 to ensure numerical stability. Variance components were estimated by REML using the REMLF90 software [50]. A homogenous variance structure was utilized to decrease model complexity and similar results were found when a heterogeneous residual variance structure was utilized (data not shown). The model for DFI Adj and DBW Avg was, where y ijkmn was DFI Adj or DBW Avg , μ was the average DFI Adj or DBW Avg , CG i was the fixed effect of contemporary group (concatenation of birth year, season and pen), Parity j was the fixed effect of parity of the dam (1,2,3+), β k was the fixed regression coefficient of age, u mk was the k th random regression for animal m , pe mk was the k th random regression for the permanent environmental effect of animal m and e ijkmn was the residual. The effect φ mnk was the k th Legendre polynomial for animal m at age n . It was assumed u~N(0, H ⊗ G), where G was a 3x3 (co)variance matrix for the animal Legendre polynomials and pe~N(0, I ⊗ P), where P was a 3x3 (co)variance matrix for animal permanent environmental Legendre polynomials. Construction of the H matrix consisted of blending a 3-generation pedigree derived numerator relationship matrix and a SNP-derived genomic relationship matrix with a weighting factor of 0.995 and 0.005, respectively [17]: where A 22 is a numerator relationship matrix for genotyped animals. The genomic relationship matrix (G) was created by weighting each marker contribution by its expected variance: where Z is a matrix of gene content containing genotype (−1, 0, 1) adjusted for allele frequencies and D is a diagonal matrix with elements containing the reciprocal of the expected marker variance [49]. In order to determine the change in heritability and genetic correlation across time, the trajectory was split into three phases. Only age classes from 90 to 175d were used when calculating the heritability within a phase and the genetic correlation across phases. This was done due to large variance component standard errors from a small sample size at the beginning and end of the trajectory. Phase 1, 2 and 3 consisted of age classes 90 to 118d, 119 to 146d and 147 to 175d, respectively. Genome-wide association mapping A single step genome-wide association study (ssGWAS) as described by Wang et al. [13] was conducted on the animal specific polynomial coefficients (i.e. intercept, linear and quadratic) for both DFI Adj and DBW Avg . Briefly, the GEBV solutions from the previous analysis were used to estimate marker effects through an iterative process. In the first round the GEBV solutions are utilized to estimate marker effects based on a G matrix weighted by the expected marker variance [51]. In successive iterations marker effects are then recalculated with a similar process but with SNP expected variance in G replaced by the realized variance obtained in the previous iteration. The reweighting process effectively increases the weight of SNP with large effect and decreases those with small effects. A detailed description of the iterative algorithm is outlined in Wang et al. [13] under the 'Scenario 1' procedure. In our study the reweighting process was repeated twice to ensure stability of the marker effects estimates [15]. Similar to Sun et al. [14,16,52], a 10-SNP sliding window approach was utilized to characterize regions that have a large effect on a specific parameter of the trajectory and to declare significance for these regions using bootstrap methods. This was done to account for marker effects potentially being shared by adjacent SNP in high linkage-disequilibrium (LD) and to remove assumptions regarding the start and stop site of a region in LD with a QTL. Furthermore, it has been shown that SNP segments are useful to discriminate important effects from statistical noise [52] and it has been shown by Beissinnger et al. [53] that either 5 or 10 SNP window sizes had the most favorable ratio of detection rate to false-positive rate. The variance of 10-SNP sliding windows GEBV (WGEBV) was computed for each individual by multiplying the estimated SNP effects with their respective genotypes and summing across all SNP within the window. The WGEBV variance was then used in a two-stage approach to identify regions with large effects. The first-stage involved isolating regions with large effects by keeping the top 5 % WGEBV regions. The second stage involved sorting the windows by chromosome and genome location. Overlapping WGEBV were aggregated into one region and the aggregated regions were ranked based on their maximum WGEBV variance. The top 10 % aggregated regions were tagged as putative QTL (n = 17) to be further investigated for significance. Declaring significance Within each trait and polynomial coefficient the significance of putative QTL regions were determined based on a bootstrap analysis with 1000 replicates. Bootstrap samples were constructed using the estimated marker effects across the 3 polynomial coefficients to construct the distribution of the test statistic (WGEBV variance) for each putative QTL window within each polynomial coefficient. A bootstrap sample was constructed according to the null hypothesis of no QTL in the identified SNP window [28]. A bootstrap sample of vector y for replicate k (y (k) ) was constructed from the estimated fixed effects, random permanent environmental effects, SNP effects across all three polynomials, excluding SNP contained within the putative QTL and adding a simulated residual for each animal and day combination. The simulated residual was generated from sampling an independent standard normal distribution that was scaled by the residual variance from the full model. Using the predicted phenotype generated from the full model for animal i on day j , a bootstrap sample for replicate k was generated by: y ij k ð Þ ¼ŷ ij k ð Þ -û ij k ð Þ þũ ij k ð Þ þ e ij k ð Þ ; where ỹ ij(k) refers to the bootstrap sample phenotype, ŷ ij(k) refers to the predicted phenotype from the full analysis, û ij(k) refers to the GEBV from the full analysis, ũ ij(k) refers to the GEBV with SNP contained within the putative QTL window excluded for a given polynomial coefficient and e ij(k) refers to a simulated residual. For each bootstrap sample the ssGWAS reweighting procedure was conducted and the resulting marker effects were again partitioned into sliding windows and WGEBV were obtained as described above. The WGEBV for each putative QTL window was accumulated across all bootstrap samples and compared to the WGEBV variance test statistic derived from the real data. The p-value of a window was reported as the number of times a bootstrap statistic from the 1000 simulated exceeded the test statistic from the real data. Significance was declared using an empirical cutoff of P < 0.001 (i.e. test static from real data was never greater than any bootstrap statistic). Furthermore, the imputation accuracy (Beagle r 2 ) for all significant SNPs within a region were checked to ensure no spurious results. The percent of the additive genetic variation explained by all significant QTL regions for each polynomial coefficient was calculated using the following formula:

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Effect of Organic Selenium-Enriched Yeast Supplementation in Finishing Sheep Diet on Carcasses Microbiological Contamination and Meat Physical Characteristics The aim of the current study was to evaluate the effect of feeding Pelibuey sheep on diet supplemented with different doses of organic selenium (Se)-enriched yeast on carcasses microbiological contamination and meat physical characteristics. The experiment was conducted during the finishing stage of 18 female sheep and lasted for 60 days. In a complete randomized design, sheep were distributed to one of three treatments: the control without Se-yeast (T1), the control supplemented with Se-yeast at 0.35 mg Se/kg DM (T2), and control supplemented with Se-yeast at 0.60 mg Se/kg DM (T3). The yeast product used was Selyeast 3000TM yeast (LFA Lesaffre, Toluca, Mexico) with a Se concentration of 3000 ppm (mg/kg). Lambs were slaughtered at the end of the experiment at an average weight of 39.5±4.41 kg and samples were taken for microbiological analysis. There were no differences between treatments (P>0.05) and the aerobic plate counts for T1, T2 and T3 had indexes of 0.10, 0.08 and 0.08 log10CFU/cm2, respectively. Total coliform counts obtained were 0.13, 0.10 and 0.09 log10 CFU/cm2 for T1, T2 and T3, respectively, and the faecal coliform counts were 0.09 log10CFU/cm2 for T1, 0.06 log10CFU/cm2 for T2 and 0.07 log10 CFU/cm2 for T3. No significant effects (P>0.05) were observed for carcasses physical characteristics of microbial growth, initial and ultimate pH and temperature, colour values and water holding capacity. It can therefore be concluded that organic Se-enriched yeast did not affect carcasses bacterial proliferation or meat physical characteristics. Introduction In recent years, much attention has been paid to meat production with physiological functions that promote health conditions and prevent disease risks. Functional meat value could be increased by adding compounds with antimicrobial and antioxidant functions to the animal's basal diet like phytogenic extracts, conjugated linoleic acid, vitamin E, n-3 fatty acids and selenium (Se) to improve animal production, carcass composition, fresh meat quality and increasing the antioxidant capacity (Grashorn 2007;Zhang et al., 2010;Yanian et al., 2011;Salem et al., 2014aSalem et al., , 2014b. The amount of Se supplementation to diets varies according to the species. In case of sheep, 0.30-0.45 mg/kg DM is the recommended level (Vignola et al., 2009) whether Se supplemented in inorganic or organic forms. Selenium is an essential trace element for both animal and human health. Selenium is present in tissues and is part of the glutathione peroxidase (GSH-Px) enzyme, which reduces lipid and hydrogen peroxides to less harmful hydroxides via oxidation, and subsequent reduction of selenocysteine and without Se, this enzyme could not act (Juniper et al., 2009;Vignola et al., 2009). Glutathione peroxidases are probably protecting neutrophils from oxygen-derived radicals, which are produced to kill invading organisms (Splettstoesser and Schuff Werner, 2002). Moreover, Se is essential for other cell mediated immunity traits, like removal of viruses and destruction of neoplastic cells (Stazi and Trinti, 2010). De Vore et al. (1983) mentioned that Se antioxidant functions have persisted after slaughter in poultry muscle tissue, via GSH-Px activity. Moreover, Juniper et al. (2009) reported that GSH-Px activity was greater in lambs that receiving Se-enriched yeast compared with those receiving a similar dose Se from an inorganic source (sodium selenite). Selenium has the ability to improve immune system as this trace element is essential for the development and expression of non-specific humoral and cell mediated immune responses (Kumar et al., 2009). The most important factors in fresh meat handling are handling speed, control of temperature and proper hygiene conditions (Ray and Bhunia, 2008). Meat quality factors such as colour and drip loss are decisive for consumer purchase decision. Discoloration of meat is believed to be related to the oxidation processes, and as a consequence sensorial changes and microorganisms proliferation (Baron and Andersen 2002;Wang et al., 2009). Researches have been done on meat, but there is no information about the effect of organic Se on microbial contamination of carcasses. The hypothesis of the current study was based on the ability of Se to improve immune system, its importance for cell removal of viruses and the destruction of neoplastic cells, which may reduce carcasses microbiological contamination. Therefore, the aim of this study was to evaluate carcasses microbiological contamination and meat physical characteristics in sheep fed diet supplemented with Se-enriched yeast at different doses. Study design The experiment was conducted during the finishing stage of 18 Pelibuey breed ewes with an initial body weight of 27.75±3.37 kg and final body weight of 39.5±4.41. Animals were randomly assigned to one of three treatments: a control without Se-enriched yeast supplementation (T1), control supplemented with Se-enriched yeast with total Se concentration of 0.35 mg/kg DM (T2) or control supplemented with Se-enriched yeast with total Se concentration of 0.60 mg/kg DM (T3). The yeast product used was Selyeast 3000 TM Se-enriched yeast (LFA Lesaffre, Toluca, Mexico), obtained from the growth of Saccharomyces cerevisiae on a rich culture medium and fixed intracellularly as seleno-methionine and seleno-cysteine yeast, which makes it a highly bioavailable source of organic Se. Selenium concentration in the product was 3000 ppm (mg/kg). For 60 days, sheep were given a balanced diet according to National Research Council (2007) requirements with an energy concentration of 3.1 Mcal/kg DM and 10.2% of crude protein/kg DM. The diet's main ingredients were: whole grain sorghum, ground corn, cracker crumbs, rolled corn, DDG (distillers dried grains), bran and molasses. Water and feed was offered ad libitum, whereas Se-enriched yeast was given individually. Carcasses sampling The non-destructive method of the European Commission Directive 2001/471/EC (European Commission, 2011) was used to evaluate the carcass for contamination. After evisceration and before chilling, samples (100 cm 2 per sampling site) were taken from the flank, thorax lateral, brisket, and breast to make a composite sample. The sample surface was delineated by an aluminium sterile template. Sterile swabs with large single-ended cotton wool tip 15 cm long (Protec™, DF, Mexico) were moistened in sterile saline peptone water (Laboratories CONDA, Madrid, Spain) (0.1% peptone + 0.85 % NaCl distilled water) and rubbed vertically, horizontally and diagonally for 20 seconds. Swabs were placed in sterile test tubes (Thomas Scientific, NJ, USA) with 10 mL of sterile saline peptone water. Samples were transported in a cooler (Coleman Company, Inc., Colorado, USA) at 4°C , and stored at the same temperature until analysing before 24 h. Microbiological analysis Test tubes with samples were shaken vigorously for uniform microorganisms distribution. Decimal dilutions of up to 10 -3 were prepared using test tubes with 9 mL of sterile saline peptone water (0.1 % buffered peptone water, 0.9 % sodium chloride solution) as recommended by NOM-110-SSA1-1994(Norma Oficial Mexicana, 1994b. Samples were analysed for aerobic plate counts (APC), total coliform counts (TCC) and faecal coliform counts (FCC). Aerobic plate count To evaluate the APC, the standard pour plate method as established by Official Mexican Standard NOM-092- SSA1-1994(Norma Oficial Mexicana, 1994c was used. All sample dilutions were inoculated in duplicates on to plate count agar (Sigma-Aldrich Co., MO, USA). After solidification plates were incubated at 35±2 °C for 48±1 h. Total coliform count The standard pour plate technique was used to quantify total coliform counts (TCC). Violet red bile agar (Sigma-Aldrich Co., MO, USA; VRBA) was poured on to 1 mL of each dilution and when the agar had solidified; approximately 4 mL of RVBA was added. Plates were incubated at 35±2 °C for 24±2 h, according to NOM-113-SSA1-1994 (Norma Oficial Mexicana, 1994d). Faecal coliform count Because Mexico does not have an official standard method for pour plate technique, the Association Française de Normalisation (AFNOR) NF V08-60 (1996) method was used. The VRBA was added to each plate with 1 mL of dilution and after solidification a double layer of VRBA was added and the plates incubated at 45±2 °C for 24±2 h. Physico-chemical characteristics For the 10 th rib, temperature and pH were recorded 45 minutes after slaughtering the sheep (pH45). The carcasses were then refrig-erated at 4 °C for 24 h and the pH (pH24) and temperature were recorded again using a potentiometer (Hanna Instruments, model HI 99163, Italy) according to Honikel (1998). Samples from the Longissimus dorsi muscle were taken at 24 h after slaughter to record colour, lightness (L*), redness (a*) and yellowness (b*) using a Minolta Chroma Meter CR-400 (Minolta, Osaka, Japan). Water holding capacity (WHC) was measured 24 h after slaughter by compression between two petri dishes as described by Cañeque and Sañudo (2005). Statistical analysis All bacterial count data were transformed to log10 CFU/cm 2 per sample before statistical analysis. Differences between treatments for APC, TCC, FCC, colour, initial and final pH, temperature and WHC were analysed by ANOVA at a significance level of 95% using the statistical package Statgraphics Plus 5.0. Microbiological profile The microbiological variables APC, TCC and FCC were not different among treatments (P>0.05). However, the APC in carcasses of sheep supplemented with 0.60 mg/kg of Se was numerically lower (P>0.05) by about 20 %. The total coliform loads in T3 were numerically lower by about 30.2%, while the faecal coliforms counts were numerically lower by about 30.1% (Table 1). Aerobic plate count is a very widely used test to estimate general contamination and is accepted as a criterion for carcasses surface microbial contamination. However, Enterobacteriaceae (E. coli, Salmonella spp., Serratia liquefaciens, Pantoea agglomerans, Klebsiella pneumonia, Enterobacter cloacae) counts are indicators of faecal contamination, and in combination, the two determinations are used as a criterion for the verification of slaughter hygiene (Zweifel and Stephan, 2003;Hauge et al., 2011). The European Commission Directive 2001/471/EC uses the total viable count (TVC) and Enterobacteriaceae as bacterial indicators of hygiene and faecal contamination on carcasses before chilling (Lenahan et al., 2010). In the current study, the mean values of TVC were within acceptable range according to the EC Commission Directive 2001/471/EC of < 3.5 log10 CFU/cm 2 . Treatments T1, T2 and T3 had TVC indexes of 0.10, 0.08 and 0.08 log10 CFU/cm 2 , respectively. Our values are lower than those reported by Sumner et al. (2003) [Ital J Anim Sci vol.14:2015] [page 445] from South Australia abattoirs with 2.8 log 10 CFU/cm 2 . Moreover, Zweifel and Stephan (2003) in Swiss abattoirs, and Salmela et al. (2013) in Finland abattoirs studied the microbiological contamination of sheep carcasses and reported APC mean values of 2.5 and 3.16 log 10 CFU/cm 2 , respectively for the carcasses. All these results were in accordance with EC Commission Directive 2001/471/EC. However, Bhandare et al. (2007) and Hauge et al. (2011) reported a mean APC of 4.82 to 6.06 log 10 CFU/ cm 2 with sheep and goat which are higher than those acceptable according to EC Commission Directive 2001/471/EC. Total coliform count values of 0.13, 0.10 and 0.09 log10 CFU/cm 2 were obtained for treatments, T1, T2 and T3, respectively. To our knowledge, there are no studies on sheep carcasses to compare these total coliforms counts to therefore, the results were compared to those of other animal species. These results are comparable with those of San Juan et al. (2007) and Nouichi and Hamdi (2009) who obtained value of TCC of 1.03 log 10 CFU/cm 2 and 2.92 log 10 CFU/cm 2 , respectively, in bovine carcasses in a slaughterhouse in Algeria. Our results of faecal coliform count are lower than the cutoff recommended by the EC Commission Regulation (European Commission, 2001). Other studies have shown higher FCC values (Bhandare et al., 2007;Nouichi and Hamdi, 2009) with mean values of 2.55 to 3.50 log 10 CFU/cm 2 for ovine carcasses in Algerian and Indian slaughterhouse, respectively. The activity of organoselenium compounds against microorganisms was evaluated by Pietka-Ottlik et al. (2008) who showed no activity with Gram-positive bacteria (Staphylococcus aureus and Staphylococcus simulans), whereas for Gram-negative bacteria (Escherichia coli, Pseudomonas aeuginosa, Klebsiella pneumonia) was substantially lower. Based on the above, it can be suggested that Se reduced the bacterial count. However, Se antimicrobial activity is not completely understood. ALQuthami et al. (2014) studied the antibacterial effect of Se and obtained cell disintegration because of cytoplasmic constituents leakage and cell dehydration. Yang et al. (2009) evaluated Se-enriched probiotics' antibacterial action in vitro and in vivo in mice and reported a strongly antagonize pathogenic of Escherichia coli in both in vitro and in vivo. Physical characteristics Carcasses physical characteristics of microbial growth, initial and final pH, temperature, colour values (L*, a* and b*) and WHC are presented in Table 2. There were no differences (P>0.05) among treatment in initial and final pH, temperature at 45 min after slaughter and after 24 h of chilling, L*, a* and b*. However, differences were observed among treatments for WHC. Treatment of T2 had lower (P<0.05) WHC at 0.35 µg/kg Se compared to other treatments (Table 2). There were no differences (P>0.05) in initial and final pH and temperature. These findings are in agreement with Vignola et al. (2009) who also did not find any difference between treatments with different Se sources and levels. In contrast, Li et al. (2011) found that pH was lower in pigs fed Se free diet. In general, the muscle pH of living animals is normally around 7.4, but after death, the pH falls to 5.5 -5.8 as a result of muscles glucose converting into lactic acid (Corry, 2007). Therefore, our results of pH falling from 7.15 to 5.53 is consistent. The pH value has effects on colour, shelf life, taste, microbiological stability, yield and texture of the meat. At a pH of 6.4, meat is tainted due to enzyme activity, thus producing large amounts of metabolic by-products, foul smell, sliminess and discolouration (Feiner, 2006). Bacterial proteolytic enzymes operate best near neutral pH, and the enzymes which attack carbohydrates tend to have an optimal pH below 6. Organisms such as lactic acid bacteria whose predominant activity is carbohydrate breakdown, have an optimal pH between pH 5.5 and 6.0 (Lawrie and Ledward, 2006). The final pH in the present study was in the range of 5.3 to 5.8 which is near to the optimal microbial growth pH. Cherry red colour (a*) is one of the most important qualities of meat for consumer purchase decision. It is an indicator of freshness Selenium and sheep meat contamination and quality (Brewer et al., 2001;Mancini and Hunt, 2005). In the current study, there were no differences between treatments for colour values of a*, b* and L*. However, Vignola et al. (2009) in lambs, found higher values for L*, a* and b* where values were 44.63, 15.44 and 6.76, respectively. In pigs, Li et al. (2011) reported that Se did not had effect on meat colour values of a*, b* and L*. Preventing ferrous myoglobin from oxidation is a critical factor for maintaining meat colour stability. A high level of GSH in meat tissues is associated with a high reducing capacity, reducing the formation of H2O2, and soon afterward oxidation of ferrous iron at the same time causing maintain meat colour stability (Zhan et al., 2007;Liu et al., 2011). They reported that selenomethionine-treatments increased redness of meat. In the current study, the treatment 0.35 mg T2 presented a lower percentage of juice released, therefore higher WHC. Zhan et al. (2007) evaluated the effect of different Se source added at 0.30 mg Se/kg to basal diet on loin meat quality in finishing pigs and reported values of 14.3, 14.0 and 12.5% for the control, sodium selenite treatment and selenomethionine-treated groups, respectively after 16 h exposure in a 25 °C room with significantly lower drip loss with the selenomethioninetreated group. Wang et al. (2009) and Li et al. (2011) mentioned that drip loss of meat decreased with the increase of dietary Se level in poultry and pigs. Generally, Se as part of GHS-Px elevates and maintains this enzyme activity, protect cell membranes from oxidation and improving meat WHC (Mateo et al., 2007;Wang et al., 2009). Conclusions Although the differences were not significant, sheep supplemented with 0.60 µg/kg Se in the diet had a 20% lower aerobic plate counts in the carcasses, 30% lower total coliform count and a 30% lower faecal coliforms count than un-supplemented sheep. Drip loss was lower for sheep fed the 0.35 mg/kg DM dose. From these results we can conclude that organic Se-enriched yeast did not affect carcasses bacterial proliferation or meat physical characteristics. More studies with larger numbers of animal are recommended to study the effect of organic Se supplementation on carcasses microbiological contamination and meat physical characteristics.

v3-fos

2018-04-03T02:37:46.849Z

2015-01-01T00:00:00.000Z

23953648

s2

The effect of pineapple core fiber on dough rheology and the quality of mantou The consumption of dietary fiber offers the health benefit of lowering the risk of many chronic diseases. Pineapple core fiber (PCF) in this study was extracted and incorporated into dough and mantou (i.e., steamed bread). The effects of PCF substitution and fiber size on textural and rheological properties of dough and mantou were evaluated by a texture analyzer. The substitution of wheat flour by PCF resulted in a stiffer and less extensible dough with or without fermentation. The hardness and gumminess of mantou significantly increased as the PCF substitution increased from 0% to 15%, but the cohesiveness, specific volume, and elasticity significantly decreased with the fiber substitution. Ten percent PCF-enriched dough and mantou with various fiber sizes had similar rheological and textural properties, except for the k1 and k2 values. By sensory evaluation, 5% PCF-enriched mantou and the control bread had better acceptability in texture, color, odor, and overall acceptability, compared to mantous enriched with 10% or 15% PCF. Significant correlations existed between the rheological properties of dough and textural parameters of mantou and between the sensory quality and textural parameters of mantou. Therefore, we suggest that fiber-enriched mantou can be prepared with 5% PCF substitution to increase the intake of dietary fiber and maintain the quality of mantou. Introduction Mantou (i.e., steamed bread) is an important staple food in Asia. Southern-style Chinese mantou has a soft, elastic, and uniformly fine-textured crumb. These characteristics are significantly affected by ingredients and processing variables [1e3]. Rubenthaler et al [1] reported that wheat flour with 10e11% protein and medium gluten strength is best suited for steamed bread. The low-steam generation rate significantly reduces the quality of bran-enriched steamed bread [3]. Dietary fiber (DF) is a group of food components that are resistant to hydrolysis by human digestive enzymes. Dietary fiber intake offers health benefits such as a lower risk for coronary heart disease, type 2 diabetes, obesity, and constipation [4,5]. The additions of some soluble DFs strengthens the structure of dough and improves the quality of bread [6], but excess amounts of insoluble DFs have an adverse effect on the formation of the gluten network [7,8] and reduces the quality of bread [9e12] because of a gluten dilution effect or because of glutenefiber interaction. The addition of apple pomace [13] and insoluble wheat fiber [14] results in a stiffer dough, probably through a filler-like effect in the dough matrix. Ahmed et al [8] report that dough incorporated with insoluble date fiber predominately exhibits solid-like behavior. Potato fiber, which contains a high level of insoluble DF, increases the hardness and gumminess of bread [11]. The addition of 11% apple pomace decreased the quality of bread by sensory evaluation [9]. Wu et al [3] reported that steamed bread enriched with 10% and 20% wheat bran had a similar sensory quality as the control bread, but 30% branenriched steamed bread had the lowest sensory quality, which was significant. Increasing the DF intake may be achieved by consuming fiber-enriched mantou. Few studies exist on the properties of dough and mantou enriched with fiber. Pineapple is one of the most important fruits in the world. In Taiwan, the annual yield of pineapple is more than 400,000 metric tons. In addition to being eaten fresh, pineapples are usually processed into canned fruit, juice, and jam. Pineapple core, the high-fiber part of the pineapple fruit, is a potential DF source [15,16]. The fruit and its pomace contain abundant phytochemicals such as fiber, polyphenols, and flavonoids; it furthermore has good antioxidant activity [17,18]. Prakongpan et al [19] report that purified pineapple core powder contains 99.8% total DF content. Hence, pineapple core is a good DF source for food enrichment. The aim of this study was to investigate the rheological and textural properties of dough and mantou enriched with different amounts and particle sizes of pineapple core fiber (PCF). Another purpose was to observe the correlations between dough and mantou properties. Materials Wheat flour with medium gluten strength was a gift from The core of fresh pineapple (Ananas comosus L. Merr.) was obtained as a byproduct from a local fruit processing factory. Pineapple core fiber was prepared in accordance with the method proposed by Chien and Kang [16], with some modifications. In brief, pineapple cores were washed, cut (<1 cm thick), blanched (100 C for 15 minutes), drained, air-dried (50 C for 24 hours), and crushed. The crushed samples were then extracted by 80% ethanol for 12 hours. The residues after centrifugation were air-dried (50 C for 24 hours) and milled to a particle size of less than 0.42 mm. Three fractions of PCF with different particle sizes were collected by sieving with 40 mesh, 60 mesh, and 100 mesh. Total, insoluble, and soluble DFs of the PCF were analyzed by AOAC methods [21]. Functional properties (e.g., swelling power and water-holding capacity) were measured by the method of Huang et al [22], with some modifications. The PCF (1 g) was hydrated with 20 mL of distilled water in a calibrated cylinder at room temperature. After equilibration (24 hours), the bed volume was recorded and the swelling power was expressed as milliliters of swollen sample per gram of dry sample. Furthermore, PCF (1 g) was soaked in 10 mL of distilled water for 24 hours and then centrifuged at 1000g for 20 minutes. The supernatant was decanted into a graduated cylinder and the volume of excess water was read. Hence, Water-holding capacity was expressed as milliliters of water held by 1 g of PCF. Extension test of unfermented or proofed dough A texture analyzer (TA-XT2i; Stable Micro Systems, Surrey, UK) was equipped with a probe of Kieffer dough and gluten extensibility rig, and operated in tension mode. The pretest and test speeds were both set at 2.0 mm/s to avoid vibrations that may occur at high speed. The resistance to the extension (mN) and extensibility (mm) of dough without yeast and proofed dough were determined by recording the maximum force and the distance at rupture. The measurements were conducted in six repetitions. Stress relaxation of mantou The stress relaxation of mantou was measured according to the method proposed by Wu et al [3]. In brief, the center crumb sample (3 Â 3 Â 4 cm 3 ) was removed by cutting. Stress relaxation test of the sample was executed by using a textural analyzer equipped with a P20 cylindrical probe (20-mm diameter). The sample was deformed by penetration to a constant strain of 20% with a test speed of 0.5 mm/s. The data acquisition rate was 10 points per second. The residual force was continuously recorded as a function of time for 480 seconds. The measurements were conducted in triplicate. The stress relaxation data were analyzed by using the PelegeNormand model (Equation 1), proposed by Peleg and Normand [23]. j o u r n a l o f f o o d a n d d r u g a n a l y s i s 2 3 ( 2 0 1 5 ) 4 9 3 e5 0 0 in which F 0 is the initial force, F(t) is the momentary force at time t, and k 1 and k 2 are constants. The k 1 and k 2 values are the intercept and slope of regressive straight line plotted by normalized force and time, respectively. Furthermore, the percent stress relaxation (%SR) was calculated by the following equation [24]: in which F 0 is the initial force and F t ¼ 20 is the force at 20 seconds after the initial strain was achieved. Texture of mantou Texture profile analysis of the center crumb sample was measured by a textural analyzer equipped with the P20 adapter moving at a rate of 2 mm/s; the penetration depth into the crumb sample was 20 mm. Hardness, cohesiveness, gumminess, and springiness were calculated using the texture profile analysis curve. The measurements were conducted in triplicate. Specific volume of mantou The cooled mantou was weighed and its volume was determined by seed displacement in a loaf volume meter. The specific volume was expressed as milliliters per gram (mL/g). Color of mantou The color of the mantou crust and crumb was measured using a HunterLab ColorFlex colorimeter (Hunter Associates Laboratory Inc., Reston, Virginia, VA), which was controlled by a computer that calculated color ordinates from the reflectance spectrum and was calibrated with a white standard tile. The mantou samples were placed in Petri dishes, and the color was recorded using the CIELab uniform color space at room temperature. Color determined by Commission Internationale l'Eclairage (CIE) classifies color in three dimensions: L, brightness; a, red to green color; and b, yellow to blue color. The measurements of every treatment were performed in six replicates. Statistical analysis Using SPSS software 13.0 (SPSS Chicago, IL), the data in triplicate, unless stated otherwise, were analyzed for different treatments by one-way ANOVA and Duncan's new multiple range tests to determine the statistical significance of differences among the values. Pearson's simple correlation analysis was also conducted for observing correlations between dough and mantou properties. Results and discussion The particle size of PCF used in this study was 104e149 mm (small), 149e250 mm (medium), and 250e420 mm (large). The total, insoluble, and soluble DFs of the PCF were 53.59%, 51.14%, and 2.45% (dry weight basis), respectively. This indicated that insoluble DF was the major fiber in PCF. Different fiber sizes did not affect the DF content of PCF. The swelling power of wheat flour and the three PCF fractions with small, medium, and large particle size was 2.23 ± 0.01 mL/g, 8.98 ± 0.03 mL/g, 11.44 ± 0.01 mL/g, and 11.96 ± 0.05 mL/g, respectively. The water-holding capacity was 1.32 ± 0.01 mL/g, 4.59 ± 0.01 mL/g, 7.58 ± 0.12 mL/g, and 7.65 ± 0.21 mL/g, respectively. The results indicated that the PCF had a higher swelling power and water-holding capacity than wheat flour, and that PCF with a small fiber size had a lower swelling power and water-holding capacity than PCF with a large fiber size. Our results were in agreement with the results in the study of Chien and Kang [16] that showed that small-particle pineapple pomace had a low swelling power and water-holding capacity. Table 1 lists the resistance to extension (R value) and extensibility (E value) of proofed and unfermented doughs enriched with various substitution levels and particle sizes of PCF. The R value was higher in the unfermented dough enriched with 15% PCF than in the dough enriched with 0e5% PCF. The E value of the dough without yeast decreased as the PCF substitution increased (0e15%). Hence, the addition of excessive PCF resulted in a stiffer and less extensible dough. The effect of PCF on the rheological properties of dough without yeast was consistent with previous research using different DFs, such as 30% apple pomace [13], 3% date flesh fiber concentrate [25], and 15e35% apple pomace [26]. In addition, insoluble wheat fiber resulted in a stiffer dough, probably through a filler-like effect in the dough matrix [14]. The negative effect on the formation of the gluten network by excess amounts of insoluble DF may be because of the dilution of gluten protein [7,8]. Substitution by PCF increases the R value and decreases the E value in proofed dough ( Table 1). Because of the fermentation of yeast, the proofed dough generally had lower R and E values than dough without yeast. However, the particle size of the PCF did not significantly affect the R and E values of unfermented and proofed doughs ( Table 1). The farinograph properties were not significantly affected by the particle size of barley fiber-rich fractions [27] and wheat bran [12]. However, Noort et al [12] also reported that dough with j o u r n a l o f f o o d a n d d r u g a n a l y s i s 2 3 ( 2 0 1 5 ) 4 9 3 e5 0 0 fine wheat bran had lower ability for the gluten protein to reaggregate (i.e., gluten yield), compared to dough with coarse bran. Because no work in the literature has described the rheological properties of proofed dough with added fiber, it is impossible to compare our data with the results of other research studies. Texture of mantou Texture is an important mantou quality. Gluten protein is a main contributor to the strength of Chinese steamed bread [2]. Fig. 1 shows the effect of PCF on the texture and specific volume of mantou. The hardness and gumminess of mantou significantly increased with increasing the PCF substitution, but the cohesiveness, springiness and specific volume significantly decreased with the substitution. Similar to the increase in the R value of the dough (Table 1), the PCF-enriched steamed bread had higher hardness and gumminess that resulted from the competition of water absorption between PCF and wheat flour components or from the rigid nature of the fiber. Because insoluble DF is the major type of DF in PCF, incorporating PCF into the dough system may interfere with the formation of the gluten network. This lowers cohesiveness, springiness, and specific volume of the PCF-enriched steamed bread. Wu et al [3] report that hardness and springiness of 20e30% bran fiber-enriched steamed breads had higher hardness than breads with 0e10% wheat bran. Sangnark and Noomhorm [28] found that bread containing 5% fiber from rice straw had higher firmness and lower springiness, compared to the control bread. Kaack et al [11] found that the hardness of bread increased with the quantity (0e12%) of potato fiber containing a high content of insoluble DF. However, date flesh fiber concentrate added at 0e3% insignificantly decreased the bread volume. [25]. The particle size tested in this study did not affect hardness, cohesiveness, gumminess, and springiness values (data not shown); however, the specific volume (2.95 mL/g) of 10% PCF-enriched mantou with the smallest fiber size (104e149 mm) was significantly higher (p < 0.05) than the specific volume (2.67 mL/g and 2.75 mL/g) with the medium fiber size (149e250 mm) and large fiber size (250e420 mm). Our results were similar to the studies of bread reported by Curti et al [29], Izydorczyk et al, [27], Lai et al [10], and Chien and Kang [16]. Different particle size of wheat bran fractions [29] and barley fiber-rich fractions [27] did not significantly affect the hardness of bread. Coarse pineapple pomace [16] and bran [10] had adverse effects on bread volume. However, the impact of the fiber size on baked bread quality is contentious at present. de Kock et al [30] found that coarse bran particles produced better baking results than finely ground bran. Noort et al [12] report that bread volume is higher when the particle size of wheat bran is increased. Hence more study using various fiber sources and sizes is needed to explain the The values are expressed as the mean ± standard deviation (n ¼ 3). The mean values with different superscripted letters in the same column and section are significantly different (p < 0.05). E ¼ extensibility; PCF ¼ pineapple core fiber; R ¼ resistance to extension. j o u r n a l o f f o o d a n d d r u g a n a l y s i s 2 3 ( 2 0 1 5 ) 4 9 3 e5 0 0 mechanism related to the effect of fiber size on dough and mantou properties. Stress relaxation of mantou Fundamental viscoelastic properties of foods have frequently been measured by stress relaxation. Table 2 lists the fitting parameters of the PelegeNormand model for mantou with various amounts and particle sizes of PCF. Results showed that the stress relaxation data of mantou in this study were well fitted (R 2 > 0.99) to the PelegeNormand model (Equation 1). Thus we could obtain the F 0 , k 1, and k 2 parameters by regression. A high k 1 value indicates a pronounced elastic behavior. The k 2 value is representative of the degree of solidity and it varies from 1 (for a material that is truly a liquid) to infinity (for an ideal elastic solid in which the stress does not relax) [23]. The F 0 (i.e., initial force) parameter of mantou increases with the increased PCF substitution. However, the k 1 and k 2 parameters of mantou significantly reduces with increased PCF substitution (Table 2). Hence, 10e15% PCF-enriched mantous were more rigid and less elastic than mantous with 0e5% PCF. The increasing and decreasing patterns of mantou in F 0 , k 1 , and k 2 in the PelegeNormand model (Table 2) were consistent with hardness and springiness, respectively (Fig. 1). The result of stress relaxation in this study was similar to the study of Wu et al [3], which showed that increasing the substitution of wheat flour by wheat bran resulted in less elasticity of steamed bread. Li et al [31] indicate that the relaxation properties of dough depend on gluten protein. Zhang et al [32] found a moderate correlation coefficient (r ¼ 0.55e0.61) between stress relaxation of steamed bread and gluten in a fraction of wheat cultivar. Furthermore, 10% PCF-enriched mantou with a small fiber size (104e149 mm) had significantly higher k 1 and k 2 values and a lower F 0 value than PCF-enriched mantou with medium and large fiber size (149e420 mm) ( Table 2). At present, no work was found in the literature in appropriating the PelegeNormand model to describe the viscoelastic properties of fiber-enriched mantou or bread with different fiber size. The %SR is a convenient and informative parameter to understand the viscoelastic properties of food products, and is obtained directly from the plot of stress relaxation versus time plot at an arbitrary time [24]. The %SR is 0 for an ideal elastic solid, and the %SR is 100 for an ideal liquid. The results showed that %SR of mantou tested ranged 22.05e29.62 (Table 2). Thus, mantou was classified as a viscoelastic solid. Furthermore, the %SR of mantou obviously increased with increasing amount of PCF. Wu et al [3] report that the addition of wheat bran increased the %SR value of steamed bread. Dough from strong wheat cultivars exhibited slower rates of stress relaxation and higher storage modulus, compared to moderate, weak, and very weak cultivars [33]. However, the particle size of PCF did not significantly affect the %SR of mantou (Table 2). Color of mantou Color is an important quality indicator for food products. Table 3 shows the effect of PCF on the color of mantou. The White index and Hunter L value, which indicate the luminosity of the sample, were significantly lower in the crust and crumb of mantou with PCF than in the control. Both a (redness) and b (yellowness) values of the crust and crumb of mantou increased with the substitution by PCF. However, the particle size of PCF tested did not significantly affect the L, a, and b values of mantou crust and crumb (p > 0.05). Therefore, the color of mantou was primarily affected by the amount of PCF. Chien and Kang [16] found that bread crumb with pineapple pomace had lower L and higher b values and that the color of the crumb was not influenced by the particle size of pineapple pomace. Borchani et al [25] reported that bread with 3% date flesh fiber concentrate was darker, redder, and less yellow in comparison to the control bread. Sensory evaluation of mantou Pineapple core fiber substitution or particle size significantly affected the quality of mantou, as measured instrumentally (Tables 1e3 and Fig. 1). In general, objective instrumental measurements have higher sensitivity, compared to subjective sensory evaluation. Table 4 shows the result of the consumers' sensory assessment of mantou with PCF. The sensory scores of the 5% PCF-enriched mantou and the control mantou were not significantly different (p > 0.05). The mantou enriched with 10% PCF and 15% PCF substitutions had significantly (p < 0.05) lower sensory scores in color, odor, texture and overall acceptability, compared to the 5% PCF-enriched Values are expressed as the mean ± standard deviation (n ¼ 3). The mean values with different superscripted letters in the same column and section are significantly different (p < 0.05). F 0 ¼ initial force; k 1 , and k 2 ¼ constant parameters in the PelegeNormand model; PCF ¼ pineapple core fiber; R 2 ¼ coefficient of determination; %SR ¼ percent stress relaxation. j o u r n a l o f f o o d a n d d r u g a n a l y s i s 2 3 ( 2 0 1 5 ) 4 9 3 e5 0 0 and control mantous. The mantou enriched with the highest substitution (i.e., 15% PCF) had the lowest sensory scores in color, odor, texture and overall acceptability. However, the tested fiber sizes did not significantly affect the consumers' sensory assessment of mantou. Therefore, we suggest fiberenriched mantou can be prepared with 5% PCF to increase the intake of DF and maintain sensory acceptability by consumers. Steamed bread with 0e20% wheat bran had higher sensory scores than bread with 30% bran [3]. Compared to the control bread, all breads containing various amounts (3e6%) and particle sizes of pineapple pomace had similar sensory scores in texture, flavor, and overall acceptability [16]. Borchani et al [25] report that the addition of 0e3% date flesh fiber concentrate did not significantly affect the overall acceptability of the bread. Table 5 lists the correlation coefficients between dough and mantou properties. The R p and R u values (resistance to extension) of the proofed and unfermented doughs, respectively, were positively correlated with hardness and the %SR, and was negatively correlated with a specific volume, sensory texture, and overall scores. The extensibility (E p and E u ) of the proofed and unfermented doughs were positively correlated with the specific volume, and negatively correlated with hardness and %SR. The results showed that the extensibility of dough is crucial to the specific volume and elasticity of fiber-enriched mantou. The hardness of mantou was positively correlated with R u , R p , %SR, and F 0 ; and negatively correlated with E u , E p , specific volume, k 1 , k 2 , and sensory texture, and overall scores. The springiness of mantou was positively correlated to E u , and specific volume. The fitting parameters (k 1 and k 2 ) were positively correlated with the specific volume, and negatively correlated with R u , hardness, F 0, and %SR. The sensory texture and overall scores were negatively correlated with R u , R p , hardness, and %SR. The results indicate a good relationship between rheological properties of dough and textural parameters of mantou and between sensory quality and textural parameters of mantou. Significant correlations existed between the textural j o u r n a l o f f o o d a n d d r u g a n a l y s i s 2 3 ( 2 0 1 5 ) 4 9 3 e5 0 0 characteristics and stress relaxation parameters of branenriched steamed bread [3]. Conclusion Pineapple core fiber substitution made unfermented and proofed doughs stiffer and less extensible. The hardness, gumminess, F 0 , and %SR of mantou significantly increased with increased PCF substitution (0e15%); however, the cohesiveness, springiness, specific volume, k 1 , and k 2 significantly decreased with the substitution. Pineapple core fiber with a small fiber size had lower swelling power and water-holding capacity, compared to the large fiber size; however, 10% PCFenriched dough and mantou with various fiber sizes had similar rheological and textural properties, except for the k 1 and k 2 values. Based on colorimetry, the color of mantou was darker, redder, and more yellow with the enrichment by PCF. The sensory quality of mantou with 5% PCF was similar to that of the control bread. Therefore, PCF could be supplied as a potential source of DF, and a good-quality fiber-enriched mantou could be produced by the substitution of 5% PCF in wheat flour. j o u r n a l o f f o o d a n d d r u g a n a l y s i s 2 3 ( 2 0 1 5 ) 4 9 3 e5 0 0

v3-fos

2016-03-01T03:19:46.873Z

2015-04-01T00:00:00.000Z

3026268

s2

Effect of the Characters of Chitosans Used and Regeneration Conditions on the Yield and Physicochemical Characteristics of Regenerated Products The objective of this study was to explore the effect of the character of chitosans used, and the regeneration conditions employed on, the yield and physicochemical characteristics of regenerated products. Different concentrations of acetic acid were used to dissolve chitosans of 61.7% and 94.9% degree of deacetylation (DD), and weight-average molecular weight (Mw) of 176 and 97 kDa, respectively; they were then precipitated with an 8 N NaOH solution, followed by washing and neutral and freeze drying to get the regenerated products. Yields of regenerated products and their physicochemical properties, such as ash content, bulk density, Mw, polydispersity index (PDI), DD, and crystallinity were measured. A higher concentration of acetic acid used resulted in a higher yield. The purity of the regenerated product increased significantly, whereas the bulk density and crystallinity decreased significantly after regeneration. The regeneration process showed its merits of narrowing down the PDI of regenerated products. The DD and structure of chitosan was changed insignificantly after the regeneration process. chitosan, with 87% and 96% DD, than that of the 75% DD counterpart, but water absorption capacity was just the opposite. Dutkiewicz et al. [26] reported that regenerated krill chitosan with weight-average molecular weights (Mw) of 800 kDa shows whole blood clotting time two times longer than that of silica gel. The above-mentioned literature indicated that those enhanced functional properties may be attributed to difference in solubility, Mr, and DD, which in turn resulted in different content of positively charged amino groups in an acid solution between original and regenerated chitosan. Protonated amino groups may result in better immune adjuvant activity and fat and anionic dye absorption. However, up to now, the regenerated conditions used and the effect of characters of chitosan, such as the Mr, DD on the yields, and physicochemical of regenerated products have not been systematically explored. The objective of this study was to explore the effects of the characters of chitosan used and regeneration conditions on the yield and physicochemical characteristics of regenerated products. The different DDs and Mw of chitosans were dissolved in 0.1 and 1.0 M acetic acid, respectively, then chitosan was precipitated with 8 N NaOH, followed by washing, neutral and freeze drying to get the regenerated products. The physicochemical properties, such as Mw, PDI, DD and crystallinity index of regenerated products, were measured. The Yield Results in Table 1 show that the yield of R-2 was higher than R-1 and the yield of R-4 was higher than R-3. This may be attributed to using a higher concentration of acetic acid (1.0 M) will dissolve more chitosan than using a lower concentration (0.1 M) [29], and the insoluble materials were filtered and discarded prior to the solution proceeding to the regeneration procedure; therefore, the yield of R-2 was higher than R-1, thus R-4 was higher than R-3. The yield of R-4 was higher than R-2, whereas the yield of R-3 was higher than R-1. It indicated that the higher the DDs of chitosan used, the higher the yield of regenerated chitosan was obtained. The result was similar to that of Chen and Liu [9]. This may be due to the fact that the solubility of lower-DD or higher-Mr chitosan was lower than that of the higher-DD or lower-Mr one, respectively [14,23]. The reasons for using higher DD chitosan that ends up with a higher yield than when using lower DD chitosan might be because the insoluble materials were filtered and discarded prior to the solution that proceeded to the regeneration procedure. The Ash Content and Bulk Density Changes of ash content and bulk density of regenerated chitosans are listed in Table 1. Ash content decreased significantly after regeneration. It decreased from 1.94% of O-1 to 0.35% and 0.33% for R-1 and R-2, respectively, and from 1.75% of O-2 to 0.29% and 0.27% for R-3 and R-4, respectively. Results indicated that the different acid concentration treatments and/or different DDs did not differ significantly in ash. Trung [21] reported there was an insignificant difference in ash content removal among different acid treatments. It is conceivable that some minerals were not removed during the extraction of the original chitosan from raw material because they were enclosed in the interior of the solid material and protected against hydrochloric acid used for deminerization. In the regeneration procedure, the chitosan is completely dissolved, the minerals will be freed and will react with acid solvent. Thus, ash measurement is an indicator of the effectiveness of the regeneration step for removal of impurities. The change in bulk density content decreased significantly after regeneration. It decreased from 0.55 g/cm 3 of O-1 to 0.49 and 0.48 g/cm 3 for R-1 and R-2 respectively, and from 0.55 g/cm 3 of O-2 to 0.49 and 0.49 g/cm 3 for R-3 and R-4 respectively. Results indicated that the different acid concentration treatments and different DDs differed insignificantly in bulk density. The results were similar to Trung [21]. The reduction in ash contained might result in lower bulk density and elevate the purity of the resulted products; therefore, the purities of regeneration chitosans were better than the original ones. This may be one of the reasons why regenerated chitosan shows better performance in medical, cosmetics and biochemical studies than the original chitosan [16,17,19,22,23,[26][27][28]. Table 1. Mw decreased after regeneration of both high and low DD chitosans. It decreased from 176 kDa of O-1 to 155 and 150 kDa for R-1 and R-2, respectively, and from 97 kDa of O-2 to 89 and 88 kDa for R-3 and R-4, respectively. After regeneration, Mw of regenerated chitosan decreased, possibly due to material loss during the filtering insoluble materials, precipitation process and sieving, as well as acid hydrolysis. Decreases in Mw after being regenerated was more significant of Low DD chitosan (R-1: 11.9%, R-2: 14.8%) than high DD ones (R-3: 8.2%, R-4: 9.3%). The reasons were similar to the previous one just mentioned, i.e., due to different solubilities of O-1 and O-2 chitosans in acetic acid solution, precipitation, and collection process. However, from the size exclusion high performance liquid chromatography (SE-HPLC) elution curve, the decrease in Mw of high DD chitosan might be more significant than the low DD one, i.e., decrease in higher Mw portion was faster in the beginning of elution curve ( Figure 1). This is illustrated from the difference of elution curve at starting increase points (arrow) between R-3 and R-4 (both at 25.0 min) to O-2 (at 24.4 min) were more significant than that of the peak in R-1 and R-2 to O-1 (all at 21.8 min). It has been assumed that a high DD chitosan molecule has an extended contour due to more electrostatic force between -NH3 + groups, and the glycosidic linkage is easier to access by H + hydrolysis reaction [30]. Table 1. Figure 1 show the molecular weights distribution of low and high DD chitosans (O-1 and O-2, respectively) and their regenerated chitosans (R-1, R-2, R-3, and R-4). The PDIs of both original chitosans were similar to each other, and their regenerated ones were also similar to each other. The distribution of 61.7% DD chitosan and its regenerated products showed that the high-Mr side (the left-hand side of peak) is steeper, whereas the low-Mr side (the right-hand side of peak) is tailing. After regeneration, the elution curves of R-1 and R-2 were shift to right-hand side of O-1 ( Figure 1A). This indicated that the high-Mr fractions (21.8-26.0 min) of R-1 and R-2 decreased, so the retention time were longer than that of O-1, consequently, low-Mr fractions (26.0-35.5 min) increased. The distribution of 94.9% DD chitosan and its regenerated products showed that the high-Mr side is steeper (24.0-28.0 min), whereas the low-Mr side (28.0-35.5 min) is tailing ( Figure 1B), similar to Figure 1A. Results in However, the proportion of medium-Mr fractions (27.0-29.0 min) increased significantly after regeneration ( Figure 1B). Furthermore, the PDI decreased both after regeneration for high or low DD chitosans used. It decreased from 2.07 of O-1 to 1.72 and 1.73 for R-1 and R-2, respectively, and from 2.09 of O-2 to 1.73 and 1.78 for R-3 and R-4, respectively. Results indicated that the PDI of regenerated products narrows down after the regeneration process. The merits of the regeneration process can narrow down the PDI have not been reported. The result is unprecedented. The reasons that PDI of chitosan narrows down after regeneration may be due to different solubilities of 61.7% and 94.9% DD chitosans in 0.1 or 1.0 M acetic acid, and different susceptibilities to acid hydrolysis of low and high DD chitosans [31]; it also may be due to the precipitation process and sieving, washing, and collection to get the regenerated products. Therefore, the elution patterns of regenerated products changes; the ratio of medium-Mr fractions increased and PDI decreased. The regeneration procedure of precipitation process and sieving, washing, and collection and it affects the Mw and PDI has not been studied. However, those steps should affect the Mw and PDI of the regenerated product. Therefore, they should be explored in the future studies. FTIR Spectrum In the literature, the OH stretching band at 3450 cm −1 [32]; the CH stretching bands within 2870-2880 cm −1 [33]; the amide I band at 1650-1655 cm −1 ; the amide II band at 1550-1555 cm −1 ; the amide III band at 1315-1320 cm −1 [34] have been reported. Results in Figure 2 show that the FTIR spectra of the above-mentioned functional absorption bands have not changed significantly after regeneration. The functional groups on the backbone of different regenerated chitosans either from lower DD chitosans (O-1 to R-1 and R-2) or higher DD chitosans (O-2 to R-3 and R-4) were the same as the original chitosans. Results imply that the regenerated process used in this report did not result in changes of functional groups and DD of polymer chains, suggesting that the acetamido groups were stable during the regenerated treatment. Therefore, the regeneration method is a very good practice to produce final products with higher purity at the same time the functional groups and DD of the chitosan can be preserved. Table 1. The Crystallinity The degree of crystallinity for chitosans (O-1 and O-2) and regenerated chitosans (R-1, R-2, R3, and R4) were evaluated through X-ray powder diffraction method. Webster et al. [35] reported that the diffraction pattern of chitin which exhibits four diffraction peaks at 9°, 12°, 19°, and 26°. The peaks at 10° and 20° are the two prominent peaks in the diffraction pattern, which confirms the partial crystallinity of the polymer. Ogawa [36] reported four crystalline polymorphs, they are tendon, annealed, L-2 and I-2. The tendon and L-2 crystals are hydrated, i.e., water molecules are incorporated. However, the annealed polymorph is anhydrous. The hydrated polymorph (tendon or L-2) showed a strong equatorial reflection spot at 2θ of around 10°, whereas, the anhydrous crystal (annealed) exhibited a strong spot at around 15°, L-2 showed a strong reflection at 2θ at 10.6° and no diffraction spot at around 15°. But I-2 showed diffraction spot at both 2θ of 10.7° and 15.4°. Chitosan after deacetylated to 61.7% (O-1), only three broad peaks appeared at 10°, 20°, and 22°, this may be due to the deacetylaction reaction occurred under high temperature and the strong alkaline condition, displacing the acetamido group to amine group [35]. The resulted showed a similar diffraction pattern that of the hydrated L-2 polymorph of chitosan. After deacetylated to 94.9% (O-2) and regenerated to R-1, R-2, R-3, and R-4, resulted showed that they are hydrated L-2 polymorph, however, only two broad peaks appeared at 10° and 20°, as shown in Figure 3. The results were similar to those of Ogawa [36]. However, Trung [21] reported the diffraction of most regeneration chitosan showed only one major peak at approximately 2θ = 20°. The crystallinity intensity decreased significantly after regeneration. Results in Figure 3 show that I020 (at 2θ = 10°) decreased from 1648 of O-1 to 1155 and 1191 for R-1 and R-2, respectively, and from 1315 of O-2 to 1111 and 1278 for R-3 and R-4, respectively, whereas I110 (at 2θ = 20°) of X-ray intensity decreased from 2528 of O-1 to 1667 and 1728 for R-1 and R-2, respectively, and from 2291 of O-2 to 1659 and 1589 for R-3 and R-4, respectively of X-ray intensity. Results indicated that the structure of regenerated chitosans (R-1, R-2, R-3, and R-4) and their counterparts (O-1 and O-2) differed significantly in crystalline intensity. This is may be due to the fact that original chitosan at 2θ = 10° or 20° decreased with the increase of DD [37] or structure change. However, the DD between original and regenerated chitosans did not change significantly ( Figure 2 and Table 1). Thus, the decreases of I020 and I110 of regenerated chitosans should be structure change. On the other hand, the X-ray spectra among regenerated chitosans were similar, which suggest that the same regeneration process including precipitation, washing, neutral and freeze drying might cause the similar structure of regenerated chitosans. Results in Table 2 shows that CrI110 decreased from 80.99% of O-1 to 66.39% and 62.33% for R-1 and R-2, respectively, and decreased from 72.53% of O-2 to 65.71% and 59.11% for R3 and R4, respectively. Results indicated that the regenerated chitosans had a lower crystallinity index than the original chitosans. Table 1. At I110, the crystallinity intensity of R-1 and R-2 those regenerated from O-1 also R-3 and R-4 that regenerated from O-2 were close. However, the crystallinity intensity of O-1 and O-2 are different from each other; furthermore, they are different from the regenerated product derived from them. The difference in I110 between O-1 and O-2 should be due to the deacetylation process. The similarity in I110 among R-1, R-2, R-3, and R-4 may be due to similar processes of dissolving by acetic acid and alkali precipitation, then washing, neutral, freeze drying, which therefore, end up with a similar molecular arrangement and, thus, similar crystallinity intensity. Preparation of Chitosan Chitin powder was passed though sieves of 40-60 mesh, and then was alkali-treated (50% NaOH) at 140 °C for 1 or 3 h to get different DD chitosan. Chitosan was washed until neutral and dried at 50 °C to get the final product [5]. Preparation of Regenerated Chitosan The modified procedure of Chen and Liu [9] was used to prepare the regenerated chitosans. Chitosans with different DD were dissolved in 0.1 or 1.0 M acetic acid to make 1% solutions, stirred for 10 h then filtered through filter paper (Toyo No. 1, 90 mm, Toyo Roshi Kaisha, Tokyo, Japan) to remove insoluble materials. One liter of 1% chitosan-acetic acid solution was added to two liters of 8 N NaOH solution to precipitate the chitosan. The precipitates were collected with a 325-mesh sieve and washed with distilled water until neutral. The precipitates were freeze dried to obtain the product that was ground and sieved through a 40-60 mesh size to get regenerated chitosans. Determination of DD Infrared spectrometry was used to determine the DD of the chitosan or regenerated chitosan [38]. Chitosan or regenerated chitosan powder was sieved through a 200 mesh and then mixed with KBr (1:100), dried at 60 °C for 3 days to prevent interference of the effect of water molecules on the peak of hydroxyl band in FTIR measurements, and pressed into a pellet. The absorbance of amide I (1655 cm −1 ) and the hydroxyl band (3450 cm −1 ) were measured using a Bio-Rad FTS-155 infrared spectrophotometer (Hercules, CA, USA). The band of the hydroxyl group at 3450 cm −1 was used as an internal standard to correct for disc thickness and for differences in chitosan concentration when making the KBr disc. Triplicate measurements were averaged and used to calculate the DD using the following equation: DD = 100 − (A1655/A3450) × 115 (1) here, A1655 and A3450 were the absorbance at 1655 and 3450 cm −1 , respectively. Determination of Molecular Weight and PDI The weight-average molecular weight (Mw), number-average molecular weight (Mn) and polydispersity index (PDI = Mw/Mn) of samples were measured by size exclusion high performance liquid chromatography (SE-HPLC) [14]. A column (7.8 mm × 30 cm) packed with TSK gel G4000 PWXL and G5000 PWXL (Tosoh Co., Ltd., Tokyo, Japan) was used. The mobile phase consisted of 0.2 M acetic acid/0.1 M sodium acetate and 0.008 M of sodium azide. A sample concentration of 0.1% (w/v) was loaded and eluted with a flow rate of 0.6 mL/min by an LDC Analytical ConstaMetric 3500 pump (Thermo Scientific, Waltham, MA, USA). The elute peak was detected by an RI detector (Gilson model M132, Gilson, Middleton, WI, USA). The data were analyzed by Chem-Lab software (SISC 3.0, Scientific Information Service, Taipei, Taiwan). Pullulan standards with different Mr values were used as markers. The Mr values of the samples were calculated from the pullulan calibration curve with Chem-Lab software. Determination of Bulk Density Chitosan or regenerated chitosan powder was assayed for its bulk density as described by Cho et al. [39]. One gram of chitosan or regenerated chitosan (40-60 mesh particle size) was placed in a 15-mL tapered graduated centrifuge tube, vibrated on a vortex mixer for 1 min, and packed by gently tapping the tube on the bench top 10 times. The volume of the sample was recorded. The procedure was repeated three times for each sample, and the bulk density was computed as grams per milliliter of the sample. Determination of Crystallinity The crystallinity of chitosan or regenerated chitosan was measured by a Miniflex Rigaku X-ray diffractrometer, using Ni filtered Cu Ka radiation generated at 30 kV and 10 mA at a scanning speed of 2° 2θ/min within a range from 5° to 30°. The crystallinity index was also used, based on the method proposed by Zhang et al. [37]. It consisted of measuring the maximum peak intensity, I110, at 2θ = 20° of the (110) lattice diffraction and that of the amorphous diffraction, Iam, at 2θ = 16°. The crystallinity index (CrI110) was calculated using the following formula: Determination of Ash The ash content was deduced from the difference in weight before and after a thermal treatment of the product in an electric furnace. The crucible containing the dry sample was placed in an electric furnace at 600 °C for 6 h. Ash content was estimated by ignition of a chitosan or regenerated chitosan sample in an electric furnace and quantization of the ash by gravimetric analysis [40]. Statistical Analysis All experiments were carried out in triplicate and average values or means (standard deviations) reported. Mean separation and significance for correlation were analyzed using the SPSS (Statistical Package for Social Sciences, SPSS Inc., Chicago, IL, USA) software package. Conclusions Higher DD of original chitosan and/or higher concentration of acetic acid used resulted in higher purity, however, the bulk density, Mw, PDI, and crystallinity intensity of regenerated chitosan were not affected. The regeneration process could increase the purity, manipulate the Mw and narrow down the PDI of regenerated chitosan and lower the crystallinity intensity, and bulk density, whereas the DD, structure and functional groups on chitosan molecules were preserved. These changes would be beneficial to chitosan for use in the biomedical field. Therefore, the regeneration process has a bright future in the developing areas of medical applications.

v3-fos

2015-09-18T23:22:04.000Z

2015-01-19T00:00:00.000Z

6231128

s2

Chemical composition and antibacterial activities of seven Eucalyptus species essential oils leaves Background In this paper, we have studied the essential oils chemical composition of the leaves of seven Eucalyptus species developed in Tunisia. Eucalyptus leaves were picked from trees growing in different arboretums in Tunisia. Choucha and Mrifeg arboretums located in Sedjnene, region of Bizerte (Choucha: E. maideni, E. astrengens et E. cinerea; Mrifeg : E. leucoxylon), Korbous arboretums located in the region of Nabeul, North East Tunisia with sub-humid bioclimate, (E. lehmani), Souiniet-Ain Drahem arboretum located in region of Jendouba (E. sideroxylon, E. bicostata). Essential oils were individually tested against a large panel of microorganisms including Staphylococcus aureus (ATCC 6539), Escherichia coli (ATCC 25922), Enterococcus faecalis (ATCC29212), Listeria ivanovii (RBL 30), Bacillus cereus (ATCC11778). Results The yield of essential oils ranged from 1.2% to 3% (w/w) for the different Eucalyptus species. All essential oils contain α-pinene, 1,8-cineol and pinocarveol-trans for all Eucalyptus species studied. The 1,8-cineol was the major compound in all species (49.07 to 83.59%). Diameter of inhibition zone of essential oils of Eucalyptus species varied from 10 to 29 mm. The largest zone of inhibition was obtained for Bacillus cereus (E. astrengens) and the lowest for Staphylococcus aureus (E. cinerea). The essential oils from E. maideni, E. astrengens, E. cinerea (arboretum of Bizerte), E. bicostata (arboretum of Aindraham) showed the highest antibacterial activity against Listeria ivanovii and Bacillus cereus. Conclusion The major constituents of Eucalyptus leaves essential oils are 1,8-cineol (49.07 to 83.59%) and α-pinene (1.27 to 26.35%). The essential oils from E. maideni, E. astrengens, E. cinerea, E. bicostata showed the highest antibacterial activity against Listeria ivanovii and Bacillus cereus, they may have potential applications in food and pharmaceutical products. Background Eucalyptus, a native genus from Australia, belongs to Myrtaceae family and comprises about 900 species and subspecies, it is one of the world's most important and most widely planted genera [1][2][3][4]. It has been introduced worldwide, including in Tunisia and mainly cultivated for its timber, pulp and essential oils that present medicinal properties and therapeutic uses [5]. In recent decades, the essential oils and their components of plants have been of great interest as they have been the sources of natural products [6]. The value of Eucalyptus oil for medicinal purposes is based largely on the content of a particular oil constituent: 1,8-cineole (cineole or eucalyptol) [7]. Hot water extracts of dried leaves of Eucalyptus citriodora are traditionally used as analgesic, anti-inflammatory and antipyretic remedies for the symptoms of respiratory infections, such as cold, flu, and sinus congestion ( [8], Eucalyptus camaldulensis and Eucalyptus urophylla are also known to contain bioactive products that showed antibacterial [9], antifungal [10], analgesic and anti-inflammatory effects [8], antioxidative and antiradical [11] activities. Various studies showed that differences in the yield and composition of the essential oils were influenced by the time of harvest, moreover, for Eucalyptus genus, the amount and composition of leaf oil may vary seasonally and diurnally for some plants, depending on environmental conditions [12]. The objectives of the present study are to determine the chemical composition of the essential oils of seven common Tunisian Eucalyptus species, namely E. lehmani; E. leucoxylon; E. astrengens; E. cinerea; E. maideni; E. sideroxylon; E. bicostata. The study also aims at investigating the antibacterial properties against some of the common pathogens bacteria. In addition, the study determines the influence of growth conditions on the chemical composition and antibacterial properties of the essential oils of Eucalyptus species. Results and discussion Eucalyptus essential oil yields The yield of essential oils ranged from 1.2% to 3% (w/w) for the different Eucalyptus species (Table 1). The highest yield was obtained from E. cinerea and E. sideroxylon (3%), followed by E. lehmani (2.8%), while E. astrengens gave the lowest yield at 1.2%. According to these results, we confirmed that there is no relationship between region and Eucalyptus essential oil yield. In fact, species from the same region show different yields (E. leucoxylon; E. astrengens; E. cinérea) while species from different regions have almost the same yield. Some parameters can influence yields such as leaves age [13], the harvest date [14], geographical origine [15], distillation method [16,17] . Ben Jemàa et al., [12] reported that essential oil yields varied according to Eucalyptus species and seasons and for all species studied, (E. camaldulensis, E. astringens, E. leucoxylon, E. lehmannii and E. rudis) high yields were obtained from leaves collected at the summer season though E. astringens gave rather constant yields during winter (1.23% for spring and 1.1% for winter). Composition of the essential oils The chemical composition of the essential oils extracted from Eucalyptus species (E. maideni; E. astrengens; E. cinerea; E. leucoxylon; E. lehmani; E. sideroxylon; E. bicostata) is presented in Table 2. All essential oils contain α-pinene, 1,8-cineol and pinocarveol-trans for all Eucalyptus species studied. The 1,8-cineol was the major compound in all species. The essential oil composition of the Eucalyptus species from the region of Bizerte showed that all of them contained 1,8-cineole, the highest content was obtained from E. maideni (83.59%) followed by E. cinerea and E. lehmani (respectively 79.18% and 49.07%), while E. astrengens gave the lowest rate (60.01%). Although these species are from the same region, they show differences in the levels of some essential oil compounds. This may be due to genetic effects. Essential oils extracted from species from Aindraham arboretum (E. sideroxylon and E. bicostata) have the same 1,8 cineole level and the species from Korbous arboretum (E. lehmani) have the lowest rate of 1,8-cineole (49.07%) and the highest level of α-pinene (26.35%). We could identify other compounds of relatively high rates such as globulol and pinacarvone. Ben jemâa et al., [12] reported that GC and GC-MS analyses showed that chemical composition varied with Tunisian Eucalyptus species and seasons. The five essential oils contained 1,8-cineole, α-pinene, and α-terpineol as major common compounds. The essential oils of twenty Eucalyptus species harvested from North West and North of Tunisia were studied and the authors identified, by GC and GC/MS, eighteen major compounds and the main ones were 1,8-cineol followed by α-pinene, p-cymene, borneol, cryptone, spathulenol, viridiflorol and limonene. The authors showed that main components were oxygenated monoterpenes, among them 1,8-cineol which was the major one in leaf essential oils of ten species, followed by trans-pinocarveol and α-terpineol. The oxygenated sesquiterpenes were the second major class represented essentially by borneol, spathulenol, viridiflorol and globulol; the third major class was the monoterpene hydrocarbons constituted by a high level of α-pinene, pcymene and limonene [16]. Antibacterial activity According to the zone diameter inhibition (zdi) values expressed in mm, results were ranked as follows: not sensitive (-) for zone diameters equal to 8 mm or below; sensitive (+) for zone diameters between 8 and 14 mm, very sensitive (++) for zone diameters between 14 and 20 mm and extremely sensitive (+++) for zone diameters equal or larger than 20 mm [17,20,21]. The results revealed that the essential oils showed antibacterial activity with varying magnitude and depending on the size of inoculums. Diameter of inhibition zone of essential oils of eucalyptus species varied from 10 to 29 mm ( Table 3). The largest zone of inhibition was obtained for Bacillus cereus (E. astrengens) and the lowest for Staphylococcus aureus (E. cinerea). The essential oils from E. maideni, E. astrengens, E. cinerea (arboretum of Bizerte), E. bicostata (arboretum of Aindraham) showed the highest antibacterial activity against Listeria ivanovii and Bacillus cereus. Conclusion The Eucalyptus species investigated in the present study show a large variation in their chemical composition. The major constituents of Eucalyptus leaves essential oils are 1,8-cineol (49.07 to 83.59%) and α-pinene (1.27 to 26.35%). The essential oils from E. maideni, E. astrengens, E. cinerea, E. bicostata showed the highest antibacterial activity against Listeria ivanovii and Bacillus cereus, they may have potential applications in food and pharmaceutical products. Collection of plant material Eucalyptus leaves were picked from trees growing in different arboretums in Tunisia: Choucha and Mrifeg arboretums located in Sedjnene, region of Bizerte (Choucha: E. maideni, E. astrengens and E. cinerea; Mrifeg: E. leucoxylon), Korbous arboretum located in the region of Nabeul, (E. lehmani), Souiniet-Ain Drahem arboretum located in the region of Jendouba (E. sideroxylon, E. bicostata). The leaves were stored at a dry place for fifteen days. Specimens were identified at the Regional Station of the National Institute of Research in Farming Studies, Waters and Forests (INRGREF). Isolation of the essential oils One hundred grams of dried leaves were submitted to water distillation (500 mL of water) for 3 hours, using a Clevenger-type apparatus. The obtained essential oils were dried over anhydrous sodium sulphate and after filtration, stored at 4 to 7°C until use. The extraction yield was calculated using the following formula: yield = (VEO × 100)/D.M (D.M: dry material; VEO: volume of essential oil). Gas chromatography analysis/mass spectrometry analysis The GC analyses were accomplished with a HP-5890 Series II instrument equipped with HP-WAX and HP-5 capillary columns (both 30 m × 0.25 mm, 0.25 mm film thickness), working with the following temperature program: 60°C for 10 min, ramp of 5°C/min up to 220°C; injector and detector temperatures 250°C; carrier gas nitrogen (2 mL/min); detector dual FID; split ratio 1:30; injection of 0.5 mL). The identification of the components was performed, for both columns, by comparison of their retention times with those of pure authentic samples and by means of their linear retention indices (l.r.i.) relative to a series of n-hydrocarbons. The relative proportions of the essential oil constituents were percentages obtained by FID peak-area normalization, without using response factors. For GC/MS detection, an electron ionization system, with ionization energy of 70 eV, a scan time of 1.5 s and mass range 40-300 amu, was used. Helium was the carrier gas at a flow rate of 1.2 mL/min. Injector and transfer line temperatures were set at 250 and 280°C, respectively. Oven program temperature was the same with GC analysis. Diluted samples (1/100 in hexane, v/v) of 1.0 μl were injected manually and in the splitless mode. The identification of the compounds was based on mass spectra (compared with Wiley 275.L, 6th edition mass spectral library) or with authentic compounds and confirmed by comparison of their retention indices either with those of authentic compounds or with data published in the literature as described by Adams [22]. Further confirmation was done from Kovats Retention Index data generated from a series of n-alkanes retention indices (relative to C9-C28 on the BP-1). Antibacterial activity detection Essential oils were individually tested against a panel of microorganisms including Staphylococcus aureus (ATCC 6539), Escherichia coli (ATCC 25922), Enterococcus faecalis (ATCC29212), Listeria ivanovii (RBL 30), Bacillus cereus (ATCC11778). All strains were obtained from Institut Pasteur de Tunis. The Bacteriological agar was from Biokar Diagnostics (Beauvais, France). Nutrient broth (NB) was from Difco (Becton Dickinson, Le Pont de Claix, France). All the other media, used in this study, were manufactured by Biorad (Marnes-La Coquette, France) and Merck. Antibacterial activity is revealed by growth inhibition in the strains to test. This activity is observed in solid medium. In the present work, we used well diffusion method described by Perez et al., [23]. Statistical analysis The data (three replicates) were statistically evaluated using the JMP SAS version 12.

v3-fos

2019-03-31T13:44:42.775Z

2015-01-01T00:00:00.000Z

88311208

s2

Acetylated Anthocyanidin 3-O-di-glycosides in Red-purple Flowers and Grayed-purple Leaves of Saintpaulia ‘Tomoko’ A new acetylated anthocyanin was extracted from the red-purple flowers of Saintpaulia ‘Tomoko’ with 5% HOAc-H2O or 5% formic acid-H2O, and determined to be peonidin 3-O-[6-O-(4-O-(acetyl)-αrhamnopyranosyl)-β-glucospyranoside] (1), by chemical and spectroscopic methods. In addition, two known acetylated cyanidin glycosides, cyanidin 3-O-[6-O-(4-O-(acetyl)-α-rhamnopyranosyl)-β-glucospyranoside] (2) and cyanidin 3-O-[2-O-(β-xylopyranosyl)-6-O-(acetyl)-β-glucopyranoside] (3), were also identified in the redpurple flowers and grayed-purple leaves of S. ‘Tomoko’, respectively. These three anthocyanins have not been reported hitherto in plant tissues in the genus Saintpaulia. Introduction Saintpaulia cultivars (Gesneriaceae) are widely cultivated as one of the most popular ornamental indoor plants bred from some wild species native to Southern Africa, with white, pink, red, red-purple, purple, and violet-blue flowers. Recently, we reported the structural determination of malvidin, peonidin, and pelargonidin 3-acetyl-rutinoside-5-glucosides in the violet-blue, purple, and pink flowers of Saintpaulia 'Thamires' (Saintpaulia sp.) along with apigenin 4'-glucuronide as 5% HOAc-H 2 O extract (Tatsuzawa et al., 2012). However, there seem to be no previous reports on acylated peonidin and/or cyanidin 3-O-di-glycosides in the flowers and leaves of Saintpaulia, except some studies of non-acylated cyanidin 3-sambubioside in the leaves of Saintpaulia ionantha (Harborne, 1966). As part of our ongoing research on various flower colors in Saintpaulia cultivars, we studied the anthocyanin components of the red-purple flowers of S. 'Tomoko' along with grayed-purple leaves of S. 'Tomoko'. In this paper, we report the isolation and structural elucidation of a new acylated anthocyanin and two known ones from the flowers and/or leaves of S. 'Tomoko'. Plant materials The Isolation and purification of pigments Dried red-purple flowers (ca. 5 g) and grayed-purple leaves (ca. 50 g) of S. 'Tomoko' were immersed in 5% HOAc-H 2 O (5 L each) at room temperature for 12 h and then extracted. The extract was passed through a Diaion HP-20 (Nippon Rensui Co., Tokyo, Japan) column (90 × 150 mm), on which pigments were absorbed. Next the column was thoroughly washed with 5% HOAc-H 2 O (20 L) and eluted with 5% HOAc-MeOH (500 mL) to recover the pigments. After concentration, the pigments were separated and purified by paper chromatography using BAW. The separated pigments were further purified by preparative HPLC, which was performed on a Waters C18 (19 × 150 mm, Waters) column at 40°C with a flow rate of 4 mL/min and monitoring at 530 nm. The solvent used was as follows: a linear gradient elution for 15 min from 60% to 70% solvent B in solution A. The fraction was transferred to a Diaion HP-20 column, on which pigment was adsorbed. Pigments were eluted with 5% HOAc-MeOH (5:95, v/v) followed by the addition of excess Et 2 O and then dried. Purified pigment 1 (ca. 25 mg), pigment 2 (ca. 5 mg), and pigment 3 (ca. 45 mg) were obtained as dried dark-red powders. Analyses of pigment The identification of anthocyanin was performed by standard procedures involving deacylation with acid, and both alkaline and acid hydrolyses (Harborne, 1984). Results and Discussion Pigments from red-purple flowers and grayed-purple leaves of Saintpaulia HPLC analyses of 5% HOAc-H 2 O or 5% formic acid-H 2 O extracts from red-purple flowers and grayedpurple leaves of S. 'Tomoko' revealed that two major anthocyanins (pigments 1 and 2) (81.3% and 10.4%, of the total anthocyanin contents calculated from the HPLC peak area at 530 nm) and one major anthocyanin (pigment 3) (85.9%), respectively, were observed in extract at retention times of 28.1 min (pigment 1), 24.1 min (pigment 2), and 28.3 min (pigment 3), along with some other minor peaks. Pigment 1 Pigment 1 was isolated from the 5% HOAc-H 2 O extract of dried petals, and purified using Diaion HP-20 column chromatography, preparative HPLC, and paper chromatography, according to the procedure described previously (Tatsuzawa et al., 2012). The chromatographic and spectroscopic properties of pigment 1 are shown in Materials and Methods. Acid hydrolysis of pigment 1 yielded peonidin as its aglycone, glucose, rhamnose, and acetic acid. Alkaline hydrolysis of pigment 1 yielded a deacylanthocyanin and acetic acid. The deacylanthocyanin was identified as peonidin 3-rutinoside by HPLC, TLC, and UV-vis spectroscopy in comparison with authentic peonidin 3rutinoside. The molecular ion [M] + of pigment 1 was observed at m/z 651 (C 30 H 35 O 16 ) using FABMS, indicating that pigment 1 is composed of peonidin with one molecule each of glucose, rhamnose, and acetic acid. The elemental components of pigment 1 were confirmed by high-resolution FABMS. The structure of pigment 1 was further elucidated by investigation of its 1 H and 13 C NMR spectra, including 2D COSY, 2D NOESY, HMQC, and HMBC spectra (Fig. 1). The chemical shifts of six aromatic protons of the peonidin moiety with their coupling constants were assigned on the basis of analysis of the 2D COSY spectrum. The signals of two anomeric protons of sugar moieties in pigment 1 appeared at δ 5.47 (d, J = 7.6 Hz, Glucose) and δ 4.62 (d, J = 1.5 Hz, Rhamnose), and the chemical shifts of other sugar protons were assigned by analysis of the 2D COSY spectrum with their coupling constants, indicating that the glucose residues of pigment 1 must be β-glucopyranose. In the rhamnose moiety, the doublet signal corresponds to an anomeric proton (δ 4.62, d, J = 1.5 Hz) and doublet signals of methyl protons (δ 0.91, d, J = 6.4 Hz) at C-5 suggested the presence of α-rhamnopyranose. The signal of the anomeric proton of glucose correlated with that of the C-3 carbon (δ 144.2) of peonidin in the HMBC spectrum and also to the signal of H-4 proton (δ 8.39) in the NOESY spectrum of peonidin. The characteristic feature revealed that the OH-3 position of peonidin is glycosylated by glucose. The signal of the anomeric proton of rhamnose correlated with that of the C-6 carbon (δ 66.4) in the HMBC spectrum and to the signal of H-6a and b protons (δ 3.57 and 3.91) in the NOESY spectrum of glucose. Therefore, rhamnose was bonded with glucose at OH-6 of glucose forming rutinose in the pigment. The proton signal of the H-4 of rhamnose (δ 4.75, t, J = 9.8 Hz) was shifted downfield, indicating that the OH-4 of rhamnose is acylated with acetic acid. This linkage was further confirmed by HMBC correlation (Fig. 1). Consequently, the structure of pigment 1 was elucidated to be peonidin 3-O-[6-O-(4-O-(acetyl)-αrhamnopyranosyl)-β-glucopyranoside] (Fig. 1), which is a new anthocyanin in plants ( Andersen and Jordheim, 2006;Harborne and Baxter, 1999;Grayer, 2008, 2011). Pigment 2 Acid hydrolysis of pigment 2 yielded cyanidin as its aglycone, glucose, rhamnose, and acetic acid. Alkaline hydrolysis of pigment 2 yielded a deacylanthocyanin and acetic acid. The deacylanthocyanin was identified as cyanidin 3-rutinoside by HPLC, TLC, and UV-vis spectroscopy in comparison with authentic cyanidin 3rutinoside. The molecular ion [M] + of pigment 2 was observed at m/z 637 (C 29 H 33 O 16 ) using FABMS, indicating that pigment 2 is composed of cyanidin with one molecule each of glucose, rhamnose, and acetic acid. The elemental components of pigment 2 were confirmed by high-resolution FABMS. The structure of pigment 2 was further elucidated by investigation of its 1 H and 13 C NMR spectra, including 2D COSY, 2D NOESY, HMQC, and HMBC spectra (Fig. 1). Pigment 3 Acid hydrolysis of pigment 3 yielded cyanidin as its aglycone, glucose, xylose, and acetic acid. Alkaline hydrolysis of pigment 3 yielded a deacylanthocyanin and acetic acid. The deacylanthocyanin was identified as cyanidin 3-sambubioside by HPLC, TLC, and UV-vis spectroscopy in comparison with authentic cyanidin 3sambubioside. The molecular ion [M] + of pigment 3 was observed at m/z 623 (C 37 H 47 O 22 ) using FABMS, indicating that pigment 3 is composed of cyanidin with one molecule each of glucose, xylose, and acetic acid. The elemental components of pigment 3 were confirmed by highresolution FABMS. The structure of pigment 3 was further elucidated by investigation of its 1 H and 13 C NMR spectra, including 2D COSY, 2D NOESY, HMQC, and HMBC spectra (Fig. 1). The chemical shifts of six aromatic protons of the cyanidin moiety with their coupling constants were assigned on the basis of analysis of the 2D COSY spectrum. The signals of two anomeric protons of sugar moieties in pigment 3 appeared at δ 5.66 (d, J = 7.7 Hz, Glucose) and δ 4.71 (d, J = 7.6 Hz, Xylose), and the chemical shifts of other sugar protons were assigned by analysis of the 2D COSY spectrum with their coupling constants, indicating that those glucose and xylose residues of pigment 3 must be β-pyranose forms. The signal of the anomeric proton of glucose A correlated with that of the C-3 carbon (δ 143.7) of cyanidin in the HMBC spectrum and also to the signal of H-4 proton (δ 8.79) in the NOESY spectrum of cyanidin. The characteristic feature revealed that the OH-3 position of cyanidin is glycosylated by glucoses. The signal of the anomeric proton of xylose correlated with that of the C-2 carbon (δ 80.8) in the HMBC spectrum and to the signal of H-2 proton (δ 3.93) in the NOESY spectrum of glucose. Therefore, xylose was bonded with glucose at OH-2 of glucose forming sambubiose in the pigment. On the basis of the result of analysis of its COSY spectra, two characteristic methylene proton signals (δ 4.05 and 4.43) being shifted to lower magnetic fields were assigned to H-6a and b protons of glucose. This result indicated that the OH-6 of glucose must be esterified 80 with acetic acid. This linkage was further confirmed by HMBC correlation (Fig. 1). Consequently, the structure of pigment 3 was elucidated to be cyanidin 3-O-[2-O-(β-xylopyranosyl)-6-O-(acetyl)-β-glucopyranoside] (Fig. 1), which was found in Saintpaulia for the first time (Andersen and Jordheim, 2006;Harborne and Baxter, 1999;Grayer, 2008, 2011), although this pigment has been found in the flowers of Camellia 'Dalicha' (Li et al., 2008). The distributions of 3-acetyl-rutinoside-5-glucosides of malvidin, pelargonidin, and peonidin have already been reported in the violet-blue, purple, and pink flowers of S. 'Thamires' as their main anthocyanins (Sato et al., 2011;Tatsuzawa et al., 2012). However, in this study, it was revealed that the red-purple flowers of S. 'Tomoko' contain peonidin 3-acetyl-rutinoside and cyanidin 3-acetyl-rutinoside as the main anthocyanins. Therefore, it is presumed that S. 'Tomoko' has two characteristic biosynthetic features different from S. 'Thamires' (Sato et al., 2011;Tatsuzawa et al., 2012) for producing the red-purple flowers as follows: (1) it is devoid of flavonoid 3',5'-hydroxylase activity and (2) 5-OH of anthocyanidin is free from glucosylation. On the other hand, anthocyanin in the grayed-purple colored leaves of S. 'Tomoko' contained cyanidin 3-acetylsambubioside as a main anthocyanin. Therefore, it was thought that the biosynthesis of anthocyanin 3glucoside moiety for the leaf anthocyanin depended on another biosynthesis system, not the flower one.

v3-fos

2018-04-03T04:02:11.988Z

2015-05-19T00:00:00.000Z

15917078

s2

Fine Mapping and Characterization of Candidate Genes that Control Resistance to Cercospora sojina K. Hara in Two Soybean Germplasm Accessions Frogeye leaf spot (FLS), caused by the fungus Cercospora sojina K. Hara, may cause a significant yield loss to soybean growers in regions with a warm and humid climate. Two soybean accessions, PI 594891 and PI 594774, were identified to carry a high level of resistance similar to that conditioned by the Rcs3 gene in 'Davis'. Previously, we reported that the resistance to FLS in these two plant introductions (PIs) was controlled by a novel gene (s) on chromosome 13 that is different from Rcs3. To fine-map the novel FLS resistance gene(s) in these two PIs, F2: 3 seeds from the crosses between PI 594891 and PI 594774, and the FLS susceptible genotype 'Blackhawk' were genotyped with SNP markers that were designed based on the SoySNP50k iSelect BeadChip data to identify recombinant events and locate candidate genes. Analysis of lines possessing key recombination events helped narrow down the FLS-resistance genomic region in PI 594891 from 3.3 Mb to a 72.6 kb region with five annotated genes. The resistance gene in PI 594774 was fine-mapped into a 540 kb region that encompasses the 72.6 kb region found in PI 594891. Sequencing five candidate genes in PI 594891 identified three genes that have several mutations in the promoter, intron, 5', and 3' UTR regions. qPCR analysis showed a difference in expression levels of these genes in both lines compared to Blackhawk in the presence of C. sojina. Based on phenotype, genotype and haplotype analysis results, these two soybean accessions might carry different resistance alleles of the same gene or two different gene(s). The identified SNPs were used to develop Kompetitive Allele Specific PCR (KASP) assays to detect the resistance alleles on chromosome 13 from the two PIs for marker-assisted selection. Introduction Frogeye leaf spot (FLS), caused by the fungus Cercospora sojina K. Hara, is a soybean disease that can significantly impact soybean yield in warm, humid regions. This disease was first reported in Japan in 1915 and then in the USA in 1924 [1]. The development of FLS depends on the growing conditions of soybean and also weather patterns [2]. Though FLS normally causes negligible to mild damage to soybean plants, it can quickly develop into a devastating epidemic if favored by warm and humid weather [3]. Warm temperatures and high levels of rainfall and humidity induced two severe outbreaks of this disease in Argentina in the growing seasons of 1999/2000 and 2009/2010 [4,5]. The epidemic of FLS in 1999/2000 affected mainly northwestern Argentina and caused yield losses from 25 to 48% in susceptible cultivars [4]. In the growing season of 2009/2010, favorable weather conditions for FLS and planting of susceptible cultivars resulted in 100% incidence of FLS in the Pampean region [5]. The yield loss caused by FLS in the latter incidence was estimated to be from 4 to 5 million metric tons, making FLS the most expensive disease in the history of soybean production in Argentina. In the USA, FLS also is a threat to soybean growers, with yield losses from 183,000 to 345,000 metric tons from 2006 to 2009 [6]. FLS has been observed more often in the southern USA, but it has been reported recently in the northern regions. Field experiments in the USA indicated that FLS can reduce yield from 17 to 31% if susceptible cultivars are used [7,8]. A yield loss due to FLS of up to 60% was reported in Nigeria [9]. The most efficient disease management practices are foliar fungicide applications and host plant resistance, although other practices have also been recommended, including seed treatment with fungicides, crop rotation, and biological control using bacteria [10]. However, repeated use of fungicides can lead to reduced sensitivity in target populations. Strains of C. sojina resistant to strobilurin fungicides, the most commonly used fungicides against this disease, were found in several states in the USA [11,12]. Thus, development and use of soybean cultivars with resistance to FLS is of great importance. Studies on pathogenicity of C. sojina revealed a complex biological feature of this pathosystem. In Brazil and China, 22 and 14 races of C. sojina were reported, respectively [13,14]. In the USA, five races of the fungus were classified in 1981 [15], but a study in 2004 showed that there are likely more than 12 different races of C. sojina found in various states [16]. Based on reactions that a collection of 93 C. sojina isolates from the USA (71), Brazil (15), and China (7) produced on a set of 38 soybean differential cultivars, Mian et al. (2008) proposed that there were at least 11 races of C. sojina [17]. Along with the characterization of the causal pathogen, identification and mapping of FLS resistance genes facilitates introgression of the genes into elite breeding lines. Four single genes conditioning resistance to FLS are currently recognized by the Soybean Genetics Committee, though other resistance genes have been reported. The Rcs1 gene that confers resistance to race 1 of C. sojina was identified in 'Lincoln' [18]. A second resistance gene was found in 'Kent' and named Rcs2 due to its contribution to resistance to race 2 [19]. Soybean line 'Davis' carries Rcs3, which has been the most durable and robust gene [20]. The Rcs3 allele not only confers resistance to race 5, but also to all other reported races in the USA as well as to Brazilian isolates [14,20]. Two additional genes from PI 594891 and PI 594774 were approved by the Soybean Genetics Committee in 2012. These two single dominant resistance alleles had been identified and reported by Hoskins (2011), and were designated by the Soybean Genetics Committee as Rcs(PI 594891) and Rcs(PI 594774) [21]. Additionally, a dominant gene conferring resistance to Chinese race 7 has been referred to as Rcs7 [22], but this gene was not officially recognized by the Soybean Genetics Committee because of insufficient evidence to show that it is not allelic to the Rcs1, 2, and 3 genes. Although Rcs3 has been effective in soybean production, the complexity of the C. sojina races and breakdown of the FLS resistance genes have increased the need to identify additional resistance genes and pyramid those in elite germplasm. Among the reported FLS resistance genes, Rcs(PI 594891) and Rcs(PI 594774) conferred a high level of resistance similar to that conditioned by the Rcs3 gene in 'Davis'. Furthermore, the resistance to FLS in PI 594891 and PI 594774 was controlled by two single dominant genes in close proximityon chromosome (Chr) 13, which is different from the Rcs3 allele on Chr18 [21]. The genomic regions harboring the resistance genes from these two PIs were found to be in a 3-4 Mbp interval that contains hundreds of genes. With Blackhawk and these two PIs being genotyped with SoySNP50k iSelect BeadChip [23], an enormous amount of SNP data obtained within the roughly mapped region could be used for fine-mapping. In addition, the utilization of KASP technology (LGC Genomics, Middlesex, UK), a closed-tube SNP detection method, has provided an effective platform to accelerate the genotyping process. The simplicity of the onestep procedure for data collection and small reaction volumes make this technology cost-effective, flexible, and the method of choice for a project that has numerous markers and samples. The objectives of this study were to: 1) fine-map the genomic region containing Rcs(PI 594891) and Rcs(PI 594774) loci using the SoySNP50k Infinium chip data; 2) understand whether the two resistance loci located close to each other on the same chromosome with different symptoms (no lesion on PI 594891, small lesion on PI 594474) were different genes or the same resistance gene with different alleles; and 3) develop robust KASP marker assays to detect the Rcs(PI 594891/PI 594774) FLS resistance alleles for marker-assisted selection. Population development Crosses between the cultivar Blackhawk and the accessions PI 594891 and PI 594774 were made during the summer of 2003 at the Univ. of Georgia Plant Sciences Farm in Watkinsville, GA. PI 594891 and PI 594774 were found to possess resistance to a broad spectrum of C. sojina isolates, while Blackhawk is a highly susceptible parent [21]. F 2 populations derived from F 1 plants of both crosses were grown in a greenhouse at the Univ. of Georgia, Athens, GA in 2003 or 2004. Each population consisted of 200 F 2 seed and was initially genotyped with SSR markers to obtain desired genotypes to advance to the F 2: 3 generation. An additional 36 seed from the same population were used for KASP assay validation. For convenience, the F 2: 3 populations of Blackhawk x PI 594891 and Blackhawk x PI 594774 were encoded as FLS-594891 and FLS-594774, respectively. Fine-mapping procedure To fine-map the genomic region containing the resistance genes in PI 594891 and PI 594774, 200 F 2 seeds from the cross of Blackhawk with either PI 594891 or PI 594774 were chipped into ¼ and ¾ seed pieces using razor blades. The ¼ and ¾ seed pieces were loaded into a micro-centrifuge tube and plate well, respectively, which were labelled with the same ID. DNA were extracted from the ¼ seed pieces and used for genotyping the F 2 generation with SSR markers flanking the genomic region containing the FLS resistance genes. The ¾ seed pieces having PI homozygous alleles at one marker and heterozygous alleles at the other marker were selected and grown to obtain F 2: 3 seed. At the same time, SNPs polymorphic between Blackhawk and PI 594891/PI 594774 from the genomic region bordered by two flanking SSR markers were identified and used for KASP assay development. The F 2: 3 seed harvested were chipped again using the method described above. DNA from the ¼ seed chips were genotyped with KASP markers to identify individuals with recombination events in the target region. The ¾ seeds that possessed a recombination event were planted and used for phenotyping with C. sojina isolates in the greenhouse. Finally, genotype data (recombination breakpoints) and data on the reactions of all F 3 plants to two C. sojina isolates were combined to narrow down the genomic regions containing FLS resistance genes in these two PIs. FLS Greenhouse Assays PI 594774, PI 594891, Blackhawk, and F 2 or F 3 recombinant plants were evaluated in a greenhouse at the Univ. of Georgia Griffin campus using a protocol described previously [15,17,24] with some modifications. Seed were grown individually in 10-cm pots on a greenhouse bench. Seedlings were inoculated at the V2 to V3 growth stage of development [24]. A single trifoliolate of each plant was inoculated with isolates 21 and 23 of C. sojina by atomizing a conidial suspension of approximately 6.0 × 10 3 spores mL -1 . These two isolates were collected from China and classified as race 8 by Mian et al (2008). Because these two isolates belong to the same race, use of both isolates for inoculating F 2: 3 plants provided confirmation of disease phenotypes. Inoculated plants were placed into a clear plastic bag for 48 h to maintain a high relative humidity. Plants were removed from bags and then placed on a greenhouse bench. Disease ratings were made 14 days after inoculation. The FLS reaction was scored as a qualitative trait (i.e. susceptible vs. resistant) for all plants. Plants were classified as susceptible when lesions were predominately large and spreading with light centers and dark margins. Plants were rated as resistant when they showed no lesions or predominately small lesions without clearly defined centers [17]. The F 2: 3 recombinant plants were first inoculated with isolate 21 (race 8) of C. sojina [17]. After the phenotypic evaluation for this isolate was finished, all of the mature leaves with or without lesions in the experimental plants were excised. When the remaining young leaves matured, the plants were inoculated again with isolate 23 (race 8) of C. sojina [17]. For the haplotype experiment, 45 soybean lines and cultivars with diverse pedigrees were evaluated in the greenhouse at the Univ. of Georgia Griffin campus along with PI 594774, PI 594891, Davis, and Blackhawk. Three seed were planted per pot and four pots were used per genotype. Approximately one week after planting, seedlings were thinned to two plants per pot, which resulted in a total of eight plants per genotype for inoculation and rating. Two weeks after planting, the plants were inoculated with a mixture of isolates 21 and 23 (race 8) of C. sojina. Two weeks after inoculation, each plant was rated as susceptible, resistant, or immune. Genotyping F 2 seed Based on the study by Hoskins (2011), the flanking SSR markers Satt114 and Sct_033 on Chr13 were selected for genotyping F 2 seed from the cross of Blackhawk x PI 594891, and Satt114 and Satt663 were used for the F 2 seeds of Blackhawk x PI 594774. A ¼ seed chip was removed for DNA extraction from each of the F 2 seed in both populations using a CTAB (Hexadecyltrimethylammonium bromide) procedure modified from Keim et al. [25]. The PCR reaction was conducted on a 384-well GeneAmp PCR System 9700 (PE-ABI, Foster City, CA) using fluorescent dye-labeled primers, following Diwan and Cregan's protocol [26]. The marker fragments were analyzed with GeneMarker software (SoftGenetics, State College, PA). Sequencing genes identified from the fine-mapping work Five genes identified from the 72.6 kb region using the FLS-594891 population were considered as candidate FLS resistance genes. The DNA sequences of these genes were obtained from the Phytozome.net website based on the sequence of 'Williams 82'. Gene-specific primers for each gene were designed using the Primer3Plus program [27]. DNA from Blackhawk and the two PIs was isolated from leaf tissue using the CTAB protocol, and 5-50 ng of DNA were used in PCRs with gene-specific primers for each gene of interest under the following conditions: 95°C for 5 min, followed by 34 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min per 1 kb of predicted product size. After PCR, the target sequence amplification was verified on a 1.5% agarose gel by electrophoresis and then sequenced at the Georgia Genomics Facility located on the Univ. of Georgia Athens campus. Sequence traces were assembled and manually evaluated for polymorphisms using Geneious software (version 5.5.7.). Putative polymorphisms were confirmed by an independent sequencing reaction. To test the association between the SNPs identified in the two candidate genes Gly-ma13g25340 and Glyma13g25350 and the FLS phenotypes, and to develop robust SNP assays for genotyping and marker-assisted selection, one SNP specific for both PI 594891 and PI 594774 from each of the two candidate genes was selected for KASP assay development, This resulted in a total of four KASP assays. A set of 36 F 2 seed from either Blackhawk x PI 594891 or Blackhawk x PI 594774 and a panel of 158 cultivars and germplasm lines were genotyped using these KASP assays. The 158 soybean lines in the panel were selected due to the availability of DNA for the genotyping experiment and they were from the list used in a previous experiment in our lab. Due to the limited availability of seeds, only 45 lines were assayed for their reactions to C. sojina. Inoculation for RT-qPCR assays The FLS phenotyping was conducted in a greenhouse at the Univ. of Georgia, Griffin campus using procedures described by Mian et al. [17] with some modifications. A total of 100 seed for each of the three parents (Blackhawk, PI 594891, and PI 594774) were planted in eight flats which were divided into two sets. Within each flat, 12 10-cm square plastic pots, four for each of the parents (Blackhawk, PI 594891, and PI 594774), were planted using a randomized complete block design. Two to three seeds were planted in each pot and then seedlings were thinned to one plant at the V2-V3 stage [25]. Four flats in the first set were inoculated with C. sojina isolate 21, while four flats in the second set were mock-inoculated with water as a control. At 3, 6, 11, and 16 days after inoculation (dai), unifoliate leaves from one inoculated plant of each genotype were excised from the stem, placed in sealable plastic bags, frozen in dry ice, and then stored in a -80°C freezer. qPCR analysis and cDNA cloning of candidate genes qRT-PCR was carried out using the LightCycler 480 Real-Time PCR System (Roche, Germany). Specific primers for each candidate gene were designed using Primer3Plus software [28]. PCR products amplified from these primers were sequenced to determine if the correct genes of interest were amplified. Only primer pairs that gave a single amplicon were selected for qRT-PCR. Primer efficiency was determined using five-fold serial dilutions using DNA of a soybean line S03-380RR. RNA was extracted using TRIZOL RNA extraction reagent by following the procedure from the manufacturer (Life Technologies, Carlsbad, CA). The qRT-PCR reactions were conducted with three technical replications in 10 μl reactions for each biological replicate using the GoTag 1-Step RT-qPCR Kit (Promega Corporation, Madison, WI). Following the reverse transcriptase reaction, amplification was conducted at 95°C for 10 min, then 35 cycles of 95°C for 10 s, 60°C for 20 s, and 72°C for 20 s. Soybean gene cons7 was used as an internal control [28]. The quantification of gene expression was performed using the relative ΔΔCT method [29]. cDNA cloning and sequencing was conducted using the QIAGEN One- Step RT-PCR Kit (Qiagen, Valencia CA) and specific primers for each gene. Haplotype analysis To understand the genetic variation at resistance loci, haplotype analysis was performed at the fine-mapped locations of the resistance genes in PI 594774, PI 594891, and Davis (Rcs3). PI 594774, PI 594891, Davis, Blackhawk and 45 soybean lines and cultivars with diverse pedigrees were used in the haplotype analysis. The phenotypic data of these lines were obtained in the greenhouse at the Univ. of Georgia Griffin greenhouse facility. SoySNP50K Infinium Chip data were obtained at Soybase (www.soybase.org) for all lines with the exception of G00-3213, G00-3880, N05-7432, N7002, N77-114, and N8001, which were fingerprinted using the SoySNP50K Infinium BeadChip at Michigan State Univ. For PI 594891 and PI 594774, the haplotype windows were defined by the 72.6 kb fine-mapped region (Figs 1 and 2). The haplotype window for Davis was defined by the interval based on the primer sequence of Satt244) and AZ573TA150 markers used to map the Rcs3 gene on chr16 [30]. Therefore, each interval was based on the estimated physical locations of the SSR or SNP markers in the soybean genome map available at www.soybase.org using the Glyma1.01 map version. Polymorphic SNP markers from the SoySNP50K Infinium BeadChip of all soybean lines and cultivars within the defined intervals were used for haplotype analysis. The graphic haplotype allele visualization of all lines compared to PI 594774, PI 594891, or Davis was performed, and the similarity for a given genotype to the target line was calculated using Flapjack software [31]. The 2.9 Mbp genomic region associated with FLS resistance in PI 594891 A total of 192 F 2 seed derived from Blackhawk x PI594891 was chipped and DNA from these F 2 seed was used for genotyping with SSR markers Satt114 and Sct_033. Based on the genotyping results, 14 F 2 seed were found to carry PI 594891 alleles at one marker and to be segregating at the other (PI-HET or HET-PI, with PI and HET standing for homozygous and heterozygous alleles from PI 594891, respectively). There were also nine seed with the genotype BH-HET or HET-BH from this population (BH indicates the Blackhawk alleles). These 23 seed were grown to maturity in the greenhouse, and each of the F 2 plants produced 30 to170 F 3 seeds, with a total of 1,544 seeds. All of the seeds were chipped for the next cycle of genotyping. Comparison of the SoySNP50K Infinium BeadChip data between Blackhawk and PI 594891 in the 2.9 Mb region bordered by the Satt114 and Sct_033 markers identified 91 single nucleotide polymorphisms (SNPs). However, these SNPs were not evenly spaced, and often occurred in clusters of 2-14 SNPs. Of these 91 SNPs, 31 were in the regions unfavorable for designing KASP assays. Eleven SNPs residing in regions suitable for KASP primer design, and spaced approximately every 250 kb throughout the targeted region, were used to develop KASP assays designated as GSM150 to GSM160. After the phenotyping data from the seedlings of recombinant F 2: 3 seeds were obtained, 13 additional SNP assays were designed in the second round to saturate and narrow down the region containing the resistance gene Rcs(PI 594891). Of 1,544 F 2: 3 seeds genotyped with markers GSM150 through GSM160, 259 F 2: 3 seeds were found to carry one or two recombination events. Due to limited greenhouse space, only 157 seeds were selected for planting, inoculation, and evaluation with C. sojina isolates 21 and 23 in March-April 2013, respectively. In the second evaluation, 55 seed of this population were then grown and phenotyped with the same isolates in May-June 2013. DNA samples extracted from the leaf tissues of these 212 phenotyped F 2: 3 plants were genotyped with all 24 KASP assays. Finally, the region containing the resistance gene in PI 594891 was narrowed down using the data of 66 F 2: 3 recombinant plants harboring six recombination break points (Fig 1). The phenotypes of FLS for these F 2: 3 plants shown in Fig 1 are the reactions to both C. sojina isolates. PI 594891 plants and 24 F 2: 3 plants with the genotype homozygous for PI 594891 alleles in the interval between GSM150 and GSM160 were resistant to FLS, and Blackhawk and 15 plants with the Blackhawk haplotype were observed as susceptible (Fig 1). In total, six recombination break points were found among these 66 F 2: 3 plants. The region containing the resistance gene Mbp genomic region associated with FLS resistance in PI 594774 The same fine-mapping strategy was employed for the population from Blackhawk × PI 594774. In this population, 17 F 2 seed with genotypes PI-HET or HET-PI for SSR markers Satt663 and Satt114 were identified from 192 F 2 seed and advanced to the F 3 generation. At harvest, 1,372 F 2: 3 seeds were obtained and the seeds were chipped to ¼ and ¾ seeds for genotyping and advancement. Using the SoySNP50K Infinium chip data for Blackhawk and PI 594774, 33 SNPs were identified in the 3.3 Mb region flanked by Satt663 and Satt114. However, most of these SNPs (22) were clustered together, and some SNPs fell in a region that is unfavorable for primer design. Therefore, only six SNPs, designated as GSM144 to GSM149, were successfully designed as KASP assays. The performance of these KASP assays was tested using DNA from leaves of the two parents and a set of 36 F 2 individuals from Blackhawk x PI 594774. DNA was extracted from the chips of 1,372 F 2: 3 seed and genotyped with the six SNPs. Of 1,372 F 2: 3 seed, we identified 395 seed that carried one or two recombination events in the interval flanked by Satt663 and Satt114. In March-April 2013, 55 seeds with recombination events from this population, along with the seeds from the FLS-594891 population were grown in the greenhouse and were phenotyped with C. sojina isolates 21 and 23. In the second evaluation in May-June 2013, 124 seed were grown and evaluated for FLS reaction. Due to germination issues, phenotypic data were only collected for a total of 103 F 2: 3 plants from both evaluations. Leaf DNA collected from these 103 F 2: 3 plants were genotyped with a set of six KASP markers. The resistance gene from PI 594774 was initially mapped to a region of Chr 13 adjacent to the one in PI 594891, and was bordered by Satt663 and Satt114 [21]. The physical locations of these two SSR markers are close to KASP markers GSM144 and GSM149, respectively. However, fine-mapping data showed that the resistance gene(s) did not reside in this region (clearly indicated by the plants with recombination event 3 and 7). In this population, PI 594774 and 10 plants with PI 594774 alleles at all of the KASP markers showed a resistance reaction, while Blackhawk and three plants with a genotype resembling Blackhawk showed a susceptible reaction (Fig 2). Representing recombination event No. 9 were four F 2: 3 plants that were homozygous for the Blackhawk allele at SNP markers GSM152, GSM154, and GSM202, and heterozygous for all other SNPs in the region. Because these four plants had a susceptible reaction with both isolates of C. sojina, the FLS resistance gene from PI 594774 was predicted to be in one of two regions: one flanked by GSM151 and GSM153 or one flanked by GSM153 and GSM204. However, there were two F 2: 3 plants that were homozygous for the Blackhawk allele with GSM152 and PI-HET for all other KASP markers (recombination event No. 10). They were also resistant to both FLS isolates. Therefore, the resistance gene in PI 594774 was estimated to reside in the region of approximately 540 kb flanked by GSM153 and GSM204. Relative expression levels of candidates genes in the presence of FLS pathogens To further pinpoint the candidate genes that contribute to the resistance to C. sojina in both PI 594891 and PI 594774, a real-time quantitative PCR experiment was performed to examine the expression levels of five candidate genes within the 72.6 kb region fine-mapped in the FLS-594891 population. Blackhawk and the two PIs were inoculated with C. sojina isolate 21 (Fig 3). Leaves for each parent were sampled at 3, 6, 11, and 16 days after inoculation (dai). Uninoculated leaves from the three parents were also sampled, but were not used for the assays. Blackhawk, PI 594891 and PI 594774 had the same steady expression level at all-time points for the gene Glyma13g25331 (Fig 3). A similar trend was also observed for Glyma13g25380 (FBP), except that at 16 dai expression of this gene was 2× higher in PI 594774 than in the other two parents. The expression levels of Glyma13g25320, Glyma13g25340 (LRR), and Gly-ma13g25350 (TPR) were 2× and 1× higher in PI 594774 and PI 594891 than in Blackhawk, respectively, but only at 3 dai. There were no differences in the expression levels of these genes among the three genotypes at the later time points. The difference in expression levels of the three genes at 3 dai between PI 594774 and Blackhawk was statistically significant, while there was no difference between PI 594891 and Blackhawk. Because the expression levels of the five candidate genes in the two PIs differed from those in Blackhawk only at 3 dai, the expression of the five genes was compared in Blackhawk leaf tissues at 3 dai. At this time point, the expression levels of genes Glyma13g25331 and Glyma13g25380 (FBP) were about 7-8 times higher than those of Glyma13g25340 (LRR), which had the lowest expression level. Tthe Glyma13g25320 and Glyma13g25350 (TPR) genes had expression levels which were 15-18x higher than that of Glyma13g25340 (LRR) (Fig 3). Sequencing cDNA and DNA of candidate genes within the fine-mapped genomic regions To further characterize the five candidate genes identified in PI 594891 and PI 594774, cDNA of the five genes was sequenced using RNA from the RT-qPCR experiment. When RNA extracted from leaves of Blackhawk and two PIs at 3 dai was used for cDNA synthesis, no PCR products were obtained for two genes Glyma13g25340 (LRR) and Glyma13g25380 (TPR). This supported the RT-qPCR results for Glyma13g25340 (LRR), which indicates that it was weakly translated. Glyma13g25380 had no difference in expression levels among Blackhawk and the two PIs until 16 dai, so no attempt was made to further sequence cDNA of this gene. Sequencing the cDNA products of Blackhawk and two PIs for Glyma13g25320 indicated a SNP A 448 G in mRNA of PI 594891 that resulted in an amino acid change of threonine (T) 150 to alanine (A). This amino acid substitution was evaluated as tolerant by the algorithm "Sorting Intolerant from Tolerant" (SIFT) [32] because the SIFT probability for the change T 150 A was equal to 0.64 (amino acids with P <. 05 are predicted to be deleterious). In addition, the presence of both threonine and alanine was observed at this residue in Weblogo images [33], indicating that this amino acid change in Glyma13g25320 protein was also observed in other species (S2 Table). For Glyma13g25331, PI 594891 had the same sequences as Williams 82, while five SNPs were identified in cDNA sequences of Blackhawk and PI 594774 including C 178 A (H 60 N), A 446 T (N 149 I), C 548 A (S 183 Y), A 657 C, and T 662 C (I 221 T). An attempt was made to evaluate the impact of the amino acid change in this gene for Blackhawk and PI 594774, but blasting SIFT did not result in any sequences. In addition, the RT-qPCR results showed no difference at any time point for this gene in Blackhawk, PI 594891 and PI 594774, so there was no further work done with this gene. Because no products were obtained in cDNA synthesis for the Glyma13g25340 (LRR) gene, the region consisting of 7.6 kb of genomic DNA and 5 kb of promoter was sequenced for Blackhawk, PI 594891, and PI 594774. Compared to the sequence of Williams 82, there was a total of 15 genotype-specific SNPs and nine common SNPs found in Blackhawk, PI 594891, and PI 594774 (S2 Table). Four of the SNPs were found in Blackhawk, including two SNPs in the promoter, one in the intronic region, and one in the exon that resulted in an amino acid change of L 636 F. This amino acid substitution was evaluated as deleterious by SIFT (P = 0.03). For PI 594774, there were two SNPs in the intronic region, two in the exonic region (only one resulted in an amino acid replacement of S 126 T), and one SNP in the 3'UTR region. The amino acid change in PI 594774 was predicted to be tolerant by SIFT. Compared to Blackhawk's sequence, PI 594891 had a 6 bp deletion in the last intron and a SNP in the 3' UTR region. Among nine common DNA changes for all three genotypes there was a big deletion of 602 bp and an insertion of 161 bps in the 12 th intron. In addition to this deletion/insertion, there was one SNP in the promoter, five in the intronic region, and one in the exonic region. However, none resulted in any amino acid change. These SNPs as well as one in the 3' UTR region were observed in all three genotypes. In the 5 kb promoter region, there were 30 changes in DNA sequences of Blackhawk, PI 594891, and PI 594774 including SNPs, insertions and deletions when compared to that of Williams 82. Among the identified SNPs, one, five, and six SNPs are unique for PI 594891, Blackhawk and PI 594774, respectively. There were four SNPs shared between Blackhawk and PI 594891, 10 SNPs shared between Blackhawk and PI 594774, and four common to all three genotypes (S2 Table). The sequence of PI 594891 for this region was conserved and had fewer differences from that of Williams 82 in comparison to those of Blackhawk and PI 594774. In the 8.8 kb genomic DNA sequence of Glyma13g25350 (TPL), there were 123 SNPs identified in Blackhawk, PI 594891, and PI 594774 when compared to the sequence of Williams 82. Of these 123 SNPs, there were 12 SNPs common to all three genotypes, with two of them in exonic regions, resulting in one common amino acid change of F 245 L. Blackhawk and PI 594891 had 83 SNPs in common, including 74 SNPs in introns and nine in exons that caused six amino acid changes. However, all of the amino acid changes were predicted to be tolerable by SIFT. Compared to the Blackhawk sequence, PI 594891 had eight SNPs in the intron and one in the 5' UTR region, and PI 594774 had 13 genotype-specific SNPs in the intron, three in exons and one in the 5' UTR region. All of the SNPs in the exonic regions in PI 594774 resulted in amino acid changes, with two changes predicted to be benign and one, E 646 K, predicted to be deleterious for the function by the SIFT program (P = 0.02). The 5 kb promoter region of this gene was sequenced and divided into two segments based on the pattern of the SNPs found in the three genotypes. In the 1,500 bp promoter region upstream and adjacent to the 5' UTR region, there were six, two, and one SNP(s) that were specific to PI 594774, PI 594891, and Blackhawk, respectively. There was also one SNP that was present in all three genotypes. In this region, the Blackhawk and PI 594774 sequences shared the highest similarity, with 30 common SNPs and one deletion/insertion. When the 3.5 kb region upstream of this 1.5 kb promoter was sequenced, it was found that Blackhawk had the greatest number of differences in DNA sequence, including 14 SNPs and three deletions of 2, 5, and 6 bp. There were two SNPs that were shared between PI 594774 and PI 594891, two common to Blackhawk and PI 594891, and one common to Blackhawk and PI 594774. There were four and nine SNPs that are specific to PI 594891 and PI 594774, respectively. Three common SNPs were seen in all of the three genotypes in this upstream region of the promoter. KASP assay development For the STRUBBELIG-receptor like gene (Glyma13g25340), the SNP G 18 A in the exonic region of PI 594774 and A>T in the 5'UTR region of PI 594891 were used for KASP development. For the transducin-like gene (Glyma13g25350), the SNP G 1828 A (causing an amino acid change) and SNP C -382 >A (in the promoter) were used for KASP development. These KASP assays were named GSM344-347 (S1 Table). The performance and accuracy of these SNPs in predicting the FLS reaction were initially tested using DNA of Blackhawk, the two PIs, and a set of 36 F 2 individuals for each of the populations FLS-594891 and FLS-594774. The phenotype of these F 2 individuals was evaluated in the greenhouse in 2012. The tight clustering of the three genotypic classes for each marker indicated that they can effectively differentiate different genotypes (Fig 4). A complete association was observed between SNP alleles and reaction to FLS in the two sets of F 2 individuals when each was run with two SNP markers (success rate 100%), which demonstrated the robustness of these SNP markers to predict the FLS phenotypes. These four SNPs were also used to genotype F 2: 3 individuals which were used for finemapping genomic regions in both FLS-594891 and FLS-594774 populations. The PI alleles from two PIs were always found in individuals with the resistant phenotype. Haplotype analysis with additional soybean lines and cultivars When GSM344 and GSM347 assays were used to genotype a panel of 45 soybean cultivars and lines with known phenotypes, both PI 594891 and PI 594774 showed unique haplotype alleles at these marker loci (S3 Table). To understand the genetic variation at this fine-mapped region, Candidate Genes for Resistance to Cercospora sojina in Soybean a haplotype and phenotype analysis was performed at the mapped locations of the resistance genes in PI 594774, PI 594891, and Davis (Rcs3) for these 45 lines. The source of inoculum used was a combination of isolates 21 and 23 (race 8) of C. sojina. PI 594774 had an immune reaction, in which no lesions were visible. PI 594891 had a resistance reaction which was indicated by predominately small lesions without clearly defined centers. The Davis plants had a combination of immune and resistance reactions, while Blackhawk was susceptible (large lesions spreading with light centers and dark margins). Of the 45 soybean lines and cultivars, 17 had a combination of immune and resistance reactions on the assayed plants, 12 had an immune reaction, five had a resistance reaction, and 11 had a susceptible reaction (S3 Table). The haplotype window defined was based on the fine-mapping of the resistance genes to a 72.6 kb region with 11 SNP markers, with physical positions of the first and last SNPs being Gm13_28559988 and Gm13_28632634, respectively. In this interval both PI 594774 and PI 594891 have unique haplotypes which are different from those of Davis, Blackhawk, and the 45 soybean cultivars and lines (S4 Table). We have also evaluated the haplotype allele variation at the Rcs3 locus from Davis based on the mapped interval of the Rcs3 gene on chr16 [31]. The physical positions of the first and last SNP in the Davis haplotype window were Gm16_32681330 and Gm16_33360539, and there were a total of 30 SNPs in the defined window (S5 Table). Similarly, Davis had a unique haplotype from PI 594774, PI 594891, and Blackhawk. However, there were four cultivars 'Young', 'Cook', 'Doles', and 'N6201' with a 100% match to the Davis haplotype (S5 Table), suggesting that these cultivars all have Davis in their pedigrees. Discussion The emergence and spread of strains of C. sojina K. Hara with tolerance to some commonly used fungicides, the breakdown of known FLS resistance genes, and the complexity of the FLS race structure have made the development of FLS-resistant soybean cultivars an important objective. The study by Hoskins (2011) indicated that the FLS resistance gene(s) in PI 594891 and PI 594774 to C. sojina (race 8) were located at a different locus compared to the Rcs3 gene in 'Davis', providing an opportunity for gene pyramiding to obtain a more durable and stable resistance against this disease [21]. The small number of candidate genes identified from finemapping would enhance the feasibility of map-based cloning of novel FLS resistance genes in PI 594891 and PI 594774. PI 495891 and PI 594774 were found having resistance reactions to 12 isolates representing all of the 12 races in a differential experiment (data not shown). In this experiment, only one race (race 8) was used as the one that was used in the QTL mapping study conducted previously to identify the resistance genes in these two PIs. As Rcs3 was found to condition resistance to all known races of C. sojina, it is likely that the genes controlling the resistance to other C. sojina isolates in these two PIs are the same gene(s). However, this question can only be answered by phenotyping the F 3 recombinant plants generated from this experiment with other isolates of the remaining 11 races. This study has demonstrated a novel approach to fine-map genomic regions containing genes of interest using the SoySNP50K Infinium chip and KASP technology for accelerated genotyping. Three key factors typically affect fine-mapping studies: marker density, recombination frequency, and accuracy of recombinant phenotypes [34]. Most fine-mapping studies in plants have used SSR markers for genotyping and identification of recombination events [35][36][37][38][39]. Although numerous SSR markers are now available [40], deployment of SSR markers for genotyping a large number of seeds is costly, time consuming, and laborious. This study took advantage of the available SoySNP50K Infinium BeadChip data and selected polymorphic SNPs based on their locations and sequences, followed by KASP marker assay development. This strategy provided us with more markers and flexibility over the genotyping process. Ninety-one SNPs were identified for Blackhawk and PI 594891 in a 2.9 Mbp-genomic region, with an average of three SNPs per 100 kb. For Blackhawk and PI 594774, the frequency is much lower, with one SNP per 100 kb. With a total of 30 SNPs being developed as KASP marker assays and nearly 4,000 samples being screened, KASP technology became the method of choice for this study. The small reaction volume (4 μL) and simplicity of PCR implementation and data acquisition made the entire genotyping process affordable, fast, and accurate. In addition, use of seed chipping saved us a lot of time, space, and labor that would have been needed for planting the entire F 2: 3 populations. It also increased the number of F 2: 3 seeds that can be phenotypically screened and enhanced the probability of finding lines with recombination events. Altogether, the combination of high SNP marker density, seed chipping method, and an inexpensive and fast genotyping platform made our fine-mapping study successful in a reduced period of time. From this study, Rcs(PI 594891) was fine-mapped to a 72.6 kb interval based on the opposite phenotype of plants carrying a same recombination breakpoint. The FLS resistance gene(s) in PI 594774 were found to be not located within a 3.3 Mbp region bordered by Satt663 and Satt114 in the previous study, but in a 540 kb region inside the 2.9 Mbp region containing the FLS resistance gene(s) in PI 594891 [21]. In the FLS-594774 population, fewer (103 plants) F 2: 3 plants were identified when compared to the FLS-594891 population. This is due to many factors including the limited number of polymorphic SSR markers and the low recombination frequency in this region. Six plants carrying interesting crossovers were identified and used to narrow down the region containing Rcs(PI 594774) to a region of 540 kb. Because this region overlaps with the 72.6 kb region found for PI 594891, all of the candidate genes in the 72.6 kb regions were characterized for both PIs. Sequencing and qPCR data of these candidate genes in PI 594774 suggested that three genes in this region were more highly expressed in PI 594774 than in Blackhawk following infection with C. sojina race 8 isolates, and that one of the genes contained a deleterious mutation. Continuing the fine-mapping effort with larger populations and more advanced generation materials may be implemented to further narrow down the 540 kb region found in this study for Rcs(PI 594774). Extensive sequencing is also needed to provide more SNPs for better resolution of recombination events if this is the method of choice. Functional characterization of these gene(s) in the two PIs (silencing or complementation) is another feasible approach to verify the true cause of the FLS resistance carried in these two PIs. In this study, RT-PCR was conducted to measure the expression of five candidate genes from the fine-mapped genomic region in PI 594891 at four time points over two weeks which is the necessary time for the lesions to be seen. However, the data indicated that only at three days after inoculation, the expression of these five genes differed between the three genotypes. This suggests that the period from 0-3 days after inoculation is a critical phase to investigate the expression level of candidate genes that control the FLS resistance in soybean. This is different from the pattern of candidate gene(s) in response to Cercospora zeae-maydis or Cercospora beticola causing leaf spot diseases in maize and sugar beet, respectively. When maize and sugar beet were inoculated with the two aforementioned Cercospora species, resistance-related genes showed the most difference in their expression levels at 7-15 dai [42,43]. Sequencing the 72.6 kb region between Blackhawk and PI 594891 may provide the most accurate information about the number of genes present in this region. The difference in the SNP and expression profiles of the five candidate genes residing in the 72.6 kb fine-mapped interval were different between two PIs, suggesting that the FLS resistance might be controlled by two different gene(s) residing in the fine-mapped regions or different alleles of the same gene. In addition, we have to take into account that the FLS resistance gene in PI 594774 may reside outside of the 72.6 kb interval. Additional phenotypic data on these lines in a study where they were compared to 45 additional soybean lines and cultivars using a combination of FLS isolates 21 and 23 indicated a difference in phenotypic reactions which fortified this hypothesis. PI 594774 had an immune reaction, while PI 594891 had resistance-type lesions (S3 Table). Haplotype analysis also indicated that these two PIs may have different resistance alleles. In the haplotype window, both PIs had a unique haplotype (55% similarity), which was also different from the haplotypes of all other tested cultivars and elite lines, suggesting that PI 594774 may carry a different resistance allele than PI 594891. The haplotype of Davis was unique from those of PI 594774, PI 594891, and the susceptible parent, Blackhawk, but identical to four cultivars (Young, Cook, Doles, and N6201) based on 30 polymorphic SNP markers in the defined haplotype window. Young was developed by the USDA-ARS in North Carolina, and is a cross of Davis x 'Essex' [41]. Cook and Doles were both developed at the Univ. of Georgia and are derived from the crosses of 'Braxton' x Young and D74-7741 x Young, respectively. Cook is resistant to the common races of C. sojina and Doles is resistant to all known races in the U.S. [42,43]. N6201 was also released by USDA-ARS in North Carolina and is a cross of 'Nakasennari' x Young. It is also resistant to C. sojina [44]. Based on this haplotype analysis, whether intentional or accidental, the Rcs3 gene identified in the cultivar Davis may also reside in these four cultivars. With such large haplotype allele variation, it could be inferred that the other lines that are resistant to C. sojina could carry genes different than Rcs3 and resistance alleles from PI 594774 and PI 594891. Further studies are planned to identify possible additional new resistance sources. The region on chr13 surrounding Satt114 marker is a resistance gene-rich region. The resistance gene Rsp8 conditioning the resistance to P. sojae isolate OH25 was positioned between the markers Satt425 and Satt114 on chr13 (Satt114 was found to be significantly associated with FLS resistance in PI 594891 and PI 594774) [45]. Aside from Rps8, other R-genes that have been mapped to chr13 near Satt114 include Rpa1, Rsv1, Rag2, rag4, and Rag5. Rpa1 confers race-specific resistance to Pythium damping-off caused by Pythium aphanidermatum in 'Archer' soybean, Rsv1 confers strain-specific resistance to soybean mosaic virus, and Rag2, rag4, and Rag5 confer biotype-specific resistance to soybean aphid (Aphis glycines) [46][47][48][49]. A fine-mapping of the Rag2 gene from PI 200538 on chr13 showed that the Rag2 gene(s) lies within a 54 kb interval containing seven genes one of which is a nucleotide-binding site-leucine-rich repeat gene. However, similar to other fine-mapping studies of disease resistance genes in soybean, this study did not further characterize all of the candidate genes within the fine-mapped region [37]. The fine-mapping study presented here is the first that fine-mapped as well as characterized the candidate genes within the fine-mapped interval containing the resistance genes. In five candidate genes annotated based on the Williams 82 DNA sequence from the 72.6 kb genomic region fine-mapped for the population FLS-594891, there were no prominent SNPs or mutations that may be linked to the FLS resistance in PI 594891. In addition, the expression level of the three aforementioned genes in PI 594891 was higher but not statistically different compared to that in Blackhawk. In this case, further study needs to be conducted to validate and pinpoint a true underlying gene that controls the resistance to FLS in PI 594891. As there were SNPs identified in Glyma13g25320, Glyma13g25340 (LRR), and Glyma13g25350 (transducin); and these three genes had higher expression levels in the two PIs compared to Blackhawk, a resistance mechanism via a combined action of multiple genes, as is the case at the Rhg1 complex locus, is possible [50]. On the other hand, if only one gene is responsible for the resistance, Glyma13g25350 (transducin or Pleiotropic regulatory gene based on the prediction in the Phytozome.net) should be the first gene of interest. In PI 594774, this gene has a mutation that resulted in an amino acid change predicted to be deleterious for the protein function. PI 594774 and PI 594891 also had two common SNPs in the promoter region of this gene. Additionally, members of this class of the gene were reported to be highly expressed as a defense mechanism against attacking pathogens, such as in potato infected with P. infestans and in Arabidopsis challenged with C. higginsianum [51][52][53][54][55]. A pleiotropic regulatory gene (PLRG) was reported to be one of the three most important components of a protein complex that is essential for plant innate immunity [55]. In Arabidopsis, PLRG is a WD40 repeat protein shown to bind directly to an atypical R2R3 Myb transcription factor and together with a nuclear protein they make up the spliceosome-associated PRP Nineteen Complex (NTC) [55]. A mutant plrg Arabidopsis line had an increase in growth of Pseudomonas syringae pv maculicola up to more than 50 times compared to wild-type Col-0 control, indicating the role of this protein in the defense mechanism against plant pathogens [55]. The lack of deleterious SNPs in candidate genes in PI 594891 may hint at other possibilities such as the presence of a causal element residing outside of the sequenced regions of these five annotated genes or the effects of epigenetics. Nevertheless, a fine-mapped region of 72.6 kb would make it more feasible for future studies to enhance our knowledge about the mechanism of the resistance to C. sojina in soybean. Conclusions This study has demonstrated a novel approach to fine-map genomic regions containing genes of interest using the SoySNP50K Infinium chip and KASP technology. Five annotated candidate genes were narrowed down, from which the SNPs that caused amino acid changes have been identified. Based on phenotype, genotype and haplotype analysis results, two soybean accessions might carry different resistance alleles of the same or different gene(s). The SNPs were used to develop KASP assays to detect the resistance alleles on chromosome 13 from the two PIs for marker-assisted selection in breeding programs. Supporting Information S1

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Growth, Nodulation and Yield Response of Cowpea to Phosphorus Fertilizer Application in Ghana Phosphorus is a major limiting nutrient in soils in Ghana. Selection of cowpea varieties that produce good seed yield under low soil phosphorus or those with high phosphorus response efficiency can be a low input approach in solving this problem in Ghana. Two seasons experiments were conducted to evaluate influence of phosphorus (P) fertilizer on growth, nodulation and yield in cowpea. The experiment comprised 12 treatment combinations of 3 cowpea varieties and 4 levels of triple super phosphate (46% P 2 O 5 ) laid out as a factorial in RCBD with four replications. The cowpea varieties were Asetenapa (IT81D-1951), Asomdwee (IT94K-410-2) and IT89KD-347-57 and levels of P were 0, 20, 40 and 60 kg ha G 1 P 2 O 5 . In the present study, Asomdwee and IT89KD-347-57 recorded the highest and lowest crop growth of 7.88 and 2.02 g m G 2 day G 1 at 45 and 60 days after planting, respectively. Growth rate was not consistent with P application; however, application rate of 60 kg ha G 1 P 2 O 5 yielded the least growth rate in the entire study period except for 60 days after planting in the minor season. Statistically, Asetenapa and Asomdwee recorded similar number, effectiveness and dry weight of nodules and were significantly different from that of IT89KD-347-57 in both seasons. Number, effectiveness and dry weight of nodules in all varieties were directly proportional to rates of P fertilizer application in both seasons. Asomdwee produced the highest seed yield of 1557.00 and 1415.00 kg ha G 1 for major and minor seasons, respectively. The rate of P fertilizer application was directly proportional to the seed yield in all three cowpea varieties. The highest seed yield of 1682.00 and 1476.00 kg ha G 1 for major and minor seasons, respectively was produced at 60 kg ha G 1 P 2 O 5 application. Farmers are, therefore, encouraged to use P fertilizer in cowpea production in Ghana. INTRODUCTION Cowpea (Vigna unguiculata (L.) Walp) yield in Africa, particularly Ghana, is estimated to be 45% of that of developed countries (IITA., 2003). Among the factors responsible for such low yield is edaphic factor (soil physiochemical characteristics) particularly phosphorus (P) deficiency which is the most limiting soil fertility factor for cowpea production 1 www.ansinet.com | Volume | Issue | 2015 | J. Agron., 2015(IITA., 2003. This occurs as a result of either inherent low levels of P in the soils or depletion of the nutrient through cultivation. Phosphorus is among the most needed elements for crop production in many tropical soils. However, many tropical soils are inherently deficient in P (Osodeke, 2005) and nitrogen (Haruna and Aliyu, 2011). The deficiency can be so acute in some soils of the Savannah zone of Western Africa resulting in cessation of plant growth as soon as the P stored in the seed is exhausted (Mokwunye and Bationo, 2002). Cowpea does not require too much nitrogen fertilizer because it fixes its own nitrogen from the air using the nodules in its roots. However, in areas where soils are poor in nitrogen, there is a need to apply a small quantity of about 15 kg of nitrogen as a starter dose for a good crop. If too much nitrogen fertilizer is used, the plant will grow luxuriantly with poor grain yield. Cowpea requires more phosphorus than nitrogen in the form of single super phosphate or SUPA (Nkaa et al., 2014). Phosphorus plays key roles in many plant processes such as energy metabolism, nitrogen fixation, synthesis of nucleic acids and membranes, photosynthesis, respiration and enzyme regulation. Phosphorus is critical to cowpea yield because it is reported to stimulate growth, initiate nodule formation as well as influence the efficiency of the rhizobium legume symbiosis (Nkaa et al., 2014). It is required in large quantities in young cells such as shoot and root tips to increase metabolism and promote rapid cell division. It also aids in flower initiation, seed and fruit development (Ndakidemi and Dakora, 2007). According to Oti et al. (2004), phosphorus decrease zinc concentration in the cowpea grain, thereby affecting its nutritional quality. It is required for the physiological processes of protein synthesis and energy transfer in plants (Nkaa et al., 2014). Application of phosphorus has been reported by several authors to improve yield of cowpea. Seed yield is, therefore, governed by number of factors which have a direct or indirect impact. Among these factors are yield components such as number of pods per plant, number of seeds per pod and 100-seed weight over a given land area (Cobbinah et al., 2011). Attempts to improve cowpea production should be approached via a good understanding and manipulation of crops and their environment (Willey, 1979). This may be achieved by a compatible management of agronomic/cultural practices such as mineral, particularly P fertilizer management strategies. On this account, this study was undertaken to determine the effect of phosphorus fertilizer on growth, nodulation and yield of three varieties of cowpea in Ghana. MATERIALS AND METHODS Experimental site: The experiment was conducted at the Kwame Nkrumah University of Science and Technology (KNUST), Plantation Section of the Crop and Soil Sciences Department, Kumasi from June to August (major cropping season) and repeated from October to December (minor cropping season). The soil at the experimental site was well drained, sandy loam overlying reddish-brown and gravelly light clay. It belongs to the Kumasi series, Ferric Acrisol developed over deeply weathered granite rocks (Asiamah, 1998). The total rainfall recorded for the major and minor cropping seasons were 431.5 and 282.8 mm, respectively and the mean daily temperatures recorded were 25.05 and 27.24°C for the major and minor seasons respectively. Experimental design and treatment details: The experiment comprised 12 treatment combinations of 3 cowpea varieties and 4 levels of P fertilizer using triple super phosphate (46% P 2 O 5 ). The cowpea varieties were Asetenapa (IT81D-1951), Asomdwee (IT94K-410-2) and IT89KD-347-57 and levels of P were 0 (Control), 20, 40 and 60 kg haG 1 P 2 O 5 . The treatments combination was factorial RCBD with four replications. A total of 48 plots were used, each measuring 3×5 m with 1 m between blocks and 0.5 m between plots within a block and 1 m between replications. The land was cleared, ploughed, harrowed and leveled using cutlass, rake and hoe. Sowing was done by dibbling 3 seeds per hole at 0.60 m within row and 0.20 m between row and seedling were later thinned to two per stand 14 Days After Planting (DAP) at density of 240 plants per plot corresponding to 166,666 plants haG 1 . All agronomic practices were carried out accordingly. The fertilizer was applied 21 DAP by side placement method. Data collection Laboratory analysis of soil: The organic carbon was determined using modified Walkley and Black wet oxidation method. The percent organic carbon was multiplied by 1.724 (Van Bemmelen factor) to get percent organic matter. Soil pH was determined by the use of a pH meter. The modified Kjeldahl method was used to determine total nitrogen. Available phosphorus was determined by the Bray-1 test method with dilute acid fluoride as the extractant. The exchangeable base cations were extracted using ammonium acetate at pH 7.0. Calcium and magnesium were determined using the Ethylene Diamine Tetra-acetic Acid (EDTA) titration method while potassium and sodium were determined by the flame photometer method. Plant height, Growth, Nodulation and days to 50% flowering: The plant height was measured from the ground level to the highest tip of the stem at 45 Days After Planting (DAP) for the five plants tagged. The average plant height was calculated for each treatment. Crop Growth Rate (CGR) was calculated using the equation described by Radford (1967) where, W 1 and W 2 are total dry weight (above ground) at sampling periods T 1 and T 2 , respectively. Nodulation was done at 45 DAP. The plants to be uprooted were watered up to saturation point. The plants were then uprooted with the help of a dibber and the root system washed gently in clean standing water. The nodules were then separated and counted per plant. Nodules were cut opened to determine apparent effectiveness, using a razor blade and hand lens. Nodules with pink or reddish colour were considered effective and fixing nitrogen, while those with green or colourless were identified as ineffective nodules. Nodules (effective and ineffective) per plot were kept in labeled envelops and oven dried at 80°C for 48 h. Average dry weight of nodules per plant was computed and expressed in grams. The number of days taken from planting to 50% flowering of the plants was recorded as days to 50% flowering. Yield and yield components: Harvesting was done at physiological maturity when about 85% of pods had turned brown and more than 75% of leaves had senescenced. It was done within one square meter area of plants from the central rows on each plot. Five plants were taken from each plot (harvested area). All the pods were counted and the average number of pod per plant calculated. The number of seeds per pod was determined by taking five randomly selected plants from each plot. Pods were shelled, seeds counted and the average number of seeds per pod for each plot calculated. Average seed weight was determined by randomly counting 100 seeds from the threshed and oven dried. These were weighed to represent the 100-seed weight. Seed yield per hectare was determined by threshing the harvested plants from the central one square meter of each plot. These were put in labeled envelopes, oven dried at 80°C for 48 h and then weighed. The resulting weights, in grams per meter square, were then extrapolated to kilogram per hectare basis to get the average seed yield per hectare. Statistical analysis: All the crop data collected was subjected to analysis of variance (ANOVA) and where the F-values were found to be significant, the treatment means were separated by Least Significant Difference (LSD) at 5% probability level using Duncan's Multiple Range Test (DMRT). RESULTS AND DISCUSSION Soil analysis: The soils was slightly acid sandy loams with low available P, total N, exchangeable cations and very low organic matter (Table 1). The available P varied between 5.65 and 5.22 mg kgG 1 , which are lower than 7.0 mg kgG 1 established by Aune and Lai (1995) as the critical soil available P level required for proper growth and development of cowpea. The soil analysis indicates that the soil was depleted. The sites have been used for arable crop cultivation whose features-regular mechanized tillage and inorganic fertilizer application practices-have been indicted for aiding soil degradation through rapid soil organic matter depletion. Soil organic matter influences physical properties that relate to water absorption, available water content and nutrient retention (Ayodele and Oso, 2014). Therefore, soil management practices must emphasize raising the level of soil organic matter and preventing its rapid depletion. Plant height: Plant height was affected by cowpea variety at the various growth periods and seasons. Asomdwee was consistently the tallest, followed by Asetenapa and IT89KD-347-57 at all sampling periods (Fig. 1). The differences in plant height could be attributed to genetic effect of individual varieties (Magani and Kuchinda, 2009). That notwithstanding, treatment effect of 20 and 40 kg haG 1 P 2 O 5 on plant height was higher than the control during the major growing season, with treatment 20 kg haG 1 P 2 O 5 recording the highest significant value; thus, the optimal rate for greater plant height. This result is in conformity to the results observed by Nkaa et al. (2014). This could be attributed to the fact that phosphorus is required in large quantities in shoot and root tips where metabolism is high and cell division is rapid (Ndakidemi and Dakora, 2007). Thus, an indication that the cowpea varieties utilized the phosphorus fertilizer applied judiciously in growth and development processes. This is, however, contrary to observation made by Sharma et al. (2002), which states that P has no significant effect on plant height. However, application of P at 60 kg haG 1 P 2 O 5 resulted in low plant height in the minor season. This could be due to over saturation of P fertilizer in the soil making the soil nutrients immobile because of inadequate water in the soil. Crop growth rate: Higher CGR values were recorded for Asomdwee and IT89KD-347-57 in the major season but were not consistent in the minor season (Table 2). This indicates that among varieties, Asomdwee and IT89KD-347-57 produced more dry matter per unit ground area than Asetenapa in the major season. This variation could be due to genotypic make-up and growing conditions indicating different growth potential (Ankomah et al., 1996). The reduction in CGR between sampling periods contrasts the report of Cobbinah et al. (2011) that UCC-Early variety of cowpea increased from the initial sampling (30 DAP) stage to the final sampling stage (51 DAP). Nodulation: Cowpea variety significantly (p<0.05) influenced number of effective nodules per plant for both seasons (Table 3). Asomdwee recorded the highest (82.9) effective nodules per plant followed by Asetenapa (80.7) and IT89KD-347-57 (45.6) for the major season but during the minor season, Asetenapa recorded 86.7 followed by Asomdwee 86.6 and IT89KD-347-57 (59.9) effective nodules per plant (Table 3). Highest value for nodule weight was also observed in Asomdwee. However, the significant variation in nodulation per varieties could be attributed to difference in the genetic makeup of the individual varieties (Ayodele and Oso, 2014). Number of nodules, number of effective nodules per plant and dry weight of nodules per plant was significantly (p<0.05) influenced by phosphorus fertilizer application for both seasons (Table 3). Significant increase in nodulation as influenced by P application was also observed by Mokwunye and Bationo (2002) and Agboola and Obigbesan (1977). The result of the nodule dry weight in this study agrees with the report of Nkaa et al. (2014), which stated that increasing P levels increased the number and size of nodules. These observations are quite true because phosphorus initiates nodule formation as well as influence the efficiency of the rhizobium-legume symbiosis thereby enhancing nitrogen fixation (Nkaa et al., 2014). Days to 50% flowering did not differ significantly (p>0.05) with cowpea variety in both seasons. This could be as a result of genetic similarities in the varieties used in the present study. Asetenapa in both seasons flowered earlier followed by Asomdwee and IT89KD-347-57. All the varieties used in the present study produced flowers between 41-47 days after planting (Table 4). Cobbinah et al. (2011) reported that 98.9% of the accessions evaluated took between 31-49 days after planting to attain 50% flowering and this compares favourably with the result in this study. However, the interaction of variety and phosphorus fertilizer application was not significant (p>0.05) on days to 50% flowering at the different planting seasons. Phosphorus fertilizer application reduced days to 50% flowering significantly (p<0.05) in both seasons when 0, 20, 40 and 60 kg haG 1 P 2 O 5 were applied, resulting in 46-47, 45-46, 42 and 42 days to 50% flowering, respectively (Table 4). According to Nkaa et al. (2014), P fertilizer application shortens the time from planting of cowpea to harvesting of green pods and hastened maturity. This report compares favourably with the results in the present study. Also, this enhancement of growth by P application induced early flowering but the effect was no longer improved below 60 kg haG 1 P 2 O 5 rate. Plants that flower earlier would utilize G 1 available soil water and nutrients for podding, seed set and sustaining the pods to maturity before the dry season when water stress could be very severe. This is particularly so given the irregular nature of rainfall in the late cropping season and unpredictable outset of the harmattan whose desiccating effect on atmospheric humidity (mist and fog) of the mornings is vital to the yield of cowpea planted in the minor cropping season (Ayodele and Oso, 2014). Therefore, lower days to 50% flowering recorded in the minor season could be attributed to the moisture stress experienced during the growing period (Refay, 2009). 42.4 b 41.6 b Values within the treatment group in the same column followed by same superscript (s) are not significantly different at (p#0.05) according to DMRT Yield and yield components: Seed yield and yield components varied significantly (p<0.05) among the varieties used. Similar observation was made on effects of P rates (Table 5 and Fig. 2). In the present study, Asomdwee recorded the highest seed yield of 1557.00 kg haG 1 (major season) and 1415.00 kg haG 1 (minor season) followed by Asetenapa (Fig. 2). In the major season, seed yield recorded by Asomdwee was significantly higher than that of IT89KD-347-57 variety only. However, in the minor season, seed yield produced by the Asomdwee variety was significantly higher than those of the other varieties. Asetenapa and IT89KD-347-57 were not consistent in seed yield (Fig. 2). Yield data available in the Council for Scientific and Industrial Research-Crop Research Institute (CSIR/CRI., 2012) ranked Asomdwee as the highest among the three varieties and this confirms the report in the present study. However, earlier studies conducted by several researchers revealed varietal differences in the seed yield of cowpea (Sanginga et al., 2000;Nirmal et al., 2001) and this accounted for the varietal variations in yield in this study. Phosphorus fertilizer application significantly (p<0.05) influenced seed yield in both seasons. Seed yield increased with increased application of P fertilizer throughout the experiments with highest yield (1682.00 kg haG 1 for major season and 1476.00 kg haG 1 for minor season) recorded at application rate of 60 kg haG 1 P 2 O 5 (Fig. 2). This is in conformity with findings of Singh et al. (2011), who reported highest yield at 60 kg haG 1 and suggested that that may be the optimum as further application of phosphorus may or may not increase yield of cowpea. This shows that P treatments support significantly higher seed yield than the control treatment. However, this report contradicts the findings of Haruna and Usman (2013) and Nkaa et al. (2014), who reported highest yield at 30 and 40 kg haG 1 , respectively in their experiments. The significant response of the measured yield characters of cowpea to phosphorus application could be attributed to the role of phosphorus in seed formation and grain filling (Haruna and Usman, 2013). Seed yield is governed by number of factors, which have a direct or indirect impact. Among them are yield components such as number of pods per plant, number of seeds per pod and 100-seed weight over a given land area (Ayodele and Oso, 2014). A good seed yield will require varieties with short flowering periods to enable them divert energy into pod and seed development. Nkaa et al. (2014) stated that the earlier a variety sets flowers, the earlier it matured. Therefore, the varieties used in the present study would be very useful in dry environments because of their ability to escape drought as a result of their early flowing and maturity. Cobbinah et al. (2011) made similar observation upon characterizing cowpea accessions in Ghana and it compares favourably with the result in the present study. Number of pods per plant was significantly higher in Asomdwee (22.2 for major season and 21.6 for minor season) than in Asetenapa and IT89KD-347-57 (Table 5). Number of pods per plant was directly proportional to P fertilizer application rates, with the control treatment producing the least number of pods while the maximum pod number was achieved by the application of 60 kg haG 1 P 2 O 5 . This compares favourably with reports by other researchers (Haruna and Usman, 2013;Ndor et al., 2012;Singh et al., 2011), who also discovered significant increase in pod number of cowpea in response to phosphorus application. However, Agboola and Obigbesan (1977) reported that phosphorus application did not significantly increase cowpea yield but rather enhanced nodulation and phosphorus content of leaf and stem. IT89KD-347-57 consistently produced the maximum number of seeds per pod followed by Asetenapa and Asomdwee but the latter two varieties were not consistent. The number of seeds per pod could be attributed to genetic make-up of the varieties used. The effect of phosphorus fertilizer application rates of 20, 40 and 60 kg haG 1 P 2 O 5 produced statistically similar number of seeds per pod but was different from the control. This indicates that number of seeds per pod of a variety can be modified by soil management practices such as fertilizer application. One of the important yield component in cowpea is 100-seed weight. Asetenapa and Asomdwee varieties produced similar 100-seed weight but significantly higher than IT89KD-347-57. Phosphorus fertilizer application rates of 40 and 60 kg haG 1 P 2 O 5 produced statistically similar 100-seed weight but different from 0 and 20 kg haG 1 P 2 O 5 . The effect of 20 kg haG 1 P 2 O 5 was also different from the control treatment. According to Cobbinah et al. (2011), the variation in 100-seed weight between major and minor season could be as a result of variation in weather conditions particularly rainfall. Difference in yield and yield components among varieties and P fertilizer application rates could be attributed to differences in nodulation parameters such as number and dry weight of nodules and effective nodules. Zahran (1999) reported that nodulation, nitrogen fixation and specific nodule activity are related to the P supply. His result corroborates the result in this work. Also, according to Padi and Marfo (2005), rate and time of specific developmental processes conditioned by location-specific conditions of temperature, rainfall and soil factors determine the final seed yield and its components. CONCLUSION The results of this study revealed that the varieties (Asetenapa, Asomdwee and IT89KD-347-57) had significantly different growth and yield components. This indicates that cowpea varieties have unequal growth potential which ultimately influenced yield and yield components. Asomdwee produced superior seed yield of 1118 and 1165 kg haG 1 for major and minor seasons respectively. It is, therefore, recommended for soils with low P status. However, IT89KD-347-57 recorded significantly least values in plant height, nodulation and seed yield. It is therefore, not recommended for commercial purposes. It could, however, be used as a bedrock for further improvement studies since it has a good growth rate. The observed variations in the performance of the cowpea varieties used could provide a basis for selecting cowpea lines with greater agronomic efficiency in phosphorus deficient soil to reduce fertilizer cost. It could also be used in an initial screening of large number of breeder lines. Asomdwee could be suitable for a range of soil phosphorus conditions as well as recommended to farmers on large scale production. The study also indicates that application of P fertilizer significantly improved yield and yield components but contrary was observed for growth. Phosphorus fertilizer significantly increased vegetative growth, nodulation and its resultant seed yield. Seed yield response to P application rates was a reflection of the increase in vegetative growth and nodulation from which optimum rate obtained was 60 kg haG 1 P 2 O 5 . It could, therefore, be concluded that Asomdwee with phosphorus application rate of 60 kg haG 1 is ideal for soils low in phosphorus and is, thus, recommended for farmers in such regions for enhancement of cowpea yield.

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Effect of Cilembu Sweet Potato Starch and Storage Times on Physicochemical and Microbiology of Synbiotic Yoghurt Ice Cream The purpose of this research was to determine the effect of adding cilembu sweet potato starch (1-6 %) and storage time (2, 4, 6 weeks) on physicochemical and microbiology of synbiotic yoghurt ice cream. The research methodology used was experiment laboratory. The first experiment was designed by Completely Randomized Design (CRD) using 7 treatments and 4 replications, while second step was designed by CRD using 4 treatments and 4 replications. Data were analized by Analysis of Variance (ANOVA) and continued by Duncan's Multiple Range Test (DMRT). Determination of the best treatment used Effectiveness Index, which has been modified. The result of first step showed the best treatment is the addition of cilembu sweet potato starch 3% with value 5.400 log10 cfu.mL-1 of total LAB, 4.322 of pH, 0.200 g.L-1 of EPS, 40.257 P of viscosity, 35.523 minutes.50g-1 of melting rate, 30.258% of overrun. For second step, the best treatment is 6 weeks stored with value 5.004 log10cfu.mL-1 of total LAB, 4.347 of pH, 0.327 g.L-1 of EPS, 114.928 P of viscosity, 48.828 minutes.50g-1 of melting rate. INTRODUCTION Yoghurt is produced from fermented milk with the addition of starter. Yoghurt starter consist of Lactic Acid Bacteria (LAB), may include Lactobacillus bulgaricus, Streptococcus thermophilus [1] and Lactobacillus acidophilus. During the fermentation process, LAB will hydrolyze lactose into its constituent compounds, glucose and galactose [2]. Metabolism of sugars will produce energy and organic acids, such as lactic acid, citric acid and others. The acid formation will cause a decrease in pH [3]. Addition of cilembu sweet potato starch can increase total LAB. The increase in total LAB will cause a decrease of pH due to a growing number of lactose-hydrolyzed LAB become acidic. The growth of pathogenic bacteria is inhibited due to the acids formed. Yoghurt can be processed into yoghurt ice cream that can be modified by the addition of cilembu sweet potato starch as a prebiotic, which is known as the synbiotic yoghurt ice cream. Synbiotic ice cream is a product that should be stored in freezing conditions, while the viability of LAB will be decreased when stored at freezing temperatures. Thus, it should be added with cryoprotectant to minimize the loss of viability of LAB. One of the cryoprotectant that can be used is cilembu sweet potato starch. Cilembu sweet potato starch contains oligosaccharides which were not digestable to the human intestine [4]. Cilembu sweet potato starch has high oligosaccharides at ±0.272%, while purple sweet potato at ±0.126% and white sweet potato at ±0.099% [5]. Sweet potato`s oligosaccharides as a prebiotic potential, so it could support the growth of Lactobacillus [6]. Streptococcus thermophilus started the fermentation process by breaks down the lactose into glucose and galactose. Lactobacillus bulgaricus will change monosaccharide into lactic acid. Lactic acid bacteria are capable of producing exopolysaccharide (EPS). Exopolysaccharide is a polysaccharide that excreted by bacteria, fungi, or algae and found on the outside of the cell wall. Exopolysaccharide composed of two polymers, heteropolysaccharide and homopolysaccharide. Heteropolysaccharide is a polysaccharide composed by several types of monosaccharides. Homopolysaccharideis a polysaccharide composed of a single type of monosaccharides [7]. Exopolysaccharide of LAB is heteropolysaccharides with straight and branched chain repeating units of tetra-heptasaccharides [8]. Starch composed of amylopectin molecules have a high water absorption capability [9] (Dewi et al.) it can increase the viscosity of the synbiotic yogurt ice cream. Synbiotic yogurt ice cream will have a low melting rate when the viscosity was high. Branching chains of amylopectin molecules OE o š šZOE}µPZ r-1.6 group that really solid [10], so its lead to decrease of overrun due to current processing and freezing in the ice cream production, the air is hard to get into the ice cream mixed (ICM). Based on this, it is necessary to make a research about the addition of cilembu sweet potato starch 1-6% (v.w -1 ) and 2-6 weeks frozen storage (-18°C). Previous research suggests that the addition of cilembu starch as much as 1-3%, with a concentration of 3% gave the best results against the total of LAB. Thus, further assumption that the addition of 3% is a maximum point for bacterial growth, thus it is necessary to increase the concentration of cilembu starch. The addition of cilembu starch limited to 6% related to the texture of synbiotic ice cream. The maximum value of total solids in ice cream is 42%. Storage for 2-6 weeks conducted to determine the quality of synbiotic ice cream, whether LAB is able to survive after being kept frozen for up to 6 weeks, and determine the decrease of LAB. Every stage of the research tested a total LAB, pH, EPS production, microstructure, viscosity, melting rate and overrun. MATERIALS AND METHODS The materials consisted of yogurt (fresh cow's milk was derived from the Mitra Bhakti Makmur Cooperative, Junrejo-Malang. Starter derived from the House of Yoghurt Junrejo-Malang. Other ingredients are cilembu sweet potato starch (sweet potato from Karangploso, Batu), sugar, full cream milk powder (Dancow brands), and emulsifiers (Quick brands). Starch Isolation Procedures Sweet potatoes was peeled, shredded, and then mixed with water (ratio 1 kg : 1 liter). Then filtered with deposited for 8-12 hours. The water was separated from the starch sediment. The wet starch dried under the sunlight, then mashed with spoon. Data Analysis The observed variables were total LAB, pH, EPS, microstructure using Scanning Electron Microscopy (SEM), viscosity, melting rate and overrun. Data were analyzed using ANOVA and Duncan's Multiple Range Test. The qualitative data were analyzed descriptively. Determination of the best treatment used Effectiveness Index [11] which has been modified. RESULT AND DISCUSSION The quality of synbiotic yoghurt ice cream in terms of physicochemical properties and microbiology can be seen in the Table 1 and 2. Total LAB ANOVA showed that the addition of cilembu sweet potato starch with different concentration on synbiotic yoghurt ice cream provide highly •]Pv](] vš ](( OE v ~WGìXìí• }( šZ š}š o > X The addition of cilembu sweet potato starch 3% produced highest value of total LAB, i.e. 5.400 log 10 cfu.mL -1 . Enhancement of total LAB occurs on the addition of cilembu sweet potato starch 1-3%. The addition of cilembu sweet potato starch 4-6% decreased the total LAB. The addition of cilembu sweet potato starch 6% produced the lowest total LAB, i.e. 5.291 log 10 cfu.mL -1 . The addition of 6% starch inhibit the activity of LAB compared to the addition of starch 3% with the same number of additional starter (3%.L -1 of milk). It indicates that LAB is less capable of hydrolyzing starch because starch concentration was higher than LAB, causing LAB urgency and decreased activity, conversely to the addition of 3% cilembu starch. Increasing total LAB happened because the addition of cilembu sweet potato starch in yoghurt fermentation that used by LAB to support their activities and growth. Cilembu sweet potato starch have a high oligosaccharides [5]. Oligosaccharides is not digestable by human because the absence of r-galactosidase enzyme. Thus intestinal bacteria ferment it and produce gas, e.g. H 2 and CO 2 [6], therefore oligosaccharides used better as a prebiotic for LAB. Previous studies mentioned that fermented yoghurt enriched with sweet potato produce fast growth speed of LAB and doubling total LAB for 0.5880.h -1 and 6.0985 times (10% orange sweet potato), 0.5589.h -1 and 5.6837 times (10% purple sweet potato), 0.4510.h -1 and 5.5671 times (10% white sweet potato) [12]. From the results of these studies, this research is fairly good because we used orange sweet potato starch as a prebiotic. Analysis of variance showed that different frozen storage times on synbiotic yoghurt ice cream provide highly significant difference ~WGìXìí• }( šZ š}š o > X 'OE}ÁšZ }( > Á]oo be slower under the temperature of 10°C [13]. The higher difference temperature of storage and optimum temperature growth of bacterial, will further slowing the growth of LAB, ultimately there is no growth anymore [14]. The longer storage time in freezing temperatures, the lower of ability of LAB to live, thus the quality of the synbiotic ice cream will decline in terms of the presence of LAB; whereas the fermentation product should contain sufficient LAB that are beneficial for health. One study suggested that the ice cream with the addition of Lactobacillus bulgaricus and Streptococcus thermophillus had average viability of LAB for 22.1x10 8 cells.mL -1 . After being frozen stored for one month, the average viability of LAB decreased into 6.15x10 8 cells.mL -1 [14]. Ice cream contained Lactobacillus acidophilus stored for 5 weeks at a temperature of -29°C, decreased viability of LAB from 1.0x10 8 cfu.mL -1 to 4x10 6 cfu.mL -1 [15]. That study showed that the storage time of ice cream at freezing temperatures can reduce the viability of LAB, because the longer LAB live in freezing temperatures which is not an optimum temperature for LAB growth. Cause of bacteria death due to low temperatures was cell damage. Cell damage arises from changes in osmotic pressure. Crystallization of water will trigger the solution becomes viscous, thereby causing osmotic damage [13]. Crystalline form of taper and large crystal size can cause bacterial death.The formation of ice crystals can destroy bacterial cell walls, thus cell death can occur. The longer storage, the more ice crystal formed so many bacterial cells are damaged and lower the viability. Cells undergoing frozen storage process requires a cryoprotectant to prevent cell damage. Cryoprotectant materials used in the research is a Cilembu sweet potato starch, its use will reduce adverse effects. During the frozen storage decreased in osmotic and the formation of ice crystals will occur around the medium but not inside the cell. Cryoprotectant can be divided into two, penetration and nonpenetration [16]. Sweet potato starch can be categorized as non penetration cryoprotectant because it is not able to penetrate into the cells, but only protects the inside of the bacterial cell membrane, thus stabilizing the membrane against freezing damage. The freezing process can reduce up to 1 log cycle of bacteria [17], but from the results obtained in total LAB does not decline until it reaches 1 log, this indicates that the starch is able to carry out their duties as cryoprotectant. produced the highest value of LAB, 5.400 log 10 cfu.ml -1 . The more number of LAB, the more acid is produced, so that the pH decreased. Lactic acid bacteria are capable of producing pectinolytic and cellulolytic enzymes that can destroy the cell walls of the starch [18], resulting differences in concentration. Lactic acid bacteria have a high concentration after releasing the enzyme and starch had low concentrations after their cell wall destroyed. This causes the LAB could hydrolyze starch and lactose. Lactose hydrolyzed into its constituent compounds, galactose and glucose, whereas raffinose and stachyose (sugar derived from starch) is ZÇ OE}oÇÌ Ç šZ vÌÇu r-galactosidase into galactose, glucose and fructose [2]. The metabolism of sugar in the form of organic acids, such as lactic acid, citric acid, and others which resulted decrease in pH. The addition of prebiotics is able to accelerate the process of acid formation, thus lowering the pH due to high activity and growth of LAB. One study suggested that fermented soy milk ranging from 4, 8, 12, 16 and 20 hours will be decreased pH. From pH control at 6.7 to 4.6 at the end of fermentation, comparable to total lactic acid produced, from 0, 11% to 0.31% [3]. Analysis of variance showed that different frozen storage times on synbiotic yoghurt ice OE u ‰OE}À] v} •]Pv](] vš ](( OE v ~WHìXìñ• of the pH values. In freezing conditions, the metabolism occur in very slow cycle, thus the change in pH that appeared was very small, only 0.003 to 0.025. Change of pH is influenced by the activity of LAB. In this research, decreased of total LAB occurs in small quantities, because of the addition of starch which act as a protective material for LAB. Changes in pH value were very small and the treatment had no effect declared, but the longer of frozen storage, the pH value increased. This corresponds to a decrease in total LAB during frozen storage, thus reshuffle lactose by LAB into lactic acid also decreases and causes increases of pH value. A study shows that the longer of the frozen storage of probiotic ice, then the lactic acid levels tend to decrease. The results showed ice cream with probiotic starter Lactobacillus casei and Bifidobacteriumbifidium (1: 1), the storage time of 10 days resulted in an average of 0.24% lactic acid, 20 days 0.23%, and 30 days 0.21% [14]. Decreased levels of lactic acid will increase the pH value. Results of other studies indicated that goat's milk yoghurt was kept frozen for 2, 4, 6 days had an average pH value of 5.51 (control), 5.94; 5.41; 5.61, respectively [19]. Changes in pH value leads to changes in the concentration of H + ions, so these changes will affect the value of the coefficient of absorption of milk due to the addition of H + ions that causes the density differences [20]. Exopolysaccharide (EPS) Statistical analysis showed that the addition of Cilembu sweet potato starch with different concentration on synbiotic yoghurt ice cream provide higly significant di(( OE v ~WGìXìí• }( the EPS values. The highest EPS produced by the addition of starch 3% (P 3 ), which is 0.200 g.L -1 . Exopolysaccharide production is generally related to the total LAB, because the EPS is produced by LAB. The highest value of total LAB is indicated by P 3 (5.400 log 10 cfu.mL -1 ), thus P 3 showed the highest EPS results. Bacterial growth is divided into four phases, the first phase is lag (phase adaptation), the current phase of the bacteria have to adjust with the environment. Second, a log phase as bacterial growth phase. Third, the stationary phase where growth rate and mortality of bacteria are in a balanced state. Fourth, the death phase when the death rate has exceeded the growth rate of bacteria. EPS production will reach a maximum point when it reaches the stationary phase [21]. One study showed that the average EPS from the fermentation process of yoghurt during 3 days have the highest value than fermentation 1 and 2 days, of which the value was 35.15 mg.L -1 (1 day), 37.25 mg.L -1 (2 days) and 56.05 mg.L -1 (3 days) [22]. Exopolysaccharide degraded because of the low activity of EPS enzymes in the next stage. The fermentation process lasts for only 24 hours in this study, thus the production of EPS has not reached the maximum value. Exopolysaccharide produced in this study was crude EPS, because it is obtained by calculating the weight after drying. Crude EPS at 0 hour was at 157. 33 Production of EPS can be affected by sources ofcarbon and nitrogen, physicochemical of LAB growth, such as temperature, pH, the presence of oxygen levels [24]. Minerals are needed for the synthesis of EPS. One study stated that the production of EPS achieve the best result (475.6 mg.L -1 ) with the addition of 0.5% sodium acetate, while the addition of magnesium sulphate lead to decreased production of EPS, the addition of potassium phosphate and citrate triamonium best produce EPS production at concentrations of 0.2% [25]. Minerals in Cilembu sweet potato such as calcium ±30 mg.100g -1 of sweet potatoes. Mineral is a component that is insoluble in water, thus the mineral of Cilembu sweet potato remain in produced starch even after undergoing a process of soaking. Microorganisms only takes few minerals, the exceeded amount of minerals can inhibit the growth of LAB [25]. The addition of starch 1-3% increase in total LAB and EPS, while 4-6% start resulted in a decrease in the number of LAB and EPS. It can be caused by too much mineral content on the addition of these concentrations. Analysis of variance showed that different frozen storage times on synbiotic yogHurt ice cream provide higly significant difference ~WGìXìí• }( šZ W^ À oµ •X ^š}OE P š]u }( ò weeks resulted in the highest EPS value, 0.327 g.L -1 . EPS production increased from 0.205 g.L -1 (2 weeks); 0.250 g.L -1 (4 weeks); and 0.327 g.L -1 (6 weeks). Formation of EPS is part of a defense of bacteria in bad conditions [26]. Frozen storage causes dormant on bacteria and viability of bacteria can be decreased [27]. The amount of EPS produced and resistance during freezing did not find any correlation [28], and freeze-dried for storage of cells [29]. Exopolysaccharide does not protect the bacteria from low temperatures. After 162 days of storage at temperature -40°C, melting at 4°C and freezing again at -40°C, viability of bacterial cell decreased 1-4% [28]. From the three statements, we can drawn a conclusion that the frozen storage causing bacteria through a phase of dormancy, so the bacterial activity is inhibited and do not affect the production of EPS. Storage temperature at 5°C can cause browning occurs due to the Maillard reaction, which can not be seen clearly using the naked eye [30]. The Maillard reaction is a reaction between carbohydrates, in particular reducing sugar and a primary amine group. Maillard reaction is a nonenzymatic browning reaction [10]. The interaction between protein and carbohydrates can cause discoloration even if it stored at a temperature of 5°C, thus the frozen storage of allow interactions between carbohydrates, although in a slow cycle. Carbohydrates can be derived from the rest of the lactose which has not fermented, oligosaccharides (raffinose and stahyose) of Cilembu sweet potato starch, glucose is added when making the ice cream. The interaction between sugars can form transgalactosidase and increase the production of EPS. Viscosity Analysis of variance showed that the addition of Cilembu sweet potato starch with different concentration on synbiotic yoghurt ice cream provide higly significant difference ~WGìXìí• of the viscosity values. The addition of 6% starch produce the highest viscosity value, i.e. 103.180 P. Value of viscosity was higher with more starch is added, the highest concentration of starch that been used in this research was 6%; that[• why the viscosity values in this level was high than the other. Starch consists of 18% amylose and 82% amylopectin [10]. Amylopectin of starch has a higher water absorption ability [9], thus increase the viscosity. The increase in viscosity occurs due to gelatinization of Cilembu sweet potato due to the heating process. The starch granules are able to absorb water. The process of water absorption occurs when the kinetic energy of water molecules is stronger than the power of attraction between molecules of starch in the water. The process of water absorption resulting in swelling. The starch granules that already swollen can not be returned as the initial size, thus resulting in increased viscosity [10]. Other constituents of ice cream also contributed to the increase in viscosity, such as a stabilizer. Carboxy Methyl Cellulose (CMC) used in the manufacture of ice cream can increase the viscosity (2.13%). It is because CMC dispersed in the liquid phase capable of binding large amounts of water and forms a gel that blocks the water framework to freedom of movement, so that the state the solution becomes more stable due to increased viscosity [31]. ANOVA results showed that different of frozen storage times on synbiotic yoghurt ice (Dewi et al.) longer of frozen storage time, the more amount of ice crystals produced, so the buildup of ice crystals will lead to increase in viscosity. Ice cream has an unstable crystal and during frozen storage, the crystal will change either in the number, size or shape, so it is called recrystallization. Recrystallization can occur naturally at a constant temperature, but most commonly affected by temperature fluctuations. Recrystallization can occur at temperature -5°C. One study showed that ice crystals can grow in size at a temperature of -5°C and kept for 5 days, (OE}u ðñ …u š} ííì …uU šZ o OEP •š ] OEÇ•š o• (}µv À v OE Z îìì …u [33]. The water is converted into ice could increase the volume until 9%, the increased volume due to lower temperature. Food products that contain a lot of water, there will be the same, but the water content, the annealing temperature and the presence of space between cells greatly affect the volume changes [34]. Increased viscosity of yoghurt can be affected by the bacterial strain producing EPS [35]. Some types of LAB were able to produce EPS are Lactobacillus acidophillus, L. casei, and L. plantarum [23]. Exopolysaccharide able to act as a thickener to increase the viscosity. The results showed when the EPS production of 0.200 g.L -1 value of the viscosity of the ice cream is 40.257 P. Exopolysaccharide achieving maximum production at 6 weeks of storage, 0.327 g.L -1 . At the same time the value of the viscosity of the ice cream also reaches the maximum value is 114.928 P. Melting Rate The addition of different concentration Cilembu sweet potato starch on synbiotic yoghurt ice cream showed a higly significant difference ~WGìXìí• of the melting rate values. The melting rate is affected by the total solids contained in the ice cream [36]. Cilembu sweet potato starch is a source of solids [9], the more amount of starch added, the lower speed of melting rate. The melting rate of synbiotic ice cream without the addition of cilembu starch was 26.150 minutes.50g -1 and with the addition of Cilembu starch 6% was 45.050 minutes.50g -1 as the highest value. It happened because of the increased solids led to the freezing point of ice cream down, water holding capacity of ice cream is getting stronger and the movement of free water is reduced, thus much water is trapped. Increasing the number of free water trapped will produce ice cream that slowly melts [37]. One study showed that a combination of ice cream with soy extract 40% and 15% cucumber pulp produces melting rate of 25.89 min [38]. Melting rate of ice cream can be affected by the constituents of ice cream. Ice cream with a high fat content will not be easy to melt [39]. Stabilizers also give effect to the melting rate. Stabilizer dispersed in the liquid phase bind large amounts of water and forms a gel framework which prevents water molecules move freely and formed membranes of ice cream that will protect components from the effects of temperature [31]. Melting rate related to the viscosity of the ice cream. High viscosity will produce ice cream with a low melting rate, consequently generated overrun value can be decreased. Observation on the melting rate of ice cream, indicates that the ice cream with low overrun value has a melting time which tended to last longer. One study showed that an ice cream with combination of the skim milk 0% and 10% sweet potato steamed produce a maximum melting speed that is 8.58 minutes, while the value of overrun was low, 22.2% [40]. Melting rate of ice cream in this study was of 25-45 minutes.50g -1 . Actually, melting rate of ice cream is about 10-15 minutes [36]. It shows that the ice cream produced in this study is good because it has a longer melting time. Different frozen storage times on synbiotic yoghurt ice cream provide high significant ](( OE v ~WGìXìí• }( šZ u oš]vP OE š À oµ •XdZ longer of frozen storage time, the more amount of ice crystals produced. That ice crystal can be changes in the number, shape and size into more numerous and larger during frozen storage causes increased viscosity of the ice cream. Along with increased viscosity, melting rate of ice cream decreases. The results showed that synbiotic yoghurt ice cream without treatments, viscosity value was lower by 40 (Dewi et al.) Melting rate of ice cream can be affected by many factors, including the amount of trapped air, ice crystals and fat globules tissue are formed during freezing [37]. The decrease of temperature of frozen storage, resulting the water will freeze back through the nucleation process (determining the cell nucleus), so the size of the ] OEÇ•š o• Á]oo ]v OE • š} Híìì…u €ð2]. Its shape becomes rough, either on the surface or the interior of ice cream. Glucose can withstand freezing so that ice cream is not easy to melt, especially after storage at low temperature (-18°C) [43]. Synbiotic yoghurt ice cream is made by adding sugar as much as 375 g.L -1 milk. The addition of high sugar was assumed to play a role in the decrease of melting rate. Use of sweeteners such as sugar as much as 25% may increase the viscosity because it is able to lower the water activity [44], thus slowing the melting rate ice cream. Overrun Analysis of variance showed that the addition of Cilembu sweet potato starch with different concentration on synbiotic yoghurt ice cream provide highly significant difference ~WGìXìí• of the overrun values. Value of overrun was lower with more starch is added. Amylopectin on starch is 82% with regard to the group branched chain ríUò €íì•. Thus, it is difficult to penetrate by air when the foaming and freezing process, so the value of overrun decreased. The highest concentration of starch that been used in this research was 6%, that[• why the overrun values in this level was low than the other, which amounted to 10.257%. Starch molecules have a very large number of hydroxyl groups, so it had the higher ability to absorb water and affect the increased of viscosity [10]. Decreasedof overrun occurred along with an increase in viscosity due to increased of adding Cilembu sweet potato starch. Solids are too high in ICM, can increase viscosity, thereby inhibiting the development of ICM and lowered overrun [31]. One study showed that a combination of skimmed milk 0% and 10% sweet potato steamed showed the lowest value of overrun, 22.22%, while the combination of 10% skim milk and sweet potato steamed 0% showed the highest value of overrun, 63.33% [40]. Microstructures Microstructure of synbiotic yogurt ice cream can be seen in Figure 1. The image of ice cream was taken with a magnification of 1000x for the visible presence of EPS. Figure 2d looks has more EPS than other images, according to the results of EPS production of 6 weeks resulted in the highest value, i.e. 0.327 g.L -1 . Figure 1d sample had a high density compared to other images. Figure 1a and 1b were still seen the empty cavities, while Figure 1c empty cavity began to decrease. Density formed in Figure 1d because the longer stored at freezing temperatures, thus the ice crystals in ice cream will be found more. During frozen storage, the ice cream will have two events, i.e. propagation and recrystallization. Propagation is the process when the volume of ice in the ice cream increases, low temperatures cause the size of the ice crystals become larger with frozen water molecules around it. Recrystallization occurs when small ice crystals melt, and then freeze again in a larger size [45]. The ice crystals which formed were more numerous with sizes getting bigger and rough texture, thus increasing the viscosity of ice cream. Viscosity in the sixth week had the highest value, 114.928 P. Viscosity of the ice cream will be increased because of two things, the low temperature and the viscosity of the suspension of solid particles increases as the volume fraction of solid [41]. The freezing process does not affect the metabolism of proteolysis. Proteolysis metabolism is still running [46]. Freezing causes of nonprotein nitrogen (NPN) increased, possibly due to damage cause by ice crystals in the case in matrix and cell bacteria liberate proteolytic enzymes into the media [47]. The formation of ice crystals during freezing cause the dehydration of the protein thus encourages the breakdown of the protein structure. This causes tiny fat droplets interact and form larger granules. Protein becomes denser or interact to form a disulfide bridge around the newly formed fat granules. After the liquefaction process, the protein can not fully bind the water back [48]. CONCLUSION The addition of 1-6% Cilembu sweet potato starch was able to increase viscosity, lowering the melting rate and overrun. The addition of starch 1-3% increased the total LAB, EPS and lowering the pH. The addition of 4-6% starch lowers total LAB, EPS and increase pH. Storage at 2, 4 and 6 weeks increased the pH, EPS, viscosity, melting rate and lowers of total LAB. We recommend increasing the storage time to know the maximum time limit of storage for synbiotic yoghurt ice cream to maintain its quality. We also recommended advancing testing of EPS to prove the truth of produced EPS, because synbiotic yoghurt ice cream has many constituent components.

v3-fos

2018-04-03T01:02:55.506Z

2015-04-15T00:00:00.000Z

27851648

s2

Fuji apple storage time rapid determination method using Vis/NIR spectroscopy Fuji apple storage time rapid determination method using visible/near-infrared (Vis/NIR) spectroscopy was studied in this paper. Vis/NIR diffuse reflection spectroscopy responses to samples were measured for 6 days. Spectroscopy data were processed by stochastic resonance (SR). Principal component analysis (PCA) was utilized to analyze original spectroscopy data and SNR eigen value. Results demonstrated that PCA could not totally discriminate Fuji apples using original spectroscopy data. Signal-to-noise ratio (SNR) spectrum clearly classified all apple samples. PCA using SNR spectrum successfully discriminated apple samples. Therefore, Vis/NIR spectroscopy was effective for Fuji apple storage time rapid discrimination. The proposed method is also promising in condition safety control and management for food and environmental laboratories. Introduction Apples possess various nutrients including vitamins, dietary fibers, amino acids, etc. [1][2][3] In recent years, apple has been one of the most popular fruits among customers for its anti-oxidation, anti-senescence, immunity strengthens, and other beneficial functions. [3][4][5] So apple has been a valuable nutrition resource in human's daily life. However, apple easily deteriorates after postharvest due to its high water content and abundant nutrients. Living cells still conduct normal respiration metabolism by consuming large amount of nutrients. In addition, fungi invasion also plays an important role in apple quality decrease. 6 Therefore, there is a great demand for a novel rapid and nondestructive apple quality discrimination technique. Vis/NIR spectroscopy, a fast-developed nondestructive detecting technique, has been widely applied in medicine, 7,8 chemistry 9,10 and food research, such as meat, 11,12 drinks, 13,14 grains, 15,16 fruits, [17][18][19] etc. Jamshidi et al managed to conduct taste characterization of Valencia oranges using reflectance Vis/ NIR spectroscopy. 20 Magwaza et al applied Vis/NIRS and chemometric analysis to predict fruit defects and postharvest behavior of 'mules Clementine' mandarin fruit. 21 Zhang et al realized subtle bruises identification on fresh jujube based on NIR spectroscopy. 22 So, Vis/NIR spectroscopy technique is frequently used in fruit quality rapid and nondestructive assessment. In this paper, Fuji apple quality discrimination using nondestructive Vis/NIR spectroscopy were performed. Spectroscopy data were recorded and processed by nonlinear SR method. PCA method was used to analyze original spectroscopy data and SNR eigen values. Vis/NIR spectroscopy was effective for Fuji apple storage time rapid discrimination. The proposed method is promising in fruit freshness rapid detection. Results and Discussion Eigen peaks extraction The original Vis/NIR diffuse reflection spectroscopy to apple samples is displayed in Figure 1. The main absorption band ranges from 400 to 1000 nm. The highest absorbance peak appears at about 600 nm and its absorbance reaches near 4000 counts. Eigen peaks corresponding to the wavelengths of 550 nm, 607 nm, 670 nm, 738 nm, 802 nm, and 789 nm are taken for subsequent principal component analysis of apple samples during storage. SNR spectrum analysis result SNR spectrum calculated by SR as a function of noise intensity is displayed in Figure 2. Each curve first rises with increase of stimulating noise intensity and then declines to a stable value. The initial SNR value in each curve ranges between ¡75 dB and ¡65 dB. Different valleys and peaks appear before eigen peak formation. And each SNR curve has its eigen peak at 190 stimulating noise intensity. SNR spectrum clearly discriminates Fuji apples with different storage days. And the maximal SNR value in each curve is taken for subsequent analysis. PCA results PCA results based on original spectroscopy data and SNR eigen value are displayed in Figure 3. As it shown in Figure 3 totally discriminate Fuji apples with different storage time just by original Vis/NIR spectroscopy data. However, the first 2 principal components include a much higher contribution of 95.77% (see Fig. 3(b)). Components from different storage days have no overlaps between each other. All apple samples in different storage days could be successfully discriminated. Results indicate that PCA on SR processed Vis/ NIR spectroscopy data was more effective in Fuji apple storage time rapid discrimination. In this paper, Fuji apple quality discrimination using nondestructive Vis/NIR spectroscopy were performed. Spectroscopy data were recorded and processed by non-linear SR method. PCA was used to analyze original spectroscopy data and SNR eigen values. Vis/NIR spectroscopy was effective for Fuji apple storage time rapid discrimination. Samples Twenty fresh Fuji apples in the same bath with near size, ripeness and color (without any pretreatments or mechanical injury) were selected and purchased from a local supermarket in Hangzhou, China. After transporting to our lab within an hour, all samples were washed with distilled water. After draining, all samples were stored at room temperature in dark place. All experiments were performed under room temperature. Vis/NIR spectroscopy system and measurement Diagram structure of Vis/NIR spectroscopy system is displayed in Figure 4. The system consists of 3 main parts including sampling device (light controller, sampling pedestal, optical fiber and special halogen lamp), detecting device (visible/NIR spectrometer, USB 2000C, America Ocean Photology Company), and desktop. In spectroscopy measurement, sample was fixed in the sampling pedestal by the optical fiber with a dip angle of 75 degrees. One end of the optical fiber was connected with the spectroscopy, while the other end was connected with the special halogen lamp. Sampling pool was a sealed and light-tight cube. By replacing the sampling terminal in the sampling pool, 5 spectroscopy data from different directions could be collected in each sample, which eliminated errors from sampling direction differences. Spectroscopy analysis software (SpectraSuite, American Ocean Photology Company, America) was applied to analyze the data. Samples were analyzed every 2 d. Five samples were randomly taken in each test. All measurements were performed under room temperature. SR SR is a typical nonlinear model and proposed by Benzi for the explanation for Earth climate periodic changes. [23][24][25] The schematic diagram of SR is displayed in Figure 5. SR phenomenon has 3 elements: a bistable system, a coherent input, and a noise source, 23 which can be described as Where x is the position of the Brownian particle, t is the time, A is periodical signal intensity, f is signal frequency. D is external noise intensity. M and D are adjustable parameters, intrinsic noise N .t/, j.t/ is the external noise, and V .x/ is the simplest double-well potential with the constants a and b characterizing the system. Noise intensity is a parameter of SR model. SR model is used as a data processing method in this research. Eq. (1) can be written as The minima of V .x/ are located at § x m , where x m D .a=b/ 1=2 . A potential barrier separates the minima with the height given by DU D a 2 =4b. The barrier top is located at x b D 0. When three elements of SR interact coherently, the potential barrier can be reduced and the Brownian particle may surmount the energy barrier and enter another potential well. 23 The intensity of signals will increase, which makes it possible that the weak signal can be detected from noise background. SNR is the common quantifier for SR and it can be approximately described as 23 Conclusions In this paper, Fuji apple storage time rapid determination method using Vis/NIR spectroscopy was proposed. Vis/NIR spectroscopy responses to Fuji apples were examined during 6 days' storage. Spectroscopy data were processed by SR technique. And PCA method was used to analyze original spectroscopy data and SNR eigen values. The results demonstrated that PCA method could not totally discriminate Fuji apples with different storage time just via original Vis/NIR spectroscopy. However, SNR spectrum calculated by SR showed different maximal values under the same noise intensity. PCA successfully discriminated all samples using SNR maximal values. This method was effective for Fuji apple storage time rapid discrimination. It presents some advantages including non-destructive, rapid response, high accuracy, etc., and is also promising in condition safety control and management for food and environmental laboratories. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.

v3-fos

2016-05-12T22:15:10.714Z

2015-12-03T00:00:00.000Z

18388418

s2

Wild Edible Fruit of Prunus nepalensis Ser. (Steud), a Potential Source of Antioxidants, Ameliorates Iron Overload-Induced Hepatotoxicity and Liver Fibrosis in Mice The antioxidant and restoration potentials of hepatic injury by Prunus nepalensis Ser. (Steud), a wild fruit plant from the Northeastern region of India, were investigated. The fruit extract (PNME) exhibited excellent antioxidant and reducing properties and also scavenged the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (IC50 = 30.92 ± 0.40 μg/ml). PNME demonstrated promising scavenging potency, as assessed by the scavenging of different reactive oxygen and nitrogen species. Moreover, the extract revealed an exceptional iron chelation capacity with an IC50 of 25.64 ± 0.60 μg/ml. The extract induced significant improvement of hepatic injury and liver fibrosis against iron overload induced hepatotoxicity in mice in a dose-dependent manner, and this effect was supported by different histopathological studies. The phytochemical constitutions and their identification by HPLC confirmed the presence of purpurin, tannic acid, methyl gallate, reserpine, gallic acid, ascorbic acid, catechin and rutin. The identified compounds were investigated for their individual radical scavenging and iron chelation activity; some compounds exhibited excellent radical scavenging and iron chelation properties, but most were toxic towards normal cells (WI-38). On the other hand, crude PNME was found to be completely nontoxic to normal cells, suggesting its feasibility as a safe oral drug. The above study suggests that different phytochemicals in PNME contributed to its free radical scavenging and iron chelation activity; however, further studies are required to determine the pathway in which PNME acts to treat iron-overload diseases. Introduction An imbalance between the efficiency of body's antioxidant defense and the free radical generation system, involving reactive oxygen species (ROS) and reactive nitrogen species (RNS), eventually gives rise to oxidative stress. These reactive species are mainly responsible for the primary initiation of oxidative damage to several important biological macro-molecules, like membrane lipids, proteins and nucleic acids [1]. Increasing evidence indicates that redoxactive metals such as iron also play a major role in the overproduction of ROS by undergoing redox cycling [2]. Although the transition metal iron is essential for important biological activities and biochemical reactions, an excess of iron is toxic, presumably via formation of highly reactive hydroxyl radicals through the Fenton reaction causing lipid peroxidation, depletion of low-molecular weight antioxidants, DNA alterations, hemochromatosis, L-thalassemia, ischemic heart disease and cancer [3][4]. Therefore, substances that are able to trap 'free iron', making it unavailable for Haber-Weiss reactions, act as antioxidants [3]. In addition to endogenous antioxidant systems, frequent consumption of foods rich in natural antioxidants also results in increased resistance to oxidative stress by altering the redox environment and is associated with a lower risk of many oxidative stress-related diseases. In recent times, edible fruits have attracted substantial interest because they contain several antioxidants and bioactive phytocompounds that may act as possible remedial agents. An important plant of interest is Prunus nepalensis Ser. (Steud) (family, Rosaceae), grown in different parts of Northeast India. Locally known as Sohiong in East Khasi hills of Meghalaya, the edible fruits of P. nepalensis are often used for making fruit juice, jam, squash and assorted wines. Moreover, the fruits of P. nepalensis are used as an astringent, the leaves are a diuretic agent and are used for edema [5]. Several bioactive compounds, such as quercetin, quinic acid, rutin, scopoletin, naringenin, palmitoleic acid and many others, have been isolated from different species of Prunus available in China [6]. However, only a few studies have focused on the preliminary quantification of phenolic compounds and antioxidant capacity of P. nepalensis [7]. A review of the literature showed that the detailed phytochemical composition, antioxidant activity and recovery effects from hepatic toxicity by aqua-methanolic extract of the fruits on iron-induced liver damage remain unexplored. Thus, this study was performed to determine the phytochemical profile and the in vitro and in vivo antioxidant properties of 70% methanolic extract of P. nepalensis fruit (PNME) and in vivo amelioration of liver toxicity due to iron overload in Swiss albino mice. Ethics The fresh fruits of P. nepalensis were collected in October 2013 from local neighboring villages in Shillong (25.6314°N, 91.8840°E), Meghalaya, India. Collection zones are not under a Government-protected area, National Park or Reserve Forest and the collection was performed only after receiving oral permission from the village headmen. The International Union for Conservation of Nature (IUCN), World Conservation Union guidelines (1994) was used to classify the conservation status and was found that this plant has not yet been assessed for the IUCN Red List. In vivo experiments were performed abiding by the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Govt. of India with due approval from the Institutional Animal Ethics Committee, Bose Institute (Registration. No. 95/1999/CPCSEA). All surgeries were done using ethyl ether as anesthetic (inside an appropriate fume hood), taking utmost care to reduce suffering. Test Animals Swiss albino mice (Male, weighing 20 ± 2 g) were acquired from Chittaranjan National Cancer Institute (CNCI), Kolkata, India. The animals were kept under a continuous 12 h light / dark cycle (temperature-22 ± 2°C). The animals were fed with laboratory diet and water ad libitum. Experimental animals were taken care every 6 h during the treatment period and it was observed that there was no unwanted animal death. Fruit Extract Preparation P. nepalensis was authenticated by the Botanical Survey of India, Eastern regional center, Shillong, Meghalaya, India (Acc. No. 52320, 45246). The collected fruits were crushed to separate the seeds from the pulp. The finely ground pulp (100 g) was mixed with 1000 ml of methanol: water (7:3) at 37°C overnight using a shaking incubator. The mixture was then centrifuged at 2850 g, and the supernatant was collected. The pellet was again suspended in 1000 ml of the same solvent and the procedure was repeated. The obtained supernatants were concentrated in a rotary evaporator under reduced pressure followed by lyophilization. The lyophilized powder was labeled as PNME and stored at -20°C. Water was used as the vehicle/solvent of choice for further experiments, as PNME dissolves easily in water. In vitro Antioxidant and free radical scavenging activity Antioxidant activity. ABTS •+ radical cation decolorization assay was used to evaluate the antioxidant capacity of PNME (0.05-10 mg/ml) with respect to the standard, trolox [8]. Standard methods were used to evaluate the Fe 3+ -reducing capacity of the extract [8]. The DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging potential of the extract was investigated according to Ghate et al, 2013 [9]. The scavenging percentage of PNME was evaluated from the values of the control and the test samples. ROS scavenging assays. The ROS scavenging activity of the extract was revealed by several in vitro radical scavenging assays, such as superoxide, hydroxyl, hypochlorous radical and singlet oxygen assays, using standard procedures [8]. RNS scavenging assays. The RNS scavenging potentials of PNME was evaluated by performing nitric oxide and peroxynitrite radical scavenging assays [8]. Metal chelating activity. The Fe 2+ chelating ability was determined as described earlier [8]. Protection efficacy of PNME in Fe 2+ -mediated supercoiled plasmid DNA (pUC18) breakdown was determined following previously described methods [9], with slight modifications. The densitometric analysis of the DNA bands was performed using ImageJ 1.47v tool by the software company NIH, USA. The DNA supercoil protection capacity of the fruit extract was expressed as the [P] 50 value, the amount of sample required for 50% protection. The degree of inhibition of Fe 2+ -mediated lipid peroxidation was assessed by estimating the TBARS by the method of Ghate et al. 2013 [9] using freshly prepared brain homogenates of Swiss albino mouse brains. The results are expressed as a percentage of inhibition. In vivo hepato-ameliorating activity Experimental design and tissue preparation. Six groups were randomly created comprising six mice each. Among them, one group was labeled the blank (B) and was administered saline. The remaining groups were injected intraperitoneally (ip) with five doses of iron-dextran of 100 mg/kg b.w. (one dose every alternative days). After the first injection oral treatment was started the next day with only saline to the iron-dextran group (C), and the remaining groups were treated with 50 mg/kg b.w. PNME (S50), 100 mg/kg b.w. PNME (S100), 200 mg/kg b.w. PNME (S200) or 20 mg/kg b.w. desirox (D) for the 21 successive days. All experimental animals were sacrificed on 22 nd day under mild anesthesia (ethyl ether) and cardiac puncture was performed to collect blood and serum was separated and stored at -80°C. After collecting the blood, the liver was quickly excised, cleaned thoroughly with cold phosphate buffer saline (PBS) to remove the remaining blood and cut into three sections. The major liver portion was dissected and homogenized using 10 volumes of 0.1 M phosphate buffer (pH 7.4) supplemented with 0.15 M NaCl and 5 mM EDTA and centrifuged for 30 min at 8000 g in the cold. The clear homogenate (supernatant) was collected and the protein concentration was quantified by Folin-Lowry method [10], where BSA was used as a standard; the remaining supernatant was then stored at -80°C. Second liver fragment was treated with a mixture of nitric acid and sulfuric acid (1:1) to analyze the iron content. The remaining portion was processed for histopathological examinations. Serum Markers. Aspartate amino transferase (ASAT), alanine amino transferase (ALAT), and billirubin levels in the serum were evaluated using commercially available kits from Merck (India). Moreover, alkaline phosphatase (ALP) levels in the serum were measured by a kit procured from Sentinel Diagnostics, Italy. Antioxidant enzymes. The suppression of the blue-colored formazan formation was assessed at 560 nm to evaluate superoxide dismutase (SOD) levels [11]. Catalase (CAT) activity was assessed by tracking the breakdown of H 2 O 2 over time at 240 nm [12]. Glutathione-Stransferase (GST) levels were estimated based on the formation of GSH-CDNB conjugate and increase in absorbance at 340 nm [13]. The level of reduced glutathione (GSH) was determined spectrophotometrically at 412 nm [14]. Evaluation of liver injury. The products of lipid peroxidation in liver were quantified as thiobarbituric acid reactive substances (TBARS) [15]. Protein carbonyl content was estimated spectrophotometrically to determine the levels of protein oxidation [16]. The measurement of hydroxyproline content in the liver allows for the quantification of collagen content, which is an important marker of liver fibrosis. The respective homogenates were hydrolyzed in 6 M HCl and hydroxyproline was measured by Ehrlich's solution [17]. The absorbance was taken at 558 nm and the results were calculated from standard curve of 4-hydroxy-L-proline (R 2 = 0.9907). The collagen content in each sample was determined by multiplying the factor of 7.69 with the quantity of overall hydroxyproline content [18]. Serum ferritin and liver iron. The manufacturer's instructions were followed to quantify serum ferritin levels using an enzyme-linked immunosorbent assay (ELISA) kit (Monobind Inc., USA). Iron content in liver was quantified using a previously reported method [19]. Briefly, samples were mixed with bathophenanthroline sulfonate and incubated at 37°C for 30 min and absorbance was recorded using a spectrophotometer at 535 nm. Histopathological investigation. PBS washed excised liver samples were fixed for two days in 10% buffered neutral formalin. Sections (5-μm thick) were cut using the paraffin-embedding technique and stained with hematoxylin and eosin (morphological examination), Masson's trichrome stain (liver fibrosis) and Perls' Prussian blue dye (iron content). The stained sections were checked microscopically for changes in histopathological condition. In vitro ferritin iron release. Iron reduction and release were determined using ferrozine, a spectrophotometric reagent for iron, as previously described [20]. Briefly, the reaction was initiated by adding different concentrations (100-500 μg/ml) of PNME in 50 mM phosphate buffer (pH 7.0) containing 200 μg of ferritin and 500 μM ferrozine and the absorbance change was measured for 20 min at 560 nm. The reaction mixture excluding PNME was used as a reference. Identification of active phytochemicals and standardization of the extract The evaluation of existing phytochemicals, such as phenols, carbohydrates, alkaloids, flavonoids, glycosides, ascorbic acid, tannins, saponins, terpenoids, triterpenoids and anthraquinones, in PNME was performed using standard methods [21][22]. Among the present phytochemicals phenols, carbohydrates, flavonoids, alkaloids, tannins, ascorbic acid were quantitatively analyzed following previously described methods [9,23]. Extract standardization was performed by HPLC analysis [24]. The samples were eluted using acetonitrile and 0.5 mM ammonium acetate in water as a mobile phase for gradient elution (flow rate of 1 ml/min) through the column (ZIC-HILIC) for 80 min at 25°C and the peaks were detected at 254 nm. Cytotoxicity assay The human normal lung fibroblast cell line (WI-38) was procured from the National Centre for Cell Science (NCCS), Pune, India. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) provided with 10% (v/v) fetal bovine serum (FBS), 50 μg/ml gentamicin sulfate, 2.5 μg/ml amphotericin B, 100 U/ml penicillin G, 100 μg/ml streptomycin and maintained in a biological CO 2 incubator. Cell viability was calculated using the WST-1 Cell Proliferation Reagent (Roche Diagnostics) as described previously [25]. Briefly, the cells (1 × 10 4 cells/well) were incubated in presence of PNME, purpurin, tannic acid, reserpine, methyl gallate, catechin, ascorbic acid, gallic acid or rutin for 48 hours with increasing doses from 0-120 μg/ml in 96-well culture plates. A total of 10 μl of the reagent was then added to each well and the samples were kept at 37°C for 2 hours. Absorbance (460 nm) was measured using a microplate ELISA reader MULTISKAN EX (Thermo Electron Corporation, USA) to calculate cell proliferation and viability. Statistical analyses All data are reported as the mean ± S.D. (n = 6). Statistical analysis was performed using Origin professional 6.0 and KyPlot version 2.0 beta 15 (32 bit). Comparisons between the groups were evaluated according to paired t-test, and a p value of <0.05 was considered significant. Results and Discussion Epidemiological studies have established that consumption of plant-based foods containing abundant sources of antioxidants is beneficial for health because these antioxidants obstruct many degenerative processes and effectively lower the incidence of oxidative stress-related diseases [26]. Antioxidants may also act as iron chelating agents because many of them consist of a range of bidentate, tridentate and hexadentate ligands in which two, three, or six atoms, respectively, are able to coordinate with iron, forming octahedral complexes [27] followed by their excretion from the body. With the intention to investigate the fundamental principle of traditional usage of the fruits as medicine, biologically active phytocomponents present in the plant need to be well extracted with different solvents. Polar solvents are frequently employed for the recovery of polyphenols from a plant matrix. Generally, extraction of bioactive compounds from various biological resources (whole plants, fruits, vegetables, etc.) is performed using aqueous mixtures (hot or cold) comprising methanol and ethanol [28,29]. Keeping this in mind, extraction of the phytochemicals from the P. nepalensis fruit pulp was performed using 70% methanol at room temperature. In vitro antioxidant potential of PNME Total antioxidant capacity. The antioxidant activity of PNME was measured using two complementary methods: TEAC through ABTS •+ radical cation scavenging and reducing power capacity, along with the scavenging assay of DPPH. A strong correlation between antioxidant activity and reducing power was revealed previously [30]. On the other hand, DPPH in its radical form has its maximum absorbance at 517 nm, but upon reduction with an antioxidant, its net absorbance decreases due to the formation of its non-radical form, DPPH-H [31]. The assays project an overall indication of the antioxidant potential of the fruit extract as shown in Fig 1A, 1B and 1C along with Table 1. Total antioxidant activity, a function of the trolox (standard)-equivalent antioxidant capacity (TEAC) of PNME was observed to be 0.46 ± 0.001. PNME can be considered a potent antioxidant agent for its exceptional DPPH radical scavenging activity (Fig 1C), in spite of its moderate reducing capacity (Fig 1B). ROS scavenging. Various ROS, such as the superoxide anion, hydroxyl radical and hypochlorous acid, are generated under numerous conditions in vivo. It is well known that superoxide anion radical primarily initiates the formation of other ROS [32]. Superoxide radicals react with cellular H 2 O 2 and generate the detrimental hydroxyl radical, which damages lipids, DNA, and proteins [8]. In the presence of H 2 O 2 , another harmful ROS, HOCl is produced in vivo by the oxidation of Cl ions catalyzed by neutrophil-derived myeloperoxidase at sites of inflammation [33]. HOCl inactivates catalase, an antioxidant enzyme causes sulfhydryl oxidation in the plasma membrane proteins to induce target cell lysis [34]. On the other side, a singlet oxygen radical is generally produced in vivo by photo-excitation upon exposure to UV radiation or by chemi-excitation. This radical also inactivates antioxidant enzymes and induces hyper-oxidation and oxygen cytotoxicity [35]. PNME showed excellent dose-dependent scavenging activities against superoxide radical as well as hypochlorous acid (Fig 2A and 2C respectively). At the same time, PNME moderately scavenged hydroxyl radical in conjunction with singlet oxygen radicals (Fig 2B and 2D respectively). The IC 50 values of PNME on different radical scavenging assays are shown in Table 1 along with their corresponding standard compounds. RNS scavenging. In normal cellular conditions, nitric oxide is essential in various inflammatory processes but also contributes to multiple sclerosis, reperfusion injury, ulcerative colitis and arthritis, etc., when produced in excess amounts [36]. The impact of NO • toxicity is critically amplified when superoxide radical reacts with it to form peroxynitrite anion [ONOO], which is highly reactive [37]. PNME moderately scavenges both RNS forms (Fig 3). The IC 50 values of PNME for both the radicals are displayed in Table 1 with their corresponding standard compounds. Metal chelation activity. Many antioxidants not only convert free radicals to more stable products but also slow the rate of oxidation by chelation of pro-oxidant metals (such as iron, copper, etc.) that promote oxidation by acting as catalysts of free radical reactions. Metal chelation by certain compounds decreases their pro-oxidant effect by reducing their redox potentials and also through steric hindrance by forming a metal hydroperoxide complex [38]. The iron chelation ability of the extract was revealed using three in vitro assays depending on the direct chelation of iron by the extract. Fig 4A suggests impressive results from PNME, as did the obtained IC 50 value, indicating the extract's effective iron chelating activity. In contrast, the most detrimental OH • , generated by the Fenton reaction (H 2 O 2 + Fe 2+ = Fe 3+ + OH+ OH • ) destroyed the lipid membrane by lipid peroxidation [39]. The extract moderately inhibited lipid peroxidation in a dose-dependent manner ( Fig 4B) compared with standard compounds, although PNME showed impressive IC 50 values (Table 1). Overall, PNME was found to be well endowed with exceptional iron chelation ability. The studies on the protective effect of PNME against OH • -mediated plasmid DNA (pUC18) slicing was also displayed a significant dose dependence (Fig 4C). An impressive [P] 50 value of 129.56 ± 1.73 μg/ml was obtained, further supporting this notion. In vivo antioxidant and hepato-ameliorating activity The liver, which is involved in numerous biochemical pathways related to nutrition and detoxification [40], is often subjected to injuries induced by various hepatotoxins. Iron, a vital constituent of countless proteins [41] becomes a well-known hepatotoxin when in excess. In ironoverloaded liver, the metal initiates and propagates several ROS, leading to the oxidative damage of many vital biomolecules, resulting in the cellular lipid peroxidation, mitochondrial damages, DNA fragmentation and, finally, cell death [42]. An effective remedial strategy should act in a dual manner by decreasing the oxidation rate: one manner sequestering and chelating the stored iron in cells [43] and other as a radical scavenger (i.e., antioxidant activity). Because PNME depicted excellent antioxidant and free radical scavenging activities along with significant in vitro iron chelation potency, the in vivo ameliorating potency of PNME on accumulation of iron and oxidative damage by iron overload in the mouse liver was studied. The hemochromatosis condition was created by the intraperitoneal injection of iron-dextran. This process will not hamper the intestinal iron absorption by fruit extract, which ultimately leads to iron overload in the liver as well as in serum [44]. Serum marker enzymes. Administration of excess iron dextran caused significant liver damage leading to the release of intracellular enzymes into the blood [45], as evidenced by the elevated level of serum parameters (Table 2). However, PNME treatment at a dose of 200 mg/kg induced a marked restoration of liver enzymes and bilirubin. Among them, the ALAT and ASAT values obtained from the PNME group were substantially superior to those from the standard drug desirox. Effect on antioxidant enzymes. Living systems, especially animals, are armed with free radical scavenging enzymes, such as SOD, CAT, GST and the small compound GSH, which are the primary defense against oxidative damage. Levels of these antioxidant enzymes indirectly indicate the pro-oxidant-antioxidant condition in tissues [46]. A significant reduction in antioxidant enzymes levels was detected in iron-intoxicated mice compared with normal mice. PNME treatment significantly increased antioxidants levels, establishing its ameliorating potential against iron-overloaded oxidative stress. It was observed that, administration of the highest extract dose resulted in levels approaching those obtained from the standard desirox, in case of SOD ( Fig 5A) and GST (Fig 5C), but not GSH (Fig 5D). Moreover, with regard to CAT (Fig 5B), the extract exhibited activity superior to the standard. Biochemical parameters of liver damage. Lipid peroxidation (LPO) has been proposed as a major factor in iron toxicity, including iron-induced hepatotoxicity. Ferrous salts undergo the Fenton reaction to form the highly reactive hydroxyl radical, which attacks all biological molecules, including cell membrane lipids, to initiate LPO [47]. Iron-overloaded liver pathogenesis causes oxidation of various important structural and functional proteins and forms protein carbonyls, which serve as markers of oxidative stress, leading to the development/onset of several diseases, including ulceative colitis and cystic fibrosis [48]. Liver damage also leads to excess extracellular proteins accumulation, especially collagen and increased hydroxyproline as observed in liver fibrosis [49]. Iron-dextran injection considerably increased lipid peroxidation (74%), protein carbonyl (155.16%) and hydroxyproline (137.99%) content in liver homogenates compared with normal mice. When treated with PNME, the level of thiobarbituric acid reactive substance (TBARS) was substantially reduced ( Fig 6A); however, two other liver damage markers were also found to be arrested significantly with increasing doses (Fig 6B and 6C) in a manner superior to that of the standard desirox. Thus, PNME treatment significantly overcomes hepatic injury/fibrosis in iron-intoxicated mice, indicating the hepato-ameliorating potency of the fruit extract. Histopathological Studies. Histological experiments, a gold standard for the assessment of the degree of hepatic injury, were performed alongside other biochemical tests. The liver sections of normal mice displayed a normal cellular architecture with a prominent nucleus inside cytoplasm and a prominent central vein without cellular infiltration (Fig 7A), whereas saline treated iron-dextran group exhibited abrupt cytological changes, including inflammation, ballooning degeneration, loss of cellular boundaries and hepatocellular necrosis (Fig 7B). In contrast, the liver sections from PNME-treated mice groups displayed evidence of reduced pathogenesis, attenuation of the pathological changes and gradual reversal to normal cytoarchitecture, with a higher dosage presenting restoration against iron overload-induced hepatic damage (Fig 7C-7E). An improved liver section morphology was observed in S200 (Fig 7F), which was quite similar to the improvement observed in the desirox-treated group. Another detrimental effect of excess iron in liver is deposition of iron in the form crystalline ferritine and amorphous hemosiderin. Iron released from denatured ferritin, ferric oxide (unused iron) as well as broken hemoglobin formed a complex to store the iron known as hemosiderin. The iron within the deposits of hemosiderin is poorly available to the body and tissue sections stained with Perls' Prussian blue is commonly used to detect its deposition in liver tissue as blue patches. Light microscopic observation of liver sections of saline treated mice (group B) exhibited elevated deposition of hemosiderin ( Fig 8B) compared with normal mice (Fig 8A). These changes also activated stellate cells in periportal zones, which enhanced the production of collagen [50]. In contrast treatment with PNME exhibited a gradual reduction of blue patches (hemosiderin deposition) (Fig 8C, 8D and 8E). PNME exhibited an effect almost parallel to that of the desirox-treated group at S200 (Fig 8F). Additionally, chronic damage in liver due to excessive iron deposition leads to the liver fibrosis characterized by the collapse of the hepatic parenchyma and its substitution with a collagen-rich tissue. A liver biopsy is considered the gold-standard method for the assessment of liver fibrosis [51]. The trichrome stain is performed to assess the degree of fibrosis in liver by staining the nuclei black; cytoplasm red and collagen blue. The microscopic observation suggested that the liver section of control mice revealed normal lobular architecture and a normal distribution of collagen ( Fig 9A). From the liver section of iron-overloaded mice it is evident that the normal architecture of the liver is destroyed and the nodules surrounded by accumulated collagen indicating fibrous cirrhotic ( Fig 9B). However, after treatment with PNME, a gradual decrease in the degree of collagen deposition was observed (Fig 9C, 9D and 9E). As evident from the other biopsy result, the highest dose of PNME (S200) revealed instances comparable to that of the desirox-treated group (Fig 9F). Overall, histopathological studied indicated that the PNME-treated group produced a dose-dependent normalization of the cyto-architecture, which signifies the in situ evidence of ameliorating effect of the extract in the iron overload-induced liver toxicity. Liver iron content and serum ferritin levels. On the basis of the fact that 5/6 of the surfeited iron in our body is settled in the liver, most procedures seek to measure liver iron levels for diagnosis. Ferritin generally stores excess iron to prevent free iron-mediated oxidative damage to cellular components [52]. The serum ferritin level is one of the main biomarkers for the detection of iron overload-induced hepatic toxicity, as the ferritin content in the blood is indirectly related to the amount of liver iron. The level of liver iron content (132.97%) and serum ferritin content (192.57%) was increased in the iron-intoxicated mice when compared with the normal mice. Upon treatment with PNME, dose-dependent reductions in liver iron content ( Fig 10A) as well as serum ferritin concentrations (Fig 10B) were observed. The values were equal to the standard; thus supporting the iron chelating potency of PNME. Iron release from ferritin. Various readily available iron chelator drugs can be administered to normalize the condition generated by iron overload but many of them suffer from limited binding capacity towards ferric iron (Fe 3+ ). Therefore, reducing agents, like ascorbic acid, are additionally supplemented to enhance the accessibility of storage iron to chelators [53]. The dose dependent increase in the formation of the ferrous-ferrozine complex [(Fe(ferrozine) 3 ) 2+ ] was measured to quantify the efficiency of PNME in releasing the reduced iron from ferritin. In the absence of PNME, minor quantities of (Fe(ferrozine) 3 ) 2+ complex formed; however, it was increased significantly with time after the addition of PNME in a dose-dependent manner (Fig 11A). The percentage of iron released from ferritin significantly correlates (R 2 = 0.9011) with the reducing power of the extract (Fig 11B), thereby confirming the efficacy of PNME as an iron chelating agent. Probable phytocompounds identification and HPLC standardization of PNME In recent years, several edible fruits with natural antioxidants have attracted considerable attention because of their potentially beneficial effects on prevention as well as cure of disease in humans. The medicinal properties of fruits are usually attributed to the active compounds acid (mg/100 mg extract L-ascorbic acid equivalent), Alkaloid (mg/100 mg extract reserpine equivalent), "+" represents presence of the phytoconstituent; "-" represents absence of the phytoconstituent. "ND" represents Not Determined. doi:10.1371/journal.pone.0144280.t003 present in them, which can easily be absorbed by our system and are safe for post-consumption symptoms. Therefore, edible fruits have become a major field of interest for the investigation of their diverse pharmacological properties. Qualitative phytochemical analysis of PNME has revealed the presence of phenolics, flavonoids, carbohydrates, terpenoids, tannins, glycosides, anthraquinones, ascorbic acid and alkaloids (Table 3). However, quantitative analysis found that among the phytochemicals, phenolics, flavonoids and alkaloids are present in higher amounts compared with the others (Table 3). Substantially higher amounts of phenolic compounds, subsequent flavonoids and even alkaloids indicate that this fruit represents a source of promising antioxidants, which, as has been well documented, could act as the basis of drug development against various ailments. To identify the existing phytochemicals in PNME, HPLC analysis was performed and the retention time of the main peaks was compared with the standard phytocompounds in the identical condition. Major peaks with retention times 2. 8 and rutin, respectively (Fig 12A). Probable identified phytochemicals were screened for their in vitro hydroxyl radical scavenging, iron chelation potentials and cytotoxicity against human normal fibroblast cells (WI-38). Free radical scavenging was assessed using a hydroxyl radical scavenging assay, as an excess iron condition induces hydroxyl radicals that result in lipid peroxidation-mediated hepatotoxicity. Except for ascorbic acid, all compounds exhibited promising hydroxyl radical scavenging activity (Fig 12B). Among the identified compounds, tannic acid was found to be the most potent iron chelator, followed by methyl gallate, gallic acid, rutin and purpurin. Reserpine, catechin and ascorbic acid failed to show any activity (Fig 12C). On the other hand, PNME exhibited excellent iron chelation activity. This result indicates that the unidentified compounds of PNME are also responsible for the improved iron chelation activity of PNME. The cytotoxicity of the identified compounds on the normal fibroblast cells indicated that gallic acid is most toxic towards normal cells followed by reserpine, methyl gallate, tannic acid, catechin, rutin and purpurin. Most importantly, PNME did not exhibit any cytotoxicity against normal cells (Fig 12D). From the reported literature, it was found that purpurin, methyl gallate, gallic acid, rutin, tannic acid, ascorbic acid, catechin are potential antioxidants, metal chelators thereby inhibit lipid peroxidation, which also support their hepato-ameliorating potentials [54][55][56][57]. On the other hand, reserpine as well as its derivatives have been reported to have antioxidant potential [58]. Rutin, a citrus flavonoid glycoside, is found in many fruits, such as oranges, grapefruits, lemons, cranberries and even other species of Prunus. Apart from its antioxidant, metal chelation and protective effects on hepatotoxicity, it also exhibits anti-inflammatory activity [59]. From the cytotoxicity point of view, some of the individual compounds are toxic to normal cells; however, when in combination, their toxic effect is nullified, possibly due to the presence of other nontoxic compounds in the extract. This possibility is supported by the fact that the extract (PNME) is a mixture of several compounds and their synergistic effect is responsible for antioxidant activity and its ameliorating activity against iron-induced hepatotoxicity. Conclusions The results demonstrated high efficacies of the 70% methanolic extract of P. nepalensis in scavenging free radicals, inhibiting reactive free radicals and chelating iron, which established its therapeutic use as a functional food that may effectively treat several diseases, with liver disease being the most important. Phytochemical screening also indicated the presence of several bioactive phytocompounds and natural antioxidants that are responsible for its overall activity. Cytotoxicity studies suggested that a single molecule may have greater activity but also may possess toxicity towards normal cells. Therefore, combination therapy has a greater impact without affecting normal cells. As, several other causes of liver toxicity such as drug induced, alcohol overloaded, viral hepatitis, nonalcoholic fatty liver follow similar pathway [60,61] for liver toxicity and PNME can be used to ameliorate them. But, presently it is hard to comment whether PNME would work similar manner on other types of hepatotoxicity or not. So, further experiments in other animal models are required to fully evaluate the antioxidant and hepatoameliorating potentials along with the complete characterization of all bioactive compounds present in this wild edible fruit. This information will establish its use in combinatorial therapy and innovative drug delivery systems, thus making it a favorable candidate to manage iron overload-mediated oxidative stress and hepatotoxicity.

v3-fos

2017-04-19T00:52:50.549Z

2015-10-27T00:00:00.000Z

17616148

s2

Biosorption of Cadmium and Manganese Using Free Cells of Klebsiella sp. Isolated from Waste Water In the present study, we evaluated a bacterium that was isolated from waste water for its ability to take up cadmium and manganese. The strain, identified both biochemically and by its 16S rRNA gene sequence as Klebsiella, was named Yangling I2 and was found to be highly resistant to heavy metals. Surface characterization of the bacterium via SEM revealed gross morphological changes, with cells appearing as biconcave discs after metal exposure rather than their typical rod shape. The effects of pH, temperature, heavy metal concentration, agitation and biomass concentration on the uptake of Cd(II) and Mn(II) was measured using atomic absorption spectrophotometry. The results showed that the biosorption was most affected by pH and incubation temperature, being maximized at pH 5.0 and 30°C, with absorption capacities of 170.4 and 114.1 mg/g for Cd(II) and Mn(II), respectively. Two models were investigated to compare the cells’ capacity for the biosorption of Cd and Mn, and the Langmuir model based on fuzzy linear regression was found to be close to the observed absorption curves and yield binding constants of 0.98 and 0.86 for Cd and Mn, respectively. This strain of Klebsiella has approximately ten times the absorption capacity reported for other strains and is promising for the removal of heavy metals from waste water. Introduction Heavy metal pollution is a global concern in the environmental field. Heavy metals, such as cadmium, copper and manganese, are among the most common pollutants found in industrial effluents (such as from mining, the surface finishing industry, energy and fuel production, and electric appliance manufacturing) [1]. Even at low concentrations, these metals are toxic to organisms, including humans. Cadmium is a metal with a small biological demand that is only slightly degraded in the environment. Additionally, it has many common industrial uses, including battery production area. We confirmed that the field studies did not involve endangered or protected species. The collected samples contained various metal ions and were stored at 4°C before analysis. Analytical-grade chemicals were used for the growth of the microorganisms, and bacterial strains were isolated from the effluents using Murashige and Skoog medium (K 2 HPO 4 1.6 g, KH 2 PO 4 0.4 g, NaNO 3 0.4 g, CaCl 2 0.02 g, MgSO 4 0.2 g, FeSO 4 Á7H 2 O 0.01 g, and 50 ppm ibuprofen). To isolate strains, the serially diluted effluents were plated onto the same agar plates described above. The inoculated plates were incubated at 30°C for 48 h. After the incubation, the distinct colonies were isolated from each plate. The strain was analyzed through morphological (Gram reaction, motility) and biochemical characterization, including carbohydrate fermentation, gelatin liquefaction test, starch hydrolysis test, and oxidation fermentation test in accordance with Bergey's Manual of Determinative Bacteriology, as well molecular identification according to the 16S rRNA sequence. Amplification of 16S rRNA was carried out using universal primers (27 F 5'-AGAGTTTGATCMT GGCTCG-3' and 1492 R 5'-GGTTACCTTGTTACGACTT-3') with genomic DNA as a template [22,23]. The purified PCR products were sequenced by Sangon Biotech (Shanghai, China). BLAST was used to find similar sequences in the GenBank database (Nucleotide Blast), followed by multiple sequence alignment and phylogenetic tree construction in MEGA 5.0 using the neighbor-joining method. Growth of Klebsiella sp. Yangling I2 strain with different heavy metals Determination of the minimum inhibitory concentration (MIC) of heavy metals. The minimum inhibitory concentration (MIC) of heavy metals was performed as follows: single clone growth was achieved on LB (Luria-Bertani Culture) agar plates, and the lowest concentration that prevented the bacterial growth was defined as the MIC. The resistant isolate was incubated in 50 mL of LB liquid medium (5 g of yeast extract, 10 g of casein enzymic hydrolysate, and 10 g of NaCl) to log phase at 30°C with shaking. Then, it was inoculated onto LB agar plates with different concentrations of CdSO 4 , ZnSO 4 , CuSO 4 Á5H 2 O, Pb(NO 3 ) 2 , K 2 Cr 2 O 7 , FeCl 3 , and MnCl 2 Á4H 2 O. The concentrations of heavy metals were initially 100 mM and were diluted from 1X to 100X. After 2 days of incubation at 30°C, the metal MIC values were estimated in terms of the first dilution at which no single clone grew. Growth curve. The strain was incubated in 50 mL of LB liquid medium (5 g of yeast extract, 10 g of peptone, and 10 g of NaCl) containing 1, 2, 3, or 4 mM of Cd(II) and 5, 10, 15 or 20 mM of Mn(II) at 30°C with shaking at 120 rpm. The optical density was measured at 600 nm (Telecomp, Shanghai, China) every 2 hours. SEM Analysis Ten milliliters of LB culture of the resistant isolate containing 4 mM Cd(II) and 20 mM Mn(II) was incubated at 5,000 rpm at 4°C for 10 min. The pellet cells were fixed for 24 h in 3% glutaraldehyde solutions, followed by dehydration with a graded series of ethanol (50, 60, 70, and 80% and absolute, 15 min each) and drying under a CO 2 atmosphere for 20 min using a critical point dryer (K850, Emitech, East Grinstead, UK). The samples were mounted on a stainless steel slab and covered with a thin layer of platinum under vacuum. The scanning electron microscopy (SEM) images and the energy dispersive spectral (EDS) analyses of the cells were obtained using a SEM instrument (S-4800, Hitachi, Tokyo, Japan). was adjusted to the appropriate value by 1 M HCl or 1 M NaOH before the addition of biomass. Using LB liquid medium, stationary stage cultures were harvested by centrifugation (8,000 rpm, 20 min). The cells were washed two times with PBS buffer and suspended in PBS buffer (1 M, pH 7.2). The concentrations of the biosorbents were calculated and expressed in terms of grams (dry weight) per liter after 18-mL biomass samples were oven-dried at 60°C. Cd(II) and Mn(II) biosorption To determine the initial pH for absorption, test solutions containing 2 mM Cd(II) and 10 mM Mn(II) with pH values ranging from 3.5 to 5.5 and 4.0 to 6.0, respectively, were used. Organisms were added at a concentration of 1 g/L. The cell-metal solutions were agitated at 120 rpm and 30°C, for 20 h or 26 h. After incubation, the supernatants were collected by centrifugation at 8,000 rpm for 20 min. Using a suitable dilution, the amount of residual ion in the solutions was determined using an atomic absorption spectrometer (AAS) (Z-2000; Hitachi, Tokyo, Japan). The ion supernatants were measured as described above. All experiments were performed in triplicate. The absorption capacity (Q e ) and the removal ratio (R e %) were evaluated by the equilibrium described by Eqs (1) and (2): [24,25] where Q e =Metal uptake (micrograms of metal per gram of biosorbent) V = Liquid sample volume (milliliters) C i =Initial concentration of the metal in the solution (milligrams per liter) C f =Final concentration of the metal in the solution (milligrams per liter) M=Amount of added biosorbent on a dry basis (milligrams) Determination of equilibrium time for Cd(II) and Mn(II) absorption The effect of contact time on the ion uptake of Klebsiella sp. Yangling I2 was investigated using solutions of 2 mM CdSO 4 and 10 mM MnCl 2 at pH values of 5.5 and 5, respectively. Samples were taken at designated time points (5,10,15,20,25,30,40,50, 60, 80, 100, 120, 150, 180, 240, and 300 min). The supernatants were collected by centrifugation at 8,000 rpm for 20 min and used to determine the amounts of the residual Cd and Mn ions by AAS. Biosorption isotherms Biosorption isotherms characterized by certain constant values indicate the surface properties and affinity of the biosorbents [26] and can be used to compare the capacities for biosorption of various heavy metals. Metal uptake by organisms can be described in terms of two stages: an initial rapid stage (passive uptake) and a much slower stage (active uptake) [27]. The biosorption isotherms of Cd(II) and Mn(II) were investigated using two isotherm models: the Langmuir and Freundlich isotherm models. Langmuir isotherm. The Langmuir isotherm is used to examine the absorption of gases on a solid surface, and sorption is considered to be a chemical phenomenon. This isotherm has been successfully applied to many pollutant biosorption processes and is the most widely used isotherm for the biosorption of a solute from a liquid solution [28]. Freundlich isotherm. The Freundlich isotherm is applied under the assumption of a heterogeneous absorption surface and active sites with different energies [28]. The model is represented in Eq (3): [29,30] where K f is a Freundlich constant relating the binding capacity and 1/n is an empirical parameter relating the biosorption intensity, which varies with the heterogeneity of the biosorbents. An efficient absorption process yields a Freundlich constant n between 1 and 10. A high value of n implies a stronger interaction between the adsorbent cell surface and divalent metals. Result and Discussion Klebsiella sp. Yangling I2 was named for the place where it was found, and the sequence generated in this study was submitted to the NCBI GenBank database under the accession number JX196956.1. The strain was analyzed in accordance with Bergey's Manual of Determinative Bacteriology, and molecular identification was achieved according to the 16S rRNA sequence, which showed 97% homology. The tolerance of Klebsiella sp. Yangling I2 to different heavy metals To examine the tolerance of Klebsiella sp. Yangling I2 to different metals, the cells were cultivated in nutrient broth with Cd(II), Zn(II) Mn(II), Fe(III), Cu(II) and Cr(VI). The organism was able to survive at metal concentrations as high as 80 mM for Mn(II), 6 mM for Zn(II) and Cd(II), 5 mM for Fe(III), 2 mM for Cu(II) and 0.1 mM for Cr(VI). Growth curve The ability of Klebsiella sp. Yangling I2 to tolerate cadmium and manganese was higher than for other metals and strains. Therefore, these two metals were selected for further metal absorption studies. Figs 1 and 2 show the growth curve of Klebsiella sp. Yangling I2 in the presence of heavy metals. The presence of heavy metal ions interferes with the growth of organisms and delays the log phase. There is evidence that the cell metabolism is inhibited mainly by ions transported into the cell, and perhaps also by ions adsorbed on the outer surface [31]. Because it resists high concentrations of cadmium and manganese, Klebsiella sp. Yangling I2 might be capable of removing these ions from effluent water heavily polluted by them. Morphology of the adsorbent according to SEM analysis Electron microscopic examination of Klebsiella sp. Yangling I2 before and after metal removal was undertaken to locate the active sites of the cell wall. Scanning electron microscopy (SEM) at 50,000X showed that the Klebsiella sp. Yangling I2 was morphologically rod shaped, as demonstrated in Fig 3A, with lengths and diameters reaching 14 μm and 3 μm, respectively. The small size of Klebsiella sp. Yangling I2, with its estimated surface area of 146 μm 2 , gives it a large contact surface [32], which should facilitate interaction with metals, and thus, biosorption [28,33]. After equilibration with a metal solution, the cell wall, shape and size of the bacteria changed. The length and size of the cell decreased (Fig 3B and 3C), and the cells began to appear as biconcave discs after exposure to 4 mM Cd(II). These bacteria typically exhibit a rod shape (Fig 3B), and this changes suggests that the metal ions were entrapped in the extracellular polymeric substances of Klebsiella sp. Yangling I2, thus causing deformation of and damage to the cell surface during Cd(II) and Mn(II) absorption. Effect of Parameters on single Ion Biosorption The effect of pH on biosorption. Previous investigations into heavy metal biosorption have shown that the pH value is an important factor for ion absorption, although pH values higher than 6.0 were not tested to prevent the precipitation of insoluble cadmium and manganese hydroxide [34,35]. The significant effect of pH on the absorption of Cd(II) and Mn(II) by Klebsiella sp. Yangling I2 was obvious. The uptake capacities of the two metals generally exhibited a similar trend, with higher pH values leading to a higher metal uptake. Regarding to Cd (II), the biosorption capability increased gradually as the pH increased, although the absorption capability of this species is higher than that of Mn(II), with a maximum value of 169.94 mg/g (Fig 4). However, Klebsiella sp. Yangling I2 was very difficult to grow in solutions with pH values of less than 4.5, which might be because of the competition between hydrogen and metal ions for the sorption sites on the biomass surface [36,37]. For Mn(II), the biosorption capability increased gradually in the range of pH 3.5 to 5.5, eventually reaching a maximum value of 105.08 mg/g. The curve of the removal ratio increased, following the trend shown by biosorption (Fig 5). The dependence of metal uptake on pH is related to both the surface functional groups on the cell walls of the biomass and the metal chemistry in solution. In a solution with low pH, the positively charged hydrogen ions may compete with metal ions to bind to the ligands on the cell wall. Increased pH results in increased availability of ligands for metal ion binding, thus enhancing biosorption [38]. In order to test this theory, 1M HCl was added into the culture medium which cells were already finished the process of biosorption. The concentration of metal ions was increased when more HCl was added into the solution. The effect of incubation temperature on biosorption. To study the effect of the incubation temperature on the binding force between glycoproteins on the membrane and heavy metal ions, we selected the following temperatures: 20, 25, 30, 35 and 40°C. The results demonstrated that an increase in the temperature in the range 20-25°C resulted in an increase in the cadmium-sorption capacity at equilibrium: 47.67 mg/g at 20°C and 153.52 mg/g at 25°C (Fig 6). At temperatures exceeding 25°C, the sorption capacities of both cadmium and manganese decreased, reaching a maxium value of 114.56 mg/g during the temperature exceeded from 25 to 30°C for cadmium (Fig 7). Within the temperature range investigated, it is possible to conclude that an increase in temperature is followed by an increase in the diffusivity of the ion [39]. Therefore, like chemical reactions, there is an optimal temperature at which the ratio of sorption to desorption is maximal, as is supported by our results. The effect of heavy metal concentration on biosorption. Several experiments were undertaken to study the effect of varying the initial ion concentration on the metal-removal process from the solution. The experimental results obtained using various ion concentrations between 0.5 and 20 mM for the biosorption of Cd(II) and Mn(II) onto Klebsiella sp. Yangling I2 are presented in Figs 8 and 9. Absorption capability increased continuously as the ion concentration increased, and saturation was achieved at 170.41 and 114.51 mg/g for Cd(II) and Mn(II), respectively. The maximum Cd(II) and Mn(II) removal percentages from the aqueous solution were 76.98% and 18.67% after 20 and 26 h at initial concentrations of 0.5 mM Cd(II) and 3 mM Mn(II). This curve clearly shows that as the initial ion concentration increased, an abrupt increase in the absorption capacity occurred and lower amounts of Cd(II) and Mn(II) were removed by the biosorbents. This behavior might be explained by the fact that the bacteria are stressed by the high concentration of heavy metals. Although the bacteria were stressed by the high heavy metal concentrations, more ions were bound to the surface, which can be explained as chemical reactions. The effect of agitation speed on biosorption. Agitation speed is the speed at which the solution is shaken during incubating. To determine the optimal agitation speed, cadmium and manganese removal experiments were shaken at rates between 60 rpm and 180 rpm. Fig 10 shows that the highest removal of cadmium at equilibrium is approximately 166.76 mg/g and was obtained with an agitation speed of 80 rpm, whereas the highest manganese removal was achieved at 120 rpm (Fig 11). At slow weak agitation speeds, we observed a reduction in the manganese sorption capacity by 114 mg/g to almost 0 mg/g. Additionally, when a high agitation speed was used, we noticed a substantial reduction, similar to the results reported by other authors [4]. The effect of biosorbent density on biosorption. The influence of biomass concentration on Cd(II) and Mn(II) absorption by Klebsiella sp. Yangling I2 was estimated using biosorbent doses ranging from 1.0 to 5.0 g/L. The ion removal efficiency was observed to be substantially enhanced as the biosorbent dosage increased, which could be attributed to increases in the absorption surface area and the availability of free absorption sites in the biomass (Fig 12). A substantial increase in the removal ratio was observed to result in a slight increase in the absorption capacity (Fig 13). This can be explained by the fact that as the mass increases, the available surface area for the sorption of cadmium and manganese also increases. The effect of contact time on biosorption The effect of equilibrium time on the absorption of cadmium and manganese by Klebsiella sp. Yangling I2 is shown in Fig 14. For both Cd(II) and Mn(II), the absorption process can be divided into 2 stages: a rapid absorption process and a long, slow uptake process. In the rapid initial absorption stage, approximately 70% of the total ion uptake can be achieved because this process is spontaneous and does not consume energy. Rather, it is a type of non-metabolic-dependent ion sequestering on the surface of the biomass, which occurs through the mechanisms of physical absorption, ion exchange, and chemical complexion with the functional groups on the surface of the organism [40]. Subsequently, nearly 30% of the Cd and Mn ion absorption occurs in the second, slower period because the ions diffuse into the intracellular area of the organism [40]. Forces between the solute molecules of the solid and bulk phases can also prevent Cd and Mn ions from occupying the remaining vacant surface sites, contributing to the lower absorption rate in the latter stage [41,42]. Approximately 90% of Mn ion uptake was accomplished at 120 min, while Cd ion uptake required 180 min. These results are in agreement with the two-phase biosorption of heavy metals using different biomaterials [43,44]. Biosorption equilibrium The Langmuir and Freundlich sorption models are commonly used to fit experimental results when solute uptake occurs by monolayer sorption. These models were tested in the present work and permitted us to determine the maximal removal capacity. Linear regression is a statistical method of modeling the relationship between two variables by fitting a linear equation to the observed data. One variable is considered to be an explanatory variable, and the other is considered to be a dependent variable. The linear regression method can be used for forecasting if it is assumed that the correlation between the variables will continue in the future [45]. However, models based on statistical regression are problematic if the data set is too small. if there is difficulty verifying that the error is normally distributed, or if there is vagueness in the relationship between the independent and dependent variables [46]. We reevaluated the Langmuir and Freundlich models based on fuzzy linear regression. We focused on models for which the data are crisp and the relationship between the variables is fuzzy, and we propose a new fuzzy linear regression method for the equilibrium model. This proposed fuzzy linear regression is based on Tanaka's approach using T W -based fuzzy arithmetic operations where the output data are fuzzy numbers [47]. Eq (4) is presented below. s:t: where H is the h-certain factor. The isotherm experimental results are shown in Fig 15A-15D. In all cases, favorable isotherms are observed. Langmuir isotherm based on fuzzy linear regression. This monolayer absorption gives the maximum absorption capacity q 0 in the linearized Langmuir expression, and the model can be described in terms of the following equation: Langmuir isotherm [29,48,49] C e . where q 0 and the constant b (absorption energy) are obtained from the slope and intercept of the plot of C e /Q e against equilibrium concentration. C e Fig 15A and 15B and the absorption energy are shown in Table 1. The statistical regression coefficients of cadmium and manganese were 0.9814 and 0.8574, respectively. In this case, R L is a dimensionless parameter related to the effectiveness of metal absorption given by Eq (6) below: R L values in the range of 0-1 indicate that the absorption process is effective [50]. The values of R L for the absorption of cadmium (C 0 = 56 mg/L) and manganese (C 0 = 164 mg/L) were found to be [0.599, 0.724] and [0.674, 0.941]. This indicates the efficacy of the interaction between the microbial cell surface and divalent cadmium and manganese ion under the optimized experimental conditions. The Langmuir model was able to fit the isotherm data with a high correlation coefficient. Comparing the q 0 and R L values (Table 1), Cd(II) biosorption is found to be superior to Mn(II) biosorption. Freundlich isotherm based on fuzzy linear regression. The model is represented as Eq (7): [29,30] log q e ¼ log K f þ 1 n log C e ð7Þ = where K f is a Freundlich constant relating to the binding capacity and 1/n is an empirical parameter relating to the biosorption intensity, which varies according to the heterogeneity of the biosorbents. An efficient absorption process yields a Freundlich constant n in the range of 1 to 10. A high value of n implies a stronger interaction between the adsorbent cell surface and divalent metals. The logarithmic plot of q e against C e (Fig 15C and 15D) gives the constants K f and n for the absorption (shown in Table 2). Comparison with other strains The absorption capacity of the developed method was compared against other bacterial and fungal strains. The comparison (Table 3) shows that Klebsiella sp. has significant absorption capacity when compared with other strains. Hence, the novel Klebsiella sp. is effective at binding cadmium and manganese on its surface, and its application is facilitated by its high resistance to heavy metals. Conclusion Because microorganisms lose viability in the presence of high concentrations of toxic heavy metal ions, the isolation of metal-reducing bacteria from contaminated environments is significant. The present study indicated that the strain Klebsiella sp. Yangling I2 has the ability to tolerate moderately high concentrations of heavy metal ions and exhibits relatively high, previously unreported biomass uptake (170.4 and 114.1 mg/g for Cd(II) and Mn(II), respectively). Klebsiella sp. is a newly discovered bacterium that was examined physically and biochemically. Based on 16s rRNA and whole genome sequencing, we believe that it is a new species. The surface characterization of the adsorbent also showed binding of the cadmium ion onto the surface of the adsorbent. Several factors, such as pH, temperature, initial metal concentration, agitation speed, and biomass density, were found to have a profound effect on Cd(II) and Mn (II) absorption. According to our experiment, we believe that the process of biosorption can be explained as a chemical reaction between ions and chemical groups on the surface of biomass. The Langmuir isotherm based on fuzzy linear regression was close to the curve and yielded binding constants of 0.98 and 0.86 for Cd and Mn, respectively. The main finding of this study is that Klebsiella sp. Yangling I2 can adsorb approximately 10 times more cadmium than previously reported adsorbents, and the biosorption equilibrium was determined based on fuzzy linear regression. Overall, this novel bacterium is able to remove cadmium at concentrations up to 400 mg/L, thus making the metal-removal process for environmental remediation both economical and green.

v3-fos

2019-04-25T13:05:27.273Z

2015-01-01T00:00:00.000Z

131171258

s2

Cost of water acquisition from forested watershed Estimation of the cost of water acquisition from two watershed, namely Sungai Teranum catchment area and Sungai Sia catchment area in Raub, Pahang, from the overall cost of managing the watershed by the Pahang State Forestry Department was conducted. Field measurements of river flow velocity and cross-sectional area of the rivers were conducted and the average flow rates (m/s) were determined using the formula Q=VA. It is estimated that the cost of 1 m of water generated from the Sungai Teranum catchment with an average water yield of 13868498.29 m/yr is RM 0.0019 and RM 0.00069 from the Sungai Sia catchment with an average water yield of 113345052.96 m/yr. © 2015 The Authors. Published by Elsevier B.V. Peer-review under responsibility of organizing committee of Environmental Forensics Research Centre, Faculty of Environmental Studies, Universiti Putra Malaysia. Introduction Water generation by a catchment area is a dynamic process that changes with time and circumstances. Natural external factors such as climate change and anthropogenic factors such as changes in land use pattern in the watershed or in the surrounding forested area, will affect the components of the hydrological cycle which will then influence the increase or decrease of the rate of water yield. Precipitation rates and high surface water runoff will increase water production while the rate of evaporation, transpiration and high infiltration rate will reduce the quantity of water produced from a catchment area [1]. In Malaysia, 97% of the country's water supply required for domestic, industrial and other purposes is derived from surface water namely rivers [2], with naturally defined forested watershed. The drastic rise in the demand for water due to growth in population, urban development, industrialisation and the increase of irrigated agriculture has placed additional pressure on the water resources of the country [3]. Annually, water demand is increasing at the rate of at 4% and is estimated to reach 20 billion m 3 by the year 2020 [4]. In order to ensure the water supplied from forested catchments is continuously in good quality to meet the demand, proper management of the catchment area is crucial [2,5]. Good watershed management practices can minimize the impact of anthropogenic disturbances on the reduction of the baseflow rate as to guarantee sustainable water generation [6,7]. In Malaysia's effort to ensure better governance of water resources, the National Water Policy was designed to manage the quantity, quality and reliability of the nation's water resources, with the view of achieving optimum, long-term, environmentally sustainable, social and economic benefits to society from their use. Aside from policy, there are also various pieces of legislation that are water related, containing provisions on good watershed management. [8]. Nevertheless, when it comes to water resources development, utilization and management, both the Federal and State Governments are involved. This is because the responsibility for water resource administration is fragmented and is shared among a number of Federal and State agencies, each of them having their own specific involvement in water related issues [9]. The Legislative Lists, which comprises of the Federal List, State List and Concurrent List, determine the jurisdiction and legislative powers relating to water management. Watershed management comes under the state jurisdiction which involves multiple agencies including the Forestry Department, Water Supply Department, Drainage and Irrigation Department, Department of Environment, local authorities and others [3]. The management of forested catchments seeks to effectively protect the source of raw water through integrative cooperation from all relevant parties, taking into account not only the entire watershed area, but also the immediate vicinities. The cost of managing the catchment area involves operational, maintenance and also emolument costs of manpower. Considering the total cost of managing the watershed in producing high quality water supply, questions arise on how to put a monetary value on the water generated from the catchment. Therefore, an estimation of the cost of acquiring water from a watershed can be relatively determined based on the cost of managing the watershed. This study was conducted with the objectives of estimating average total water yield from two forested water catchment area in Raub, Pahang namely, the Sungai Teranum catchment area and Sungai Sia catchment area and to relatively estimate the value of water generated based on the management costs of both catchments. This research will benefit the Forestry Department and other relevant bodies and stakeholders by giving a direct, tangible value of the raw water supplied by the forested catchments. This will be an indirect incentive to relevant bodies to sustainably manage the forested catchment as to produce an abundant amount of good and clean quality water to meet the future demand. Study area Sungai Teranum catchment area and Sungai Sia catchment area are both located in a permanent forest reserve, 10 km to the south and about 20 km to the north respectively, of Pekan Raub in the district of Raub, Pahang ( The flow rate measurement of a river depends greatly on the river width [11]. Sub-compartments were constructed accordingly along the river width. The measurements of the velocity of the river flow for each compartment were measured using a current meter depending on the water depth. For the depth ≤ 1.0 m, the velocity was measured at 60% of the total depth and for the depth > 1.0 m; two depths were measured at 20% and 80% of the total depth. Cross-sectional area of each sub-compartments were calculated and the discharge data represent the whole river was achieved through the summation of the data from the sub-compartments. The total discharge value in cubic meter per second calculated was then extrapolated to achieve the estimated value of total water yield from the catchment area in cubic meter per year. Estimation of the cost of water acquisition The cost of water generated from the catchment area was estimated based on the management cost of the watershed [12]. The management cost data for both catchment areas were obtained from the Pahang State Forestry Department. The Pahang State Forestry Department has no specific management budget for every catchment area in its domain. Rather the budget allocated was for the management of the whole forest reserve. Therefore, to relatively calculate the cost of water generated from a specific watershed, the total budget allocated was divided by the total land area of the forest reserve and multiplied by the total land area of the watershed. Estimation of the water cost was then obtained by dividing the cost of managing the catchment with the total water yield achieved. Hydrological characteristics and total water yield Hydrological characteristics of Sungai Teranum and Sungai Sia Catchment area are depicted in Table 1. Soil surface pressure, determines how penetrable the soils in the area are, as to allow water to infiltrate, while the infiltration rate determines how fast the water can infiltrate into the soil. The shear stress is the measure of the erodibility of the soil in the area. All parameters contributes to the increase or decrease of total water yield in the catchment area. The average total water yield estimated from Sungai Teranum and Sungai Sia catchment area is at the rate of 13868498.29 m 3 /yr and 113345052.96 m 3 /yr respectively, making the total water yield of Sungai Sia catchment about 88% higher than Sungai Teranum. This could be the result of many hydrological factors [1]. The characteristics of Sungai Sia catchment that have higher soil surface pressure and shear stress with lower infiltration rate as compared to Sungai Teranum, contribute to the higher total water yield derived from the watershed 1 . However, the amount estimated from both watershed can be an underestimation or overestimation as no direct correlation were made with the rainfall input. Nevertheless, the results portrayed the baseflow rate; the minimum amount of water yield, which are assumed to be available all throughout the year [1] in the respective watershed. According to Hinrichsen and Tacio [13], fresh water scarcity is being experienced globally due to the increasing demand from population growth and urbanization, especially in developing countries and more apparent during the dry season. As water exhibit properties of both renewable and non-renewable resources, they are both rate and stock limited whereby the rate of replenishment and the stock saved can be inadequate due to the high demand imposed [14]. Focusing on Malaysia, the water demand in 1996 was 28, 183 m 3 per capita [13] and is extrapolated to be around 49, 602 m 3 per capita in year 2015 at the increasing rate of 4% per year. To meet this growing demand, it is crucial to protect and conserve the watershed as to ensure sustainable water production and supply can be achieved. Hence, cooperation of all relevant bodies is important to manage and maintain the catchment area. Cost of water acquired from the forested watershed Raub, Pahang has an overall forest reserve area of about 130735 ha, whereby Sungai Teranum and Sungai Sia catchment occupies 2701 ha and 8126 ha of the area, respectively. The overall budget allocated to the management of the forest reserve is about RM 1,249,000, thus the estimated management cost for 1 hectare of land area is RM9.55/ha. The estimated cost for each catchment area and the estimated cost of water acquisition from each watershed is detailed out in Table 2. The huge 64% difference between the costs of water acquisition from both catchments could simply be explained based on the total land area of the catchment and the budget allocated for managing the catchment. As Sungai Sia catchment has a bigger total land area, with a higher rate of total water yield, thus the cost of acquiring 1 m 3 from Sungai Sia is much cheaper. Therefore, estimating roughly the cost of water demand based on the average per capita water demand of the country in 2015, the cost of water demand from Sungai Teranum and Sungai Sia catchment would be at the rate of RM94.00 and RM34.00, respectively. In 2012 the government had started the Pahang-Selangor Interstate Raw Water Transfer project as an initiative to solve water shortage issues in the state of Selangor, Kuala Lumpur and Putrajaya [15]. Relevant stakeholders are still doubtful on the aspect of the project in solving the water shortage issue as a comprehensive water conservation policy is still lacking [16]. Therefore, giving a tangible monetary value to the raw water supply from forested catchment can be an indirect indicator and incentive in developing a comprehensive policy for watershed management and protection as well as water conservation and distribution, especially in the state of Pahang which has the most abundant forest cover and watershed in Malaysia. Conclusion The average total water yield estimated from Sungai Teranum and Sungai Sia catchment area is 13868498.29 m 3 /yr and 113345052.96 m 3 /yr and the overall cost of water acquired were estimated to be RM0.0019/m 3 and RM0.00069/m 3 , respectively. Though the figure seem negligible, the impact of putting a monetary value on raw water acquisition could help in water policy development and benefit all relevant parties in tackling the inevitable water shortages in the future.

v3-fos

2018-12-13T04:53:00.118Z

2015-10-12T00:00:00.000Z

56042848

s2

Phelipanche aegyptiaca Management with Glyphosate in Potato Two years field and greenhouse studies were carried out to evaluate the efficacy of sub-lethal doses of glyphosate (Round upR), ammonia gas, phosphoric acid and sulfuric acid against Phelipanche aegyptiaca in potato. Results showed that sequential application of sub-lethal doses of glyphosate at all tested rates significantly reduced P. aegyptiaca shoot number and shoot dry weight. While, the use of ammonia gas, phosphoric acid and sulfuric acid had no significant effect on the total level of P. aegyptiaca infection as compared to the control. The best results considering both P. aegyptiaca control and selectivity in potato were obtained by sequential application of sub-lethal doses of glyphosate at 60 and 80 g·ai·ha−1. Sequential application of glyphosate at 60 g·ai·ha−1 reduced P. aegyptiaca infection by 100% after 100 days after potato emergence (DAPE). Except for sequential application of glyphosate at 60 and 80 g·ai·ha−1, all tested rates enhanced the maturity rate of potato plants and decreased the number of marketable potato tubers. Introduction Potato (Solanum tuberosum) is considered one of the most important strategic crops in the Mediterranean region. In Lebanon, the Beq'aa and Akkar provinces are the main potato producing areas in the country, with about 68% and 19% of the total production, respectively [1]. Potato is susceptible to several pests among which P. aegyptiaca. This parasitic weed parasitizes summer, spring and autumn planted potatoes across Lebanon and the Mediterranean region. Phelipanche aegyptiaca Forsk (Branched broomrape) is an aggressive root holoparasite that infects roots of various dicotyledous plants. This parasite is an invasive insidious higher plant in Lebanon and the Mediterranean region; it causes severe yield losses in Solanaceae and Fabaceae crops [2]- [4]. Severe reduction in potato yield quantity and quality were observed due to high levels of P. aegyptiaca field infestations in Lebanon [4] [5]. Management of P. aegyptiaca remains challenging for potato producers. With variable degrees of success, several control methods have been tested against Phelipanche spp. infection including physical, cultural, chemical, soil solarization, trap and catch crops, resistant varieties, synthetic germination stimulants and soil amendments [3] [6]- [13]. One of the most promising methods for controlling Phelipanche spp. is the use of sub-lethal doses of glyphosate on crops that show tolerance to glyphosate [3] [7] [8]. The plan is to spray sub-lethal doses of glyphosate on the potato leaves (POE) early in the season, so that the glyphosate would move through the crop phloem to underground Phelipanche spp. attachments on the crop roots and inhibit Phelipanche spp. growth prior to its above ground shoot emergence. Sub-lethal doses of glyphosate were found to be effective against Phelipanche spp. in broad bean [14], sunflower [15], tomato [16], carrot and celery [8] and vetch [17]. Thus, the use of sub-lethal doses of glyphosate as potential and economical method to control Phelipanche spp. has become increasingly important. To our knowledge, the use of sub-lethal doses of glyphosate against P. aegyptiaca has not been confirmed in potato, most likely because of its limited selectivity, rate, and timing of application. Inorganic compounds have also been shown to reduce Phelipanche spp. growth. Many researchers showed that high nitrogen fertilizers (NH 4 NO 3 ) and sulfur powder reduced Phelipanche spp. growth and development [10] [18] [19]. However, our literature search revealed no published work on the effect of ammonia gas, phosphoric acid and sulfuric acid against P. aegyptiaca. Acidifying irrigation water may aid in creating optimum soil pH for various crop growth [20] by mitigating a small zone from where the crop roots can better obtain nutrients. A pH range of 5.5 to 6.5 is considered ideal for most crops including potato [20], but may not be for Phelipanche spp. because the parasite is commonly distributed in alkaline soils [3]. Most soils in the Beq'aa plain are calcareous in nature with a pH ranging between 7.5 and 8.5 and are thus amenable to P. aegyptiaca infestation. The main objectives of the present work were to examine: a) P. aegyptiaca control using ammonia gas, phosphoric acid, sulfuric acid or sub-lethal doses of glyphosate; b) optimal rates of glyphosate application for P. aegyptiaca control; and c) response of potato to sub-lethal doses of glyphosate. Greenhouse Experiment A single greenhouse experiment was done at the greenhouse area of the Faculty of Agricultural Sciences at the Beirut Coastal area, during September 2009 and April 2010. Phelipanche aegyptiaca seeds were collected in 2008 from various potato fields in the Beq'aa plain and stored at room temperature. Potato tubers (cultivar Spunta) were spread on paper sheets and kept moist in the lab at room temperature three weeks prior to planting. Boxes were filled with a mixture of potting soil, perlite and peat moss at a rate of 1:1:1, inoculated with 100 mg of P. aegyptiaca seeds, and irrigated with water for a period of two weeks prior to planting potato to allow for conditioning of P. aegyptiaca seeds. Two tubers were planted in plastic netted boxes 30 × 40 × 30 cm. Methyl bromide was injected in the boxes prior to planting potato tubers. Methyl bromide was selected as another 100% free P. aegyptiaca control (comparison with inorganic chemicals). Four moist boxes containing P. aegyptiaca seeds were placed in an area of 10 m 2 covered with nylon sheet and fumigated with methyl bromide at a rate of 900 g per 10 m 2 . Sheets were removed 24 hrs after fumigation and boxes placed back in the greenhouse. Potato tubers were planted 20 days after fumigation. Glyphosate (Round up R , 48%) was sprayed on potato leaves (POE) at a rate of 125, 135, and 150 g·ai·ha −1 (no surfactant was added) in a spray volume of 1000 L·ha −1 . Spraying rates were selected based on preliminary studies performed in our laboratory. Each rate was tested for single and sequential applications at 20, 40, and 60 DAPE (20, 20 + 40 and 20 + 40 + 60 DAPE). Potato plants were 12 -18 cm tall at 20 DAPE. Spraying dates were selected based on previous experience with the species as well as on various other considerations. The underground stage of development of P. aegyptiaca in the soil is unknown [6], and seeds of the parasite in the soil keep on germinating continuously. Additionally, the best control strategy is to eliminate the parasite before it emerges above the soil [21]. Sulfuric acid or phosphoric acid was mixed with water to have a final solution of pH 2. Diluted solutions of sulfuric or phosphoric acid were then applied at a volume of one liter per box (irri-gated) every other week starting at the time of potato planting. Field Experiments General Procedure During the 2009 and 2010 spring seasons, field experiments were carried out at the Advancing Research Enabling Communities (AREC) of the American University of Beirut. The AREC is located in the Northern Central Beqa'a plain of Lebanon. A naturally infested field with P. aegyptiaca was used for both years (same field) but at different locations. The soil is a silty clay loam with a pH of 7.41, Electrical Conductivity of 0.24 ms/m and 2.4% organic matter. In both years, all plots were tilled twice with a mouldboard and disked two weeks before planting potato. The experimental area received a uniform application of 2.5 t·ha −1 of NPK (15:15:15) during planting. Nitrogen (Ammonium nitrate, 33.5%) was applied in bands 40 days after planting at a rate of 300 kg·ha −1 . All plots were sprinkler-irrigated every six days to bring the soil back to field capacity (measured by using tensiometers at 0.33 bars suction). The standard certified potato cultivar "Spunta" was planted in both years at 3.0 t·ha −1 . Potato rows were 0.70 m apart and within-the-row spacing was around 0.25 m. Plots were hilled 6 weeks after planting (standard practice by Lebanese farmers). To eliminate all weeds other than P. aegyptiaca, the entire experimental areas received a standard application of metribuzin (Sencor R , 70%, PE) at 0.75 kg·ai·ha −1 two weeks after potato sowing with a boom sprayer at a rate of 400 L·ha −1 . Glyphosate was applied POE on potato plants at 20, 40, and 60 DAPE ((20, 20 + 40 and 20 + 40 + 60). Glyphosate was sprayed by a hand held CO 2 -pressurized backpack sprayer that delivers 310 L·ha −1 at 138 Kpa through a Teejet 8002 flat fan spray tips. Irrigation water was withheld for two days after each spraying. Field Experiment in Spring 2009 Ammonia gas and sub-lethal doses of glyphosate were the only treatments tested against P. aegyptiaca. Ammonia gas was injected in the soil (at field capacity) at 30 kg·ha −1 one week prior to planting potato to a depth of 30 -35 cm. A tractor pulling a cylinder full with pressurized ammonia and connected to the injection system which released the ammonia gas at a pressure of 2 -3 bars in the soil was used. The field was then lightly irrigated to avoid evaporation of the gas. During the initial potato growth and in other plots, sequential application of foliar sub-lethal doses of glyphosate at a rate of 125 g·ai·ha −1 was applied on potato plants at 20,40, and 60 DAPE (20, 20 + 40 and 20 + 40 + 60). Experimental plots (20 m long and 10 m wide) were arranged in a randomized complete block design with three replications. Each replicate or plot consists of 12 rows. Field Experiment in 2010 Sub-lethal doses of glyphosate at 60, 80, and 100 g·ai·ha −1 were the only treatments tested against P. aegyptiaca. These rates were selected according to the results obtained from previous field and greenhouse experiments. All rates were tested for single and sequential application at 20, 40, and 60 DAPE (20, 20 + 40 and 20 + 40 + 60). A factorial experiment was used. Experimental plots (6 m long and 2.1 m wide) were arranged in a randomized complete block design with four replications. Experimental Measurements and Statistical Analyses Phelipanche aegyptiaca infection in the greenhouse experiment was assessed by counting emerged shoots and underground attachments and measuring total dry weight per box. At the end of the experiment, emerged P. aegyptiaca shoots (above ground) were counted and pulled out. Underground shoots were counted by washing the potato roots with water and separating remaining P. aegyptiaca attachments from potato roots. Phelipanche aegyptiaca infection in the field experiments was assessed by only counting emerged shoots (above ground) and measuring their total dry weight per m 2 of the middle row in the field experiments. Potato data included number of plants per box/row (5 m), phytotoxicity visual rating according to the European Weed Research Council scoring system, height/two plants/box or ten plants/plot, total and marketable yield. Potato yield was determined by harvesting potato/box or part of the middle row in each plot (5 m). Yield quality was determined by separating harvested tubers into two classes: marketable (>6 cm diameter) and non-marketable tubers (<5 cm in diameter). Statistical analyses were performed using SAS 9.2. (SAS Institute Inc., Cary, North Carolina USA). Data were analyzed statistically using ANOVA and Protected Least Significant Difference (LSD) Test at p = 0.05 level of probability was used to determine significant differences between treatments means. Greenhouse Experiment 2009/10 Methyl bromide and single or sequential application of sub-lethal doses of glyphosate at 125, and 150 g ai/ha applied 20, 40, and 60 DAPE significantly reduced P. aegyptiaca total shoot number (above and underground) and total shoot dry weight 100 DAPE compared to the control (Table 1) All these treatments reduced P. aegyptiaca infection by 100%. Sequential application of glyphosate at all tested rates significantly reduced shoot and dry weight of P. aegyptiaca 100 DAPE compared to the control. Sulfuric acid and phosphoric acid had no significant effect on P. aegyptiaca shoot number or total dry weight in comparison to the controls ( Table 1). Using sub-lethal doses of systemic herbicides as single or sequential applications have been found very effective against Phelipanche spp. in various crops. Split application with low rates of sulfunylurea herbicides inhibited P. aegyptiaca growth in potato [22] and tomato [12]. Sequential application of sub-lethal doses of glyphosate inhibited Phelipanche spp. growth in many crops [8] [14] [23]. The significance of using sequential application of systemic herbicides such as glyphosate on potato early in the season is to inhibit P. aegyptiaca growth prior to shoot emergence. Phelipanche aegyptiaca seeds are continuously induced to germinate by potato roots and develop the attachment organ, the germ tube or radicle. Sequential application of systemic herbicides may prevent the attachment of the organ or its differentiation and allow for early season control of the parasite. Injection with methyl bromide was suggested as a control strategy for Phelipanche spp. by various scientists, showing effectiveness of methyl bromide in controlling and eradicating P. ramosa [24] [25] [26]. Although methyl bromide is very effective in eradicating and killing Phelipanche spp., it is not economical for broad application, and poses significant environmental impacts [27]. The observed low efficiency of phosphoric and sulfuric acid in mitigating against P. aegyptiaca infection could be because of low concentrations applied to the boxes or because of leaching from the boxes. However, these results contradict previous findings that report that soil pH affects seed germination and development [20]. Jain and Foy [28] found that preconditioning of P. aegyptiaca seeds with phosphoric acid (21 ml of 1 Molar stock solution) decreased the parasite seed germination in tomato. Table 1. Effect of glyphosate, sulfuric acid, phosphoric acid, and methyl bromide on Phelipanche aegyptiaca above and underground shoot number (SN) and total shoot dry weight (TSDWT) 100 DAPE. Data represent average of four replicates (boxes). Means followed by the same letter, within each column, do not significantly differ at the 5% level according to the LSD test. DAPE: Days after potato emergence. Single or sequential application of glyphosate at all tested rates were toxic to the potato plants and significantly reduced potato shoot height at 30 and 60 DAPE, compared to the control ( Table 2). Sulfuric acid, phosphoric acid, and methyl bromide had no significant effect on potato plants compared to the control. Visual potato injury appeared 10 days after the first glyphosate application and included leaf yellowing, necrosis, plant stunting, and compact potato shoots. Phytotoxicity was clearly reflected in the tuber quality of potato yield grown for fresh market with a high incidence of deformed (cracked) and small tubers (Figures 1(A)-(D)). It is well known that glyphosate translocates sympoplastically and apoplastically in plants [29] [30]. During initial tuber development, tubers accumulate photosynthetic assimilates produced by the leaves and other exogenous compounds such as glyphosate. Thus, the glyphosate effect may appear as yellowing or necrosis in young leaves and malformed tubers. Field Experiment 2009 Data on the total number of P. aegyptiaca shoots indicate that sequential applications of glyphosate at 125 g·ai·ha −1 was very effective in reducing its infection as compared to ammonia gas and/or the control (Figure 2). Unlike ammonia gas, glyphosate reduced P. aegyptiaca shoot number by 97% and 99%, after 60 and 80 DAPE respectively, compared to the control. In addition, the observed shoots were stunted and almost dead. It was shown that frequent spray of glyphosate completely controlled Phelipanche spp. in parsley [23] faba bean, tobacco and tomato [14]. The reasons for the low efficiency of ammonia gas in reducing P. aegyptiaca infection are not easily discernible. They could be because of escape of volatile ammonia from the soil upon injection or because of improper injection and distribution of the gas in the soil. Results in Table 3 show that glyphosate had significantly increased total potato yield compared to the ammonia treatment and the control. Moreover, the total number of potato tubers was also significantly higher by 63% in glyphosate treated plots than both the ammonia and the control plots. However, most potato tubers were small in size (non-marketable). Table 2. Effect of glyphosate, sulfuric acid, phosphoric acid and methyl bromide on potato shoot height (PHT) and vigor (PVR). Data represent average of four replicates. Means followed by the same letter, within each column, do not significantly differ at the 5% level according to the LSD test. DAPE: Days after potato emergence; PVR: Crop phytotoxicity visual rating with 0 indicating complete death and 10 no injury. Field Experiments in 2010 Except for single application of glyphosate at 60 and 80 g·ai·ha −1 applied at 20 DAPE, all glyphosate treatments significantly reduced P. aegyptiaca infestation as compared to the control ( Table 4). Single application of glyphosate at 100 g·ai·ha −1 at 20 DAPE resulted in significant reduction of P. aegyptiaca by 65% as compared to the control after 100 DAPE. Sequential application of glyphosate at 60, 80, or 100 g·ai·ha −1 applied at 20, 40, and 60 DAPE were the most effective treatments in reducing P. aegyptiaca shoot number after 100 DAPE and shoot dry weight. These treatments reduced P. aegyptiaca infestation by 100%, compared to the control ( Table 4). These results demonstrate the previous observations that split application of sub lethal doses of glyphosate is recommended for the eradication of Phelipanche spp. Castejon-Munoz et al. [3] showed that glyphosate at 20 -40 g·ha −1 at 12 and 14 days interval eradicated more than 80% of Orobanche cemua in sunflower. Similar to the results of the green house experiment, all applications of glyphosate (60, 80, and 100 g·ai·ha −1 ) were injurious to potato plants early in the growing season (after 60 DAPE) when compared to control ( Table 5). Injuries appeared 10 days after first spraying and comprised of leaf yellowing, plant stunting, and compact plants. However, injury symptoms disappeared by 75 DAPE. Single or sequential application of glyphosate at all tested dosages had no significant effect on potato shoot height 60 DAPE. However, sequential application of glyphosate at 100 g·ai·ha −1 was toxic to potato plants and significantly reduced potato shoot height 75 DAPE. Although single and sequential applications of glyphosate at 60 and 80 g·ai·ha −1 were injurious to potato plants early in the growing season, both rates had no negative effects on total potato quantity and quality ( Table 6). While, glyphosate at 100 g·ai·ha −1 (Single or sequential) significantly reduced total potato yield and produced non-marketable tubers. Phytotoxicity was mostly reflected in the tuber quality of potato yield grown for fresh market with a high incidence of deformed and small tubers. These results are similar to the observations by Gilreath et al. [31] who observed that marketable yield of pepper declined with glyphosate dosages at 100 g·ai·ha −1 . Previous studies by Nadal et al. [32] concluded that sequential application of glyphosate at 67 g·ai·ha −1 completely controlled Orobanche crenata infestation and increased seed production of narbon bean (Vicianar bonensis L), compared to the control. Thus, selectivity remains the main obstacle in various crops and it could be mediated by the rate and time of application. Conclusion Our results suggest that sequential foliar application (20 + 40 and 20 + 40 + 60 DAPE) of glyphosate at 60 -80 g·ai·ha −1 could be used selectively to reduce P. aegyptiaca infection in potato. The timing of glyphosate application is crucial for successful control of P. aegyptiaca and could vary among potato varieties and growing seasons. Additional research is required to determine the optimum timing of glyphosate application and duration of control based on estimated P. aegyptiaca phenological underground growth stages in the field.

v3-fos

2017-04-20T02:28:05.539Z

2015-10-23T00:00:00.000Z

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Effects of exogenous proteases without or with carbohydrases on nutrient digestibility and disappearance of non-starch polysaccharides in broiler chickens The objective of the current study was to evaluate the effect of a subtilisin protease, without or with inclusion of carbohydrases, on digestibility and retention of energy and protein, as well as the solubilization and disappearance of non-starch polysaccharides (NSP) from corn-soybean meal based diets fed to broiler chickens. Two hundred eighty-eight Ross 308 male broiler chickens were used for the experiment. On d 14, the birds were weighed and allocated to 6 treatments and 8 replicates per treatment with 6 birds per replicate. Treatments were: 1) corn-soybean meal based control diet; 2) control diet plus supplemental protease at 5,000 (P5000) protease units (PU)/kg); 3) control plus 10,000 PU/kg protease (P10000); or control plus an enzyme combination containing xylanase, amylase, and protease (XAP) added to achieve protease activity of: 4) 2,500 PU/kg (XAP2500); 5) 5,000 PU/kg (XAP5000); or 6) 10,000 PU/kg (XAP10000). The enzymes in XAP were combined at fixed ratios of 10:1:25 of xylanase:amylase:protease. Data were analyzed by ANOVA and specific orthogonal contrasts between treatments were performed. Addition of xylanase and amylase increased (P < 0.05) the ileal digestibility of protein by 4.2% and 2.1% at XAP5000 and XAP10000, respectively (relative to P5000 and P10000, respectively), exhibiting a plateau after the XAP5000 dose. Increment (P < 0.05) in AME due to protease was evident, particularly in P10000. At the ileal level, XAP reduced (P < 0.05) the flow of insoluble xylose and arabinose, which indicates an increase in the solubilization of arabinoxylan polymers in the small intestine. Protease on its own reduced (P < 0.05) the flow of insoluble arabinose but did not affect the flow of insoluble xylose. XAP reduced (P < 0.05) the pre-cecal flow of insoluble and total glucose and galactose. It was concluded that whereas protease by itself improved nutrient utilization and increased solubilization of NSP components, at the lower dose, a combination of xylanase, amylase, and protease produced effects greater than those of protease alone. INTRODUCTION The use of exogenous proteases in poultry diets has gained momentum during the last decade. The first commercial protease was introduced into the poultry feed market in the 1990s in combination with other enzymes, with the aim to increase the energy and protein digestibility of grain and oilseed meal based diets (Simbaya et al., 1996). A number of proteases are now available commercially and their use has signifi-cantly increased as a result of significant pressure on the price of soybean meal, which has motivated nutritionists to further evaluate proteases for their ability to improve protein and amino acid digestibility of diets. Several studies have documented increments in the ileal digestibility of protein and amino acids of diets fed to broiler chickens with protease supplementation (Romero et al., 2013. The use of protein and amino acid matrices for protease containing products in diet formulation is becoming common practice. Nonetheless, the understanding of the mode of action of proteases in the gastrointestinal tract of chickens is still limited. Although the link between exogenous protease activity and digestion of dietary proteins is obvious, the intestinal system is complex and the effects of proteases cannot be limited to hydrolysis of dietary proteins only. Interactions between the digestions of other nutrients in the feed matrix are possible, as well as changes 2662 in the microbial communities due to modifications in the availability of easily accessible proteins in different parts of intestinal lumen (Morita et al., 1998;Scott et al., 2013). Additionally, mild interactions with the intestinal mucosa such as an increment in the thickness of the mucus layer in the intestinal lining of young chickens (Peek et al., 2009) have been reported, and ascribed potentially beneficial effects in conditions of coccidiosis challenges, which have not been fully demonstrated. Proteins are an important part of the structure of cell walls of vegetable ingredients, where they are embedded in complex matrices with carbohydrates (Parker et al., 1999). Research in ruminant in-vitro and in-vivo models has demonstrated the ability of exogenous subtilisin proteases to increase the solubilization and fermentation of hemicellulose in grains and alfalfa (Colombatto and Beauchemin, 2009). The authors postulated that these enzymes acted by removing structural proteins in the cell wall, allowing faster access of ruminal microbes to digestible substrates. To our knowledge, the effect of exogenous proteases on the disappearance of non-starch polysaccharides (NSP) has not been tested in commercially relevant diets in broiler chickens. The objective of the current study was to evaluate the effect of a subtilisin protease, without or with inclusion of carbohydrases, on ileal digestibility and total tract retention of energy and protein, as well as the solubilization and disappearance of NSP from corn-soybean meal based diets fed to broiler chickens. MATERIALS AND METHODS All the animal experimentation procedures were approved by Scotland's Rural College's Animal Experiment Committee. Birds, Diets and Sample Collection Two hundred eighty-eight Ross 308 male broiler chickens at 14 d old were used for the experiment. The birds were raised together in a group and received a standard diet that met the nutrient and energy recommendations for the breed (Ross, 2007). On d 14, the birds were weighed and allocated to 48 metabolism cages which consisted of 6 treatments and 8 replicate cages per treatment with 6 birds per replicate cage. The 6 treatments were a corn-soybean meal based control diet that met the nutrient specifications for Ross 308 broilers but was marginally low in energy (95%) and the control diet plus supplemental protease at 5,000 (P5000), or 10,000 (P10000) protease (P) units/kg, or a commercial enzyme combination (Axtra XAP; Danisco Animal Nutrition, DuPont Industrial Biosciences, Marlborough, UK) with fixed ratios (10:1:25) of xylanase (2,000 xylanase units/kg), amylase (200 amylase units/kg), and protease (5,000 protease units/kg; XAP) which was dosed to supply protease activities at 2,500 (XAP2500; 50% XAP); 5,000 (XAP5000; 100% XAP); or 10,000 (XAP10000; 200% XAP) protease units/kg. The xylanase originated from Trichoderma reesei; the amylase originated from Bacillus licheniformis; and the protease originated from Bacillus subtilis. One xylanase unit was defined as the amount of enzyme that releases 0.48 μmol of reducing sugar as xylose from wheat arabinoxylan per minute at pH 4.2 and 50 • C. One unit of Bacillus licheniformis α-amylase was the amount of enzyme required to release, in the presence of excess αglucosidase, 0.20 μmol of glucosidic linkages expressed as p-nitrophenol equivalents from a maltoheptaoside substrate per minute at pH 8.0 and 40 • C. One protease unit (PU) was defined as the amount of enzyme that releases 1.0 μg of phenolic compound, expressed as tyrosine equivalents, from a casein substrate per minute at pH 7.5 and 40 • C. Diets were manufactured in one batch, which was subdivided into 6 experimental diets, 5 of them containing enzymes. Concentrates of the test enzymes were sprayed into a wheat carrier, activity was measured and standardized, and the enzyme preparations were added to the respective diets in dry form at 0.5 g/kg after being premixed with 5 g of maize or wheat/kg of the diets. Titanium dioxide (0.5%) was added to all the diets as an indigestible marker. The ingredients and chemical composition of the diets are presented in Table 1. Excreta were collected from the birds on d 19 Table 2. Nutrient utilization (%) by broilers receiving diets supplemented with graded levels of enzymes containing protease activity alone or in combination with Xylanase and amylase activities. NC -negative control; P5000 -protease at 5,000 PU/kg; P10000 -protease at 10,000 PU/kg; XAP2500 -XAP with protease at 2,500 PU/kg; XAP5000 -XAP with protease at 5,000 PU/kg; XAP10000 -XAP with protease at 10,000 PU/kg. DM -dry matter; EE -ether extract; GE -gross energy. and 20 whereas ileal digesta were collected on d 21 following euthanasia of the birds by an overdose of sodium pentobarbital. Contents of the lower half of the ileum were obtained by gentle flushing with distilled water. The ileum was defined as the portion of the small intestine extending from the Meckel's diverticulum to a point approximately 40 mm proximal to the ileocecal junction (Ravindran et al., 2005). Digesta from birds within a cage were pooled, resulting in 8 samples per dietary treatment. The digesta samples were frozen immediately after collection, lyophilized, and processed. Chemical Analysis Chemical analyses were done according to AOAC (2006) methods unless otherwise indicated. Diets, ileal digesta, and excreta were analyzed for Ti, DM, N, ether extract, minerals, GE, NSP components, and resistant oligosaccharides (RO). Dry matter was determined by drying the samples in a drying oven (Uniterm, Russel-Lindsey Enginering Ltd., Birmingham, England, UK) at 105 • C for 24 hours (Method 934.01). Total N content was determined by the combustion method (Method 968.06) whereas ether extract was determined in a soxhlet extractor (Method 922.06). Gross energy value was determined in an adiabatic bomb calorimeter (Model 6200, Parr Instruments, Moline, IL) using benzoic acid as an internal standard. Titanium concentration in the samples was determined using the method of Short et al. (1996). Mineral content was determined using Inductively Coupled Plasma -Optical Emission Spectroscopy (Method 990.08) following digestion, in turn, in concentrated HNO 3 and HCl. Analyses for the sugar components of NSP and for RO were done using the methods of Englyst et al. (1994). Calculations and Statistical Analysis Digestibility and nutrient retention values were calculated using the index method as described previously (Olukosi et al., 2007). Calculations of the flow of NSP components were done using the concentrations of Ti in both the diet and the digesta or excreta as well as the concentration of the NSP component in digesta or excreta. All the data were analyzed using the mixed model procedure of SAS 9.2 (diets are fixed effects and the blocks are random effects) as appropriate for a Randomized Complete Block Design with pens as the experimental unit. Significantly different means were separated by orthogonal contrast tests. RESULTS The expected nutrient content of the experimental diets were met, however analyzed protease activity for XAP2500 was approximately 50% of the expected value. The analyzed PU/kg were 3,687 (for treatment P5000), 9,851 (for treatment P10000), 1,243 (for treatment XAP2500), 5,515 (for treatment XAP5000), and 10,203 (for treatment XAP10000). The anticipated activities were 2,500 PU/kg for treatment XAP2500; 5,000 PU/kg for treatments P5000 and XAP5000; and 10,000 PU/kg for treatments P10000 and XAP10000. The effect of the treatments on ileal digestibility and total tract nutrient retention are shown in Table 2. There were significant diet effects (P < 0.01) for all the nutrients and gross energy utilization at both the ileal and total tract level, except for fat digestibility at the ileal level. Supplementation of protease or XAP improved (P < 0.01) DM and N utilization relative to the control at both the ileal and total tract levels. Ileal and total tract DM and N utilization were greater (P < 0.01) in XAP5000 compared with P5000. Gross energy retention was greater (P < 0.01) in diets containing XAP or protease compared with the control and in the diet containing XAP compared with the diet containing protease alone. Phosphorus retention was greater (P < 0.01) in the diets with XAP or protease alone compared with the control and in P10000 compared with XAP10000. The pre-cecal flow of NSP components in response to the dietary treatments are shown in Table 3. Supplementation of protease or XAP reduced (P < 0.05) the pre-cecal flow of insoluble arabinose relative to the Table 3. Pre-cecal flow (g/100g dry matter intake) of components of non-starch polysaccharides in response to feeding diets supplemented with graded levels of enzymes containing protease activity alone or in combination with xylanase and amylase activities. NC -negative control; P5000 -protease at 5,000 PU/kg; P10000 -protease at 10,000 PU/kg; XAP2500 -XAP with protease at 2,500 PU/kg; XAP5000 -XAP with protease at 5,000 PU/kg; XAP10000 -XAP with protease at 10,000 PU/kg. Table 4. Post-cecal flow (g/100g dry matter intake) of NSP components in response to feeding diets supplemented with graded levels of enzymes containing protease activity alone or in combination with xylanase and amylase activities. NC -negative control; P5000 -protease at 5,000 PU/kg; P10000 -protease at 10,000 PU/kg; XAP2500 -XAP with protease at 2,500 PU/kg; XAP5000 -XAP with protease at 5,000 PU/kg; XAP10000 -XAP with protease at 10,000 PU/kg. control but had no effect on other components. Supplementation of XAP alone reduced (P < 0.05) the prececal flow of all NSP components except soluble xylose, soluble glucose, and soluble arabinose on which the enzyme had no effect. The pre-cecal flow of insoluble and total xylose, total glucose, as well as insoluble and total arabinose was lower (P < 0.05) in XAP5000 compared with P5000. However the pre-cecal flow of NSP components was not different between XAP10000 and P10000. The data of post-cecal flow of NSP components are shown in Table 4. Diets supplemented with protease or XAP had lower (P < 0.05) post-cecal flow of total glucose, soluble galactose, and total galactose relative to the control; XAP supplementation reduced (P < 0.05) post-cecal flow of insoluble xylose, insoluble glucose, and insoluble galactose relative to the control diet. Treatment XAP5000 had lower (P < 0.05) post-cecal flow of insoluble and total glucose, as well as insoluble and total galactose compared with P5000. In addition, XAP10000 had lower (P < 0.05) post-cecal flow of insoluble glucose and insoluble galactose than P10000. The data on flow of the total NSP are shown in Table 5. Supplementation of XAP reduced (P < 0.01) pre-cecal flow of insoluble and total NSP relative to the control and protease supplementation decreased (P < 0.05) post-cecal flow of soluble NSP relative to the control. XAP5000 treatment had lower (P < 0.05) pre-cecal flow of insoluble and total NSP compared with P5000. The flow of RO (Table 6) was lower (P < 0.05) in diets supplemented with XAP compared with the control. In addition, pre-cecal and post-cecal flows of galactose and glucose in the RO, as well as the total RO were lower (P < 0.05) in diets supplemented with protease compared with the control. The pre-cecal flow of glucose in the RO fraction was lower (P < 0.05) in XAP5000 compared with P5000. The pre-cecal flow of galactose and total RO was lower (P ≤ 0.05) in XAP10000 compared with P10000 treatment. DISCUSSION The objectives of the current study were to assess the ileal digestibility and total tract retention effects of protease without and with carbohydrases and the effects of the enzymes on the disappearance of NSP component sugars in 21-day-old broiler chickens fed a Table 5. Pre-and post-cecal flows (g/100g dry matter intake) of non-starch polysaccharides in response to feeding diets supplemented with graded levels of enzymes containing protease activity alone or in combination with xylanase and amylase activities. NC -negative control; P5000 -protease at 5,000 PU/kg; P10000 -protease at 10,000 PU/kg; XAP2500 -XAP with protease at 2,500 PU/kg; XAP5000 -XAP with protease at 5,000 PU/kg; XAP10000 -XAP with protease at 10,000 PU/kg. Table 6. Pre-and post-cecal flows (g/100g dry matter intake) of resistant oligosaccharides in broilers in response to feeding diets supplemented with graded levels of enzymes containing protease activity alone or in combination with xylanase and amylase activities. NC -negative control; P5000 -protease at 5,000 PU/kg; P10000 -protease at 10,000 PU/kg; XAP2500 -XAP with protease at 2,500 PU/kg; XAP5000 -XAP with protease at 5,000 PU/kg; XAP10000 -XAP with protease at 10,000 PU/kg. corn-based diet. Diets contained 10% corn-Distillers' Dried Grains with Soluble (DDGS), which increased the presence of total NSP compared with simple cornsoybean meal based diets. The experimental design allowed direct comparison of 2 doses of protease (5,000 or 10,000 PU/kg) versus enzyme combinations containing protease at the same doses, along with xylanase and amylase at fixed ratios with protease. An additional treatment tested the XAP combination at a lower dose, corresponding to 2,500 PU/kg. The single subtilisin protease increased the apparent ileal digestibility of N particularly at the high enzyme dose (10,000 PU/kg), which agreed with previous reports with the use of mono-component alkaline proteases in broilers fed corn-based diets (Angel et al., 2011). Nitrogen digestibility was 4.2% points higher in diets containing XAP relative to diets with protease alone even when protease was at 5,000 PU/kg in both diets. At 10,000 PU/kg, N digestibility was 2.1% points higher with XAP compared with diets containing protease alone. This likely indicates a plateau of the effect of XAP on N digestibility between XAP5000 and XAP10000. Interestingly, N digestibility values in XAP5000 and P10000 were not different. It can be inferred from this that the effect of carbohydrases on top of protease was not additive after a certain threshold of protein digestibility. Recently, Romero et al. (2013Romero et al. ( , 2014 studied the apparent amino acid and protein digestibility effects of protease applied on top of carbohydrases in 21-day-old broilers, and reported increased protein digestibility as a result of the addition of protease, in contrast with more inconsistent effects of carbohydrases on protein digestibility. In the present study, the use of carbohydrases increased protein digestibility beyond that of protease only at a specific dose. These findings suggest that additivity of protein and amino acid matrices of proteases and carbohydrases cannot be generally assumed in diet formulation. In the current study, a significant increment in the AME due to protease was evident, particularly at 10,000 PU/kg. Interestingly, no additivity of proteases and carbohydrases on energy utilization was evident at this dose, but this was evident at 5,000 PU/kg. Romero et al. (2013) reported marginal increments of AMEn with the addition of 5,000 PU/kg of the same protease on top of xylanase and amylase in corn-based diets fed to 21-day-old chickens, which was explained by the caloric content of increments of protein digestibility due to the enzyme. In another study, Romero et al. (2014) reported increments in AMEn with the addition of this protease on top of xylanase and amylase in cornand wheat-based diets using 21-day-old and 42-day-old chickens, with greater increments in older birds, and in wheat-based diets, particularly with inclusion of corn DDGs and canola meal. In some particular diets of that study, the increments in energy digestibility with the addition of protease were not explained by increments in the digestibility of starch, fat, and protein; therefore, the authors suggested a possible effect on the ileal disappearance of NSP which was not measured in that study. The current study measured the flow of individual sugars within the soluble and insoluble NSP fractions. The flow of NSP components is an indication of the concentration of NSP components in the dry matter. Logically, greater hydrolysis will reduce the amount of NSP components that is detected in the digesta or excreta and consequently lower values for NSP flow is an indication of greater NSP hydrolysis in the particular diet. Generally NSP flows, pre-and post-cecal, were lower in diets with XAP compared with diets containing protease alone. This is an indication that combination of enzymes with carbohydrase and protease activities was more effective in reducing NSP flow than enzyme with protease activity alone. Others have similarly reported improved NSP degradation following the use of exogenous enzymes (Korcher et al., 2002;Ouhida et al., 2002;Barekatain et al., 2013). At the pre-cecal level, XAP was able to reduce the flow of insoluble xylose and arabinose, which are the most important components of hemicellulose in corn and corn-DDGS. This observation indicates an increase in the solubilization of arabinoxylan polymers in the small intestine. The xylanase used in the current study was an endo-1-4xylanase of the GH11 family, which has been reported to cleave the xylose backbone of the arabinoxylan chain (Courtin and Delcour, 2002;Hu et al., 2008). Therefore, it would be expected that the presence of xylanase would reduce the flow of insoluble xylose, which would also bring arabinose substitutions into the soluble fraction. There is close relationship between fiber and protein in corn (Rybka et al., 1992;Parker et al., 1999) and hence the use of protease by itself may have a limited effect on NSP hydrolysis by attacking the structural protein or glycoprotein as suggested by Cowieson (2010). The corn endosperm proteins are of 2 types, one of which is the matrix that also envelops the starch granules (Parker et al., 1999). Close fiber-protein interactions have been reported for cereals and oilseeds (Ouhida et al., 2002;Duodu et al., 2003). Interactions such as cross-linking is a physico-structural barrier to hydrolysis of structural and storage carbohydrates and the hydrolysis of the structural proteins by protease will in itself increase NSP hydrolysis. Consequently, it can be expected that the use of protease along with the carbohydrase will increase the efficacy of the carbohydrase as evidenced through decreased NSP component in the current experiment. It is interesting to note that the only NSP component which protease reduced its pre-cecal flow is insoluble arabinose, and this may be related to the ability of protease to increase access of the digestive enzyme to structural carbohydrate and thus reduce cellular integrity. The XAP combination caused a reduction of the total flow of xylose and arabinose, which indicates disappearance, likely due to fermentation in the small intestine, which was not evident with the use of protease on its own. It appears that protease contributed to opening the tri-dimensional structure of hemicellulose, without a significant solubilization of arabinoxylo-oligosaccharides, for bacterial fermentation. Evidence for effects of xylanases in the solubilization and fermentation of fiber in the ceca of 21-day-old broiler chickens have been reported in corn and wheat based diets (Kiarie et al., 2014). However, reports on the disappearance of fiber in the small intestine are conflicting and have been limited to wheat based diets. Choct et al. (1999) reported that a combination of xylanase and protease reduced the production of short chain fatty acids (SCFA) in the ileum, but increased it in the cecum of 29-day-old broilers. In contrast, Wang et al. (2005) reported that a carbohydrase combination increased SCFA production in the ileum of broilers at 21 d, but had no effect at 42 d, whereas the production of SCFA in the cecum was increased at both 21 and 42 d. Logically, the activity of exogenous xylanases that are active in the gastrointestinal tract of chickens would be initiated as soon as the enzyme is released and moisture is sufficient to allow enzymatic activity, and would potentially solubilize hemicellulose in the small intestine. The effect of protease alone on NSP flow was more apparent at the post-cecal level. This activity of the protease was likely indirect, perhaps involving activity of intestinal bacteria. Evidently, protease does not have the same ability as xylanase to solubilize xylose but appeared to allow a preferential solubilization of arabinose substituted side chains. It is established that microorganisms are both more abundant and of greater variety post-ileal (Gong et al., 2007;Yeoman et al., 2012) and hence it is reasonable to conclude that this contributes more to the hydrolysis of NSP observed postileal. There is the possibility that the action of microorganisms that are capable of hydrolyzing NSP, even if partially, will further enhance the ability of protease to hydrolyze structural carbohydrates that are, very likely, already partially weakened by microbial activity. In addition, at the post-cecal level, protease reduced the flow of many of NSP constituents, which may be the result of the joint action of both the protease and microbial activities. Previous research using in-vitro and in-vivo ruminant models demonstrated the ability of exogenous subtilisin proteases to increase the solubilization and fermentation of hemicellulose (Colombatto and Beauchemin, 2009). The authors postulated that these enzymes acted by removing structural proteins in the cell wall, allowing faster access of ruminal microbes to digestible substrates. Taken as a whole, these results confirm the hypothesis of an indirect effect of this subtilisin protease in the solubilization and disappearance of different NSP sugar components. Those effects were certainly evident at the post-cecal level as confirmed by a significant effect in the disappearance of glucose and galactose from the NSP fraction. However as expected, effects on solubilization and the disappearance of NSP due to protease were not as vigorous compared with a combination of protease with xylanase and amylase. The experimental design did not allow a precise evaluation of additivity or synergy between these 3 enzymes, but certainly demonstrated that protease played a role in the solubilization and disappearance of fiber in this enzyme combination, which was enhanced when xylanase and amylase were present. Based on the extensive research of exogenous xylanase in poultry feed (Adeola and Cowieson, 2011;Slominski, 2011) and its biochemical activity, it can be inferred that xylanase, as opposed to amylase, was the major activity to produce these effects. The most important RO in corn-soybean meal based diets are raffinose and stachyose, which together can make up to 5% of soybean meal (Choct et al., 2010). Additionally, β-mannans are also present in soy, but at lower concentrations compared to raffinose plus stachyose (Choct et al., 2010). Effects of enzymes in the flow of sugars from RO were analyzed with the understanding of the lack of specificity of the enzymes used to target the direct hydrolysis of bonds from these α-galactosides and from β-mannans. Protease reduced the flow of glucose and galactose from the RO fraction only post-cecal whereas XAP reduced the flow of these RO sugars in both pre-and post-cecal digesta. These effects were likely the result of fermentation resulting from the solubilization of the major fiber component of chicken feed, i.e., arabinoxylans from grains. These RO have been ascribed both prebiotic (Spring et al., 2000;Choct, 2006) as well as negative effects on gut health (Coon et al., 1990) depending on what responses have been measured in specific studies and what diets and animal models were used. The nature of these effects is likely dependent of the concentration of the specific RO as well as the health status of the flock. In any case, a reduction of the flow of RO through fermentation would probably have a neutral or beneficial effect in animal performance and gut health if it avoids negative effects of high concentrations of RO in the diet, or contributes to prebiotic effects of the same RO. What is not yet clear is the additional value of exogenous prebiotics in chicken diets, which naturally contain high levels of potentially prebiotic NSP, particularly when exogenous fibrolytic enzymes are used, some of which are capable of increasing the production of prebiotic oligosaccharides in-situ. It can be concluded from the current study that whereas protease by itself improved nutrient utilization in corn-soybean meal diets and increased solubilization of NSP components, at the lower dose, a combination of xylanase, amylase, and protease produced effects greater than those of protease alone. However, the current data do not indicate an additivity in the effect of the enzymes when used in combination at higher doses.

v3-fos

2018-05-29T17:12:32.116Z

2015-10-09T00:00:00.000Z

44130228

s2

Fruit Phenology of Tree Species and Chimpanzees’ Choice of Consumption in Kalinzu Forest Reserve, Uganda One hundred and eighteen (118) tree species were identified, among which 58 species produced fruit within the two-year study. Fruit of only 26.3% of the latter is eaten by chimpanzees. The consumption of each of these fruits was generally low, with only two species constituting more than 25% consumption. Only about 1.7% of woody biomass is relied upon by chimpanzees in Kalinzu for food. The major tree species in chimpanzee diet monitored showed that fruit production varies monthly and seasonally. Apart from Musanga leo-errerae and Ficus spp. whose fruiting was consistent throughout the year, general fruit phenology was positively correlated with rainfall. Only three species namely: Craterispermum laurinum, Aframomum angustifolium and Beilschmiedia ugandensis produced fruit in the dry seasons. Correlation between fruit availability and consumption was significantly positive for only one species, Landlophia dawei. This indicated that frugivory of chimpanzees in Kalinzu was not opportunistic; they search for what they like to eat. Chimpanzees would have to range furthest in periods of scarcity and asynchronous fruiting hence a lot of energy expenditure in the food search alone. Therefore, diversity in fruit phenology is important for chimpanzees’ energy conservation, health and survival. Selective logging and other selective human activities that involve cutting down trees that are palatable would in future affect the food diversity and consequently the health of frugivores if not done sustainably. Since patterns of fruit phenology are also linked to patterns of rainfall, changes in the former can assist in predicting the influence of climate change on food availability for big frugivores like chimpanzees. Introduction Knowing the dynamics of fruit production is important for understanding great apes' (chimpanzees, gorillas, and bonobos) ecology, as fruit provides a nutritious resource for most of them in tropical African regions. Plant species composition, density, distribution and temporal fluctuation affect apes' ecological features such as diet [1]- [5], ranging patterns [6]- [8], group size [9] [10], reproductive parameters [11], patterns of social interaction [12] and tool-using behavior [13]. Comparison of fruit phenology is also important for understanding the ecological differences in great apes among different study sites. The patterns of fluctuation in party size of chimpanzees are different among four chimpanzee study sites and it was suggested that the differences in dynamics of fruit production among sites were the cause [9]. The annual and seasonal fluctuations in fruit production are distinct, and a period of fruit scarcity exists at all study sites [14]. In Central Africa, fruits are more abundant in the rainy season than in the dry season. In Lopé National Park (Gabon), fruit abundance peaks in the early rainy season and fruits are scarce during the long dry seasons. Fruit abundance peaks may also occur during the mid-rainy season and become scarce during the dry season [2] [6] [15]. In East Africa, climatic factors influencing fruit phenology are varied among sites. In Mahale Mountains National Park (Tanzania), the number of plant species in fruit declines in December, January and February during the rainy season [16]. In Kibale Forest (Uganda) and Kahuzi-Biega (DRC) National Parks, fruit abundance is negatively correlated with monthly rainfall, and more fruits are available during the dry season [8] [17]. In contrast, peaks in fruit abundance occur during the rainy season in Budongo [18]. In the two sites of West Africa, Taï National Park (Cotê d'Ivore) and Bossou (Guinea), fruits tend to be abundant during the dry season [13] [19]. Even within the same habitat, patterns of fruit production differ among species, plant life forms, and vegetation types, and therefore periods of fruit scarcity also vary. Frugivorous animals respond to periods of fruit scarcity by changing their dietary composition and/or ranging patterns [6] [8] [14] [20]. Several primate populations like vervet monkeys (Cercopithecus aethiops) [21] and baboons (Papio anubis) in Amboselli, Kenya, and Toque macaques in Sri Lanka [22] have been documented to decline in number with a natural decline in their food resources. In Kalinzu forest, selective commercial logging and pit sawing continue to occur, especially in the eastern part [23] [24]. The former Nkombe saw mill, a logging company, had been mechanically logging in the north eastern part of the forest and exclusively harvested Parinari excelsa. Local people have been harvesting/logging some useful trees, such as Carapa grandiflora and Funtumia africana, in some areas. These logging activities created some patches of secondary vegetation. Phenological patterns are linked to factors that govern forest health such as population biology of pollinators, seed dispersers and predators, herbivores, interspecific competition among trees and other processes of primary production [25] and assist conservation scientists in predicting consequences of adverse climatic events. In this paper, overall fruit abundance and patterns of fruiting of major species are analyzed in relation to rainfall and chimpanzee feeding patterns. This will provide baseline data for management options occurring in Kalinzu Forest Reserve especially during periods of tree harvests and habitat-wide fruit scarcity since this has a dietary implication on population dynamics of frugivores like chimpanzees. Study Area In Uganda, chimpanzees are restricted to the western part and the total number is estimated at 4950. Of these, 230 occur in Kalinzu at a density of 1.55 km −2 ranking third after Kibale at 2.32 km −2 and Bugoma at 1.9 km −2 ( Table 1) [26]. Kalinzu Forest Reserve lies in south-western Uganda (0˚17'S and 30˚07'E). It covers 137 km 2 and borders the Maramaganbo forest and the two form part of the Queen Elizabeth National Park. Kalinzu forest is one of the richest forests in Uganda, with more tree species recorded here than anywhere else. Three important timber species from this forest are considered endangered. These include Cordia milleni, Entandrophragma angolense, and Lovoa swynnertonii [27]. It is inhabited by six species of primates including the largest population of l'hoest's monkeys (Cercopithecus l'hoest), a species considered vulnerable to extinction. Kalinzu together with Maramagambo forests represent the largest tract of forests at this altitude in Uganda. Kalinzu covers an exceptional altitudinal range, which combined with its topographical, climatic and geological diversity, and its location in western Albertine rift valley close to the believed upper Pleistocene forest refugia, gives rise to a great variety of forest habitat. The Forest occupies a shallow saucer-shaped depression in the rift valley escarpment with its floor at about 1463 m above sea level from which rise a few grassy hills that reach up to 1845 m. The vegetation of Kalinzu is broadly classified as medium altitude moist evergreen tropical rain forest [27]. According to the most recent classification, Kalinzu forest vegetation can be simplified into four vegetation types: mixed mature forest, Parinari dominated mature forest (Parinari mature), Parinari dominated secondary forest (Parinari secondary), and Musanga dominated secondary forest (Musanga secondary) [28]. The rainfall pattern has two peaks-in April and October. The June-July dry season is more severe than the one in January. The temperatures of Kalinzu Forest Reserve range from the minimum of 13˚C to the maximum of 28˚C. Methods Ten parallel transects 5 km long and 5 m wide running east to west of Kalinzu forest were used to collect data. Distance between transects was 500 m. Each of these transects was divided into ten 500-m sections totalling to 100 plots. All trees above 10 cm in diameter at breast height (DBH) within 2.5 m of each side of the transect were recorded. The number of trees of each species in 5 × 500 m block was used as a variable for the analysis of Species Important Value Index (SIVI). A total of 25 km 2 of vegetation was sampled. SIVI values were calculated to give a comparable index incorporating measures of tree species distribution, relative density and abundance. The SIVI value is therefore, the summed value of relative density, relative frequency and relative dominance. The sum of these three values for the particular species, the species importance value index (SIVI) never exceeds 300 because each parameter is a percentage ranging from 0 to 100 [29]. Tree species were ranked on the basis of their SIVI values. A fruit phenology census was conducted along each of these 10 transects for two years. During each census, fallen fruit was counted. Fallen fruit from one tree, 2.5 m of each side of the transect, was considered as one fruit cluster. When more than one tree of similar species contributed to the cluster of fruit on the ground, it was divided amongst the number of fruiting trees. The number of clusters thus matched with the number of fruiting trees that dropped fruit within the census belt. The tree species, the numbers of fruit in each cluster and whether the fruit would be ripe or unripe (determined by majority) was recorded. The numbers of fruit in each cluster was grouped as 1 -4, 5 -9, or >10, and assigned a frequency score of 1, 3, or 9 respectively, then a fruit abundance index (FAI) was determined from the total number of scores per hectare as explained in [1]. The FAI of fruits eaten by chimpanzees was calculated separately. To examine the relationship between number of fruit in the tree and number of fallen fruit, major trees whose fruit was recorded to be eaten by chimpanzees during the preliminary faecal analysis were randomly chosen on the transects and monitored for fruit production. A record of ripe and unripe fruit was taken. The pattern of fruit consumption by chimpanzees reported in this study was determined by faecal analysis [2]. It was supplemented by direct observations of feeding where possible, to confirm the consumption of foods that were seen in faecal sample remains. Fresh chimpanzee faecal samples were collected but those that scattered on the ground or among branches were ignored due to the difficulty in picking a representative sample of such. After a day's collection, the faecal samples were preserved in plastic bags with 100% ethanol and the following noted: date of collection, location, time of collection, and the visible contents of the faecal sample by majority constituent. A total of 2635 faecal samples were collected in 26 months (monthly range 24 -206; mean = 101). Each month, the faecal samples were placed in a metal sieve with 1mm mesh and washed in running water. Once the soluble solution had gone, the seeds were sorted. The samples were then sun dried and divided into categories: seed, skin, and pulp of fruit, leaf matter, pith, ant, mammal, entire leaf, or others. We evaluated the percent volume of each food category in each sample, where the smallest unit would be 5%. We recorded species names except where more than one species in the same genus could have indistinguishable seeds e.g. Ficus spp. Indistinguishable seeds in this genus were treated as a single fruit "species group". One identifiable fruit species meant one fruit species group. Kalinzu Forest Tree Composition A total of 16,778 trees belonging to 118 species (6 of which remained unidentified) and 44 families were recorded. The identified tree species and their Species Importance Value Indices are as shown in Table 2. Of these Fruit Phenology and Availability The fruit availability indices (FAI) of all species indicate that Celtis durandii produced most fruit followed by Strombosia scheffleri (Alocaceae) which produced 16% of total fruit abundance ( Table 2). Fruit monitoring produced different categories of fruiting phenology. Only major trees whose fruit were eaten by chimpanzees were monitored. The fruit trees exhibited different fruiting peaks. The presence of young and mature fruit of these species is shown in Figure 1. Generally most mature fruit was abundant in the rainy season, except for Afromomum angustifolium where the fruiting peak appears during the late dry season. There was almost no ripe fruit for Myrianthus holstii, and Drypetes bipidensis in the months of August and September. Pseudospondias microcarpa and Bielschimedia ugandensis did not fruit in March, January and November respectively. Amount of fruiting also varied between years and some trees like Bielschmiedia ugandensis were observed to fruit once in the two years. Musanga leo-errerae and Ficus spp showed perennial fruiting. For purposes of illustration, phenologies of the six ficus species (Ficus sur, Ficus thoningii, Ficus saussreana, Ficus vallis choudae, Ficus bubu and Ficus natalensis) that were monitored are combined and taken as one species group. The highest diversity in fruiting of trees eaten by chimpanzees was recorded in the short (January-February) and the long rainy season (September-December). There was a positive correlation between fruiting and rainfall within Kalinzu Forest Reserve (r = 0.603; P = 0.008). Seasonality in Fruit Availability and Consumption Availability of fruit eaten by chimpanzee fluctuated both monthly and seasonally and so did the frequency of occurrence of fruit seed in chimpanzee faeces. After the analysis of 2635 faecal samples, seeds of 33 fruit species were identified. Of these, five species were found in very minimal amounts in chimpanzee diet. These include Vangueria apiculata, Alchonea hitera, Antiaris toxicaria, Fagaropsis angolensis and Syzigium guinense. The trees that produced the largest crop of fruit like Celtis durandii and Craterispermum laurinum did not occur prominently in chimpanzee diet. Craterispermum laurinum fruits however supplemented chimpanzee diet for two months during the dry season (period of scarcity). The tree species, whose fruit were eaten in greater amounts and frequencies, had medium fruit abundance. Musanga and Ficus species occurred in faeces all year round. Myrianthus holstii, Afromomum angustifolium, Landolphia dawei, Pseudospondias microcarpa, Mimusops kummel and Uvariopsis congensis occurred in faeces in the rainy season. Celtis durandii, Beilschmiedia ugandensis, Drypetes bipidensis and Phytolacca dodecandra occurred in mid season between the rainy and dry seasons. Craterispermurm laurinum and Monodora myristica's occurrence in faecal samples coincided exactly with their availability during the dry season. The occurrence of seeds of some fruit species in chimpanzee faeces was positively correlated with their availability in the habitat. These included Myrianthus holstii, Uvariopsis congensis, Monodora myristica, Bielschmiedia ugandensis, Landolphia dawei and Craterispermum laurinum, suggesting that these fruits were consumed as they were available. This correlation was however significant only for Landolphia dawei. The monthly mean of fruit availability index of Musanga leo-errerae, Drypetes bipidensis, Pseudospondias microcarpa, Ficus spp. and Celtis durandii did not correlate with their monthly frequency in chimpanzee faeces. Only tree species were considered in this analysis, therefore fruits of Afromomum angustifolium (herb) and Phytolacca dodecandra (shrub) were not included in this analysis (Figure 2). Discussion Tree species diversity and production associated with high altitudes have been documented to be low [30]. Kalinzu forest has a medium altitude that is between montane and lowland categories. This may explain the lower density of tree species whose fruit is eaten by chimpanzees in Kalinzu than other forests at lower altitudes. Of the total 118 recorded species only 33 (26.3%) were recorded to be eaten by chimpanzees. Of these 33, only two species were eaten in percentage abundance of more than 25%. This shows that about 1.7% of woody biodiversity supports chimpanzees in terms of diet Kalinzu forest. The same trend occurs worldwide. According to [31] and [32] less than 1% of the plant diversity in the tropics sustains almost all frugivore communities. Therefore there is an urgent need to sustainably maintain and improve the already existing food source for the endangered chimpanzees and other endangered frugivorous species of the tropics. Human beings through their various forms of forest utilisation are one of the factors that can affect tropical forest reproductive patterns. One of such utilisations is logging. Despite the continued logging, the top timber forest species still greatly dominate in Kalinzu forest. The tree species whose fruits are eaten by chimpanzees are poor in terms of timber provision; therefore cutting of timber species would not affect chimpanzees greatly in terms of food availability. Timber harvesting leads to creation of gaps that cause colonisation of secondary tree species that are important sources of food for chimpanzees. Secondary vegetation may certainly help chimpanzee survival because of the high density of important foods such as Terrestrial Herbaceous Vegetation (THV), Ficus and Musanga genera [23] [33] [34]. In Budongo, however, there was no evidence that chimpanzees benefited from logging, although some other primates did [35]. In Kibale, the density of chimpanzees was significantly lower in the logged plot [36] [37]. In Kalinzu, the chimpanzee density was very low in the Parinari secondary forest, although the density in the Musanga secondary forest was high. It is indicated that where log-ging has taken place, the density of chimpanzees is often lower than that in mature undisturbed forests [35] [37]. The value of secondary vegetation may therefore depend on the original vegetation type, the manner and frequency of logging and time elapsed since latest logging. Logging is one form of disturbance that affects fruiting patterns in tropical forests because it alters the microhabitat and distribution of conspecific trees, consequently influencing fruit and seed production [38]. Duration, frequency and intervals of fruiting episodes also differ among trees of different successional status. Pioneer species produce fruit every year and have extended fruiting periods. In contrast, primary forest species have irregular fruiting periods and sometimes experience masting. Masting, a phenomenon in which synchronous reproduction within a plant in one year is followed by an interval in which few or no fruits are produced was not seen in Kalinzu. Some of the primary forest fruit species eaten by chimpanzee like Beilschmiedia ugandensis and Pseudospondius microcarpa showed only one peak of fruit production during the long rainy season. Craterispermum laurinum and Monodora myristica were uni-modal and important for the chimpanzees in the dry season. Uvariopsis congensis was unimodal and fruited during the long wet season. Therefore, different fruit species have different seasons of fruiting and the interval period between fruiting cycles differ from species to species. Musanga leo-errerae and most of the Ficus sp. (except Ficus natalensis), on which chimpanzees depended mostly, did not show marked seasonality. There was however an increase of fruit production of both genera with increase in rainfall. The continued occurrence of the fruit of Ficus species can be explained by the presence of aganoid wasps responsible for pollination in Ficus species [39]. This ensures all year round production of fruit. When one species/tree is running out of fruit, another starts ripening while another has immature fruits and others flowering. Ficus sur had three high peaks of fruit production and short-lived low peaks which meant an almost continued existence of this fruit throughout the year [40]. Fruiting among other major figs was largely bimodal. Similarly, in Budongo, the availability of figs was important especially in the dry season, because other fruiting trees tended to have fruits in the wet season [41]. Figs were also important for chimpanzee social interaction because they have wide canopies and produce large crops when in fruit [42]. Figs that occurred in Budongo also occur in Kalinzu except Ficus mucuso, Ficus varifolia, Ficus asperlifolia and Ficus polita. F. mucuso and F. varifolia have 2 -3 years interval [40] without any fruit production and similar findings were recorded in other tropical forests. These could therefore have not accounted for major differences in Ficus fruit availability if they were present in Kalinzu forest. In Musanga, the type of inflorescence ensures continued growth and production of fruit throughout the year as the preliminary study by [43] also documented. The ants of genus Azeteca found in the internodes of Musanga stem may also play a big role in pollination. General fruiting patterns were distinctly seasonal, with different peaks in both ripe and unripe fruit production. In most lowland tropical rain forests, there is seasonal variation in the phenological patterns of tree species during the annual cycle [44]. Factors including temperature, rainfall, humidity levels, and day length were found to be important in determining such patterns [45]- [48]. In Kalinzu forest, a higher number of species fruited during the wet season than during the dry season indicating that rainfall is one of the major factors that influence fruiting. This was also indicated by [49] that the monthly number of species that fruited in each forest condition was significantly related to monthly rainfall. The tree species whose fruit were eaten by chimpanzees during the wet season did not greatly influence fruit abundance. Chimpanzees relied on them when they are abundant, but they did not play a significant role since the difference between periods of abundance and periods of scarcity as a result of fruiting in wet season was small. Similar seasonal patterns in plant production in Kibale forest with almost same altitude as Kalinzu and where climate is similarly seasonal were documented [44]. The lack of correlation between fruit availability index and occurrence of fruit seed in faeces of some species suggests that chimpanzees are not opportunistic feeders, and they actively search for these fruits. Fruit production of trees constitutes the major food sources for frugivorous primates, and its abundance and seasonal availability may have great influences on the density of these primates [50]. On the other hand, a combination of various types of trees with both seasonal and annual fruiting may certainly benefit chimpanzees [51] [52]. Therefore the pattern and length of the fruiting period of these species play an important role in survival of chimpanzees in Kalinzu where only a limited number of species offer ripe fruit at the same time in both the dry and the long rainy seasons. Chimpanzees have been documented to range most in areas of high fruiting and in areas with important foods especially during fruit scarcity [53] [54]. This means that chimpanzees range furthest during fruit scarcity in search of food that is not readily available. Fruit availability is therefore important in the conservation of chimpanzees in terms of energy costs during fruit search. With abundant availability of fruit, chimpanzees will range less and conserve energy.

v3-fos

2019-03-20T13:04:11.689Z

2015-04-16T00:00:00.000Z

83594408

s2

Relationship between antioxidant properties and GC-MS component composition of extracts from flowers , leaves and fruits of Crataegus Oxycanta The aim of this study was to perform a comparative investigation of the antioxidant effect of Crataegus oxycanthas’ flowers, leaves, green and ripe fruits probes and to relate the obtained results with the ones of the accomplished qualitative GC-MS analysis of the antioxidant components participating in the studied standardized ethanol extracts. We established that the three extracts have well manifested antioxidant properties in three biological relevant model systems with different mechanism of generation of ROS: Fe-induced lipid peroxidation, UV induced deoxyribose damage and horse-radish peroxidase (HRP)-H2O2 chemiluminescent system. In the UV system the strongest effect is observed for the extract from flowers and leaves (C-50 = 0.109±0.009 mg/ml), followed by the ones from ripe fruits (C-50 = 0.259±0.015 mg/ml) and finally – by green fruits (C-50 = 0.557±0.014 mg/ml). In the Fe-induced lipid peroxidation system the antioxidant effect of the studied samples decreases in the following order: effect of flowers and leaves (C-50 = 0.287±0.018 mg/ml) > effect of green fruits (C-50 = 0.495±0.021 mg/ml) > effect of ripe fruits (C-50 = 0.704±0.035 mg/ml). Similar results were obtained in the HRP-H2O2 system. The generalized results show that the strongest antioxidant effect is observed for the plant extract from flowers and leaves of Crataegus. The GC-MS analyses we carried out indicate that the observed differences are due to the presence of a bigger number of constitutes possessing antioxidant properties in flowers and leaves extracts, compared with the fruits extracts. Indexing terms/ INTRODUCTION Contemporary pharmaceutics and cosmetic industry turn nowadays more frequently towards natural products when preparing drugs. This is connected to the fact that many patients prefer to be treated with herbal drugs, as it is believed that such preparations have less side effects than their synthetic analogues. The usage of drugs for medication purposes however requires numerous investigations of their composition and biological effect. Crataegus species have been known with their beneficial therapeutics effects since ancient times. [1,2,3]. The evaluation of the evidences in the literature reviews clearly demonstrates the immense effect of several hawthorn species used mainly for cardiovascular disease which are among the leading causes of mortality, disability and reduced quality of life in developed countries [1,4]. Crataegus oxycantha possesses valuable medical application in releasing the symptoms of cardiovascular diseases such as hypertension, hyperlipidemia, and in particular -congestive heart failure [5]. There are scientific proofs that the Hawthorn may induce anti-ischemia/reperfusion-injury and possess anti-arrhythmic effects [6,7,2,8]. These beneficial effects may be due, in part, to the presence of antioxidant components in its flowers, leaves and fruits extract [1,9,10]. During the years several pharmaceutical products containing standardized extracts from Crataegus oxycanthas' flowers, leaves and deep red fruits have been developed [11]. Several authors have suggested possible connection between the observed therapeutic effects of extracts from Crataegus species and their antioxidant properties they have indicated the need of comparative aviation of their antioxidant potential [1,4]. The performed in vitro and in vivo investigations of the medical effects of the obtained by different methods Crataegus species leaves, fruits, green fruits and flowers extracts have proved antioxidant capability. For example, leaves extract of Crataegus monogyna, Crataegus pinnatifida fruits, Crataegus aronia, and Crataegus sinaica decrease the process of lipid peroxidation and have scavenging effects against hydroxyl and nitroxyl radicals [9,12,13,14,15]. The purpose of the present study was to perform a comparative investigation of the antioxidant potential of Crataegus oxycanthas' flowers, leaves, and green and ripe fruits probes and to relate the obtained results with ones of the accomplished qualitative GC-MS analysis of the antioxidant components participating in the studied standardized ethanol extracts. Free radicals are generated upon natural function of plants and other organisms. Due to the fact that plants use peroxides and other reactive species in redox signaling and defense processes they expose themselves to high levels of these reagents. The observed rates of nitroxyl radicals' generation, basal levels of H2O2 and lipid hydroperoxides in plant tissues are much higher compared to animals' samples [16]. ROS are produced as a normal product of plant cell metabolism. They are always formed by the unavoidable leakage of electron onto O2 from the electron transport activities taking place in chloroplasts, mitochondria and plasma membrane or as a product of the metabolic pathways. Investigating the main trigger and processes associated with ROS generation in plants we have come to the conclusion that the most suitable biological relevant model system for comparative evaluation of the antioxidant properties of the plant extracts would be the one based on the main damaging mechanisms of in vivo radical generation. To perform the experimental work we have chosen two spectrophotometric assays -Fegenerated lipid peroxidation and UV induced ROS generation, and one chemiluminescent assay -HRP-H2O2 system. The Fe-induced lipid peroxidation and UV ROS generation assays are among the most commonly used model systems for evaluation of the antioxidant properties of plant extracts. The reason is that during the photosynthesis processes plants are exposed to risk of solar UV radiation induced photo oxidative destruction of the main cellular components. The oxidative breakdown of biological phospholipids occurring in most cellular membranes including chloroplast, mitochondria, peroxisomes and plasma membrane is the main harmful mechanism implicated in oxidative stress cellular damage. To these both methods we have decided to add a sensitive chemiluminescent assay which measures the oxidation of luminol in the presence of H2O2 by horse-radish peroxidase. H2O2 is generated in the plant cell under normal and stressful conditions such as drought, UV irradiation and exposure to intense sunlight. It has been proved that cell walls peroxidase is implicated in the catalyzed the formation of H2O2 in the presence of NADH. The obtained results from the chosen model system will give us the possibility to investigate the efficiency of the studied extracts upon different mechanism of free radical induction. Crataegus oxycantha is used successfully for relief of cardiovascular diseases. It is known that these pathological conditions are associated with increased levels of oxidative stress markers [17]. Due to this fact numerical experiments performed by other authors have shown increased plasma levels of oxidized LDL. Our investigation includes oxidation in model lipid system of a classic type. Preparation of ethanol extract: Fresh flowers, leaves and green and deep red (ripe) fruits from Bulgarian Crataegus oxycantha (Rosaceae) were extracted with ethanol according to Long et al. (2006) [18]. The obtained extracts were standardized based on the content of flavonoid [11]. Three different plant extracts were obtained: from flowers and leaves; from green fruits and from deep red (ripe) fruits. All extracts were prepared strictly following the same procedure which is essential for correct comparison of the obtained by the used analytical methods results. A p r i l 16, 2 0 1 5 Registration of TBA-RS induced by Fe 2+ : The TBA-RS of lipid peroxidation were measured in liposomal suspension of phospholipids from egg yolk extracted according to Folch et al. (1957) [19]. Each sample comprises 1ml PBS, рH 7.4, containing 1 mg lipid/ml, the studied extracts or buffer for the controls. After addition of 0.1 mmol/l FeCl2 samples were incubated at 37 ºC for 30 min. Subsequently 0.5 ml of 2.8% trichloroacetic acid and 0.5 ml of 0.5 % TBA were added. The solution was heated at 100 ºC for 20 min. The absorption was measured at 532 nm. UV induced deoxyribose damage: The deoxyribose assay was performed as given by Halliwell et al. (1987) [20] with small modifications. The final volume for all probes was one ml. The samples mixture comprised PBS. pH 7.4 containing: 0.3 mmol/l 2-deoxy-D-ribose and the tested extracts at concentrations between 0.01 and 1 mg/ml. In control samples plant extracts were omitted. After 25 min of UV irradiation (UV 220-400 nm) 0.5 ml of 2.8% trichloroacetic acid and 0.5 ml of 1 % TBA were added. The mixture was vortexed, heated in a 1000C water bath for 20 min and cooled in a cold water bath. The chromophore absorption was measured at 532 nm and the antioxidant activity (AOA) was calculated by the following equation: where A0 is the absorption of the control sample and A the absorption the studied probes. HRP-H 2 O 2 assay: The horse-radish peroxidase (HRP) -luminol-hydrogen peroxide system is a sensitive assay for monitoring antioxidant activity. The assay was carried out using 1 ml samples of 50 mmol/l PBS containing: 100 µmol/l luminol, 0.5 IU/l HRP enzyme and the tested extracts at concentrations from 0.01 to 1 mg/ml. In the control samples the extract solutions were omitted. The reaction was started by adding of 50 µl H2O2 (1 mmol/l). Chemiluminescence (CL) was measured for 5 min at temperature of 25 ºC. We calculated chemiluminiscent scavenging index -CL-SI (%) as a ratio of CL in the presence and in the absence of the extract. Calculation of C-50: The value concentration that provide AOA = 50 % was termed C-50 and was calculated by the equation: AOA =100/[1 + 10 B (lgC -lg (C-50))] The calculations used fitting of the data to the "sigmoid" model, where B is the coefficient (hill slope) and C is the substance concentration [21]. RESULTS AND DISCUSSION The results of the experiments carried out in a system with Fe-induced LP are shown on Fig. 1. In this system we have established that the extract of flowers and leaves had the strongest antioxidant properties, followed by the extracts of green and ripe fruits. In order to compare quantitatively AOC of the three extracts we calculated C-50. The smaller is the C-50 valuethe stronger is the antioxidant effect of the investigated extract. The obtained C-50 data decrease in the following order: of flowers and leaves(C-50 = 0.287 ± 0.018 mg/ml) > green fruits (C-50 = 0.495 ± 0.021 mg/ml) > ripe fruits (C-50 = 0.704 ± 0.035 mg/ml). The values for green and ripe fruits are respectively 2 and 2.5 times bigger, compared to the C-50 values for flower extracts. Fig 1: Effect of Crataegus oxycanthas' extracts on TBARS production in a model system of Fe2+ -induced lipid peroxidation. In later experiments we used UV radiation as a free radicals source. This method causes formation of OH • , leading to degradation of deoxyribose molecules. The performed experiments prove the protective efficiency of the studied extracts in the investigated model system. The obtained results let us establish strong AOA and diminished DNA oxidative damage for the three investigated extracts (Fig. 2). They demonstrated different efficiency in this process, though the effect as a whole was stronger in comparison to the previous model system. The investigated concentration range includes concentration above 0.1 mg/ml which indicates stronger efficiency of the action upon elimination of • OH. Like in the previous studied model system the flower extracts have demonstrated the strongest AOA (C-50 = 0.109 ± 0.009 mg/ml). They were followed by ripe fruits (C-50 = 0.259 ± 0.015 mg/ml) and finallyby green fruits (C-50 = 0.557 ± 0.014 mg/ml). Compared to the previous model system of Fe-induced lipid peroxidation there is an increase in the effectiveness of ripe fruits and slight decrease in green fruits extracts' antioxidant properties. Recent publications prove radio protective effect of Crataegus extract in in vivo mouse model [22] and in vitro human lymphocytes test system [23]. Given the fact that the hydroxyl radical is considered to be the main damaging factor of ultraviolet irradiation the presented in vitro results from the UV deoxyribose model system and the chemiluminescent assay, which are in agreement with the date of the mentioned reviews, could be considered as an independent experimental proof of the observed by the authors radio protective effect. The extracts of flowers and leaves that we have studied could prove most effective in this process. The peroxidase activity of the extracts is studied by HRP-H2O2 assay. Luminol dependent CL is used for registration of the process. In this method HRP is used as an electron donor of luminol upon destruction of H2O2. This method gives us the possibility to determine the total antioxidant effect of extracts in an enzyme system, using wide variety of substrates to initiate the decomposition of H2O2 to water. According to the obtained results the addition of extracts has led to dramatic reduction of CL (Fig. 3). The values measured at concentrations above 0.03 mg/ml show a 100 times decrease of CL with regard to control. Again the effect is the strongest for extracts from flowers and leaves (C-50 is out of tested concentration range) and weaker, but still well expressed, for green (C-50 = 0.014 ± 0.005 mg/ml) and for ripe (C-50 = 0.020 ± 0.003 mg/ml) fruits samples. Fig 3: Effect of Crataegus oxycanthas' flowers, leaves, green and ripe fruits extracts on luminol-dependent CL induced by HRP/H2O2. From the generalized experimental data it is evident that extracts from flowers and leaves possess the strongest antioxidant potential. The obtained values for green and ripe fruits suggest considerable antioxidant effect. Many of the authors who have investigated the antioxidant properties of plant extracts have tried to establish relationship between the detected antioxidant effect and the amount of total phenols and procyanidins in the investigated samples. In previously performed experiments we have determined the procyanidin composition of the tested samples using HPLC and LC/MS analysis, but the obtained results could not explain the observed antioxidant-induced changes [24,25]. Additional GC-MS analysis was performed in order to determine other constituents (beyond the procyanidins) determining samples' effect. The TIC-mass chromatographs obtained for the three extracts shows that each extract contains over 50 different components. From the obtained results we can conclude there is difference in the component composition between the three studied extracts. Constituents with proven in literature AOA are shown on Table 1. According to the performed analysis the 4 types of tocopherols, phytol, α,β,γ,δamyrines, ursenal, campesterol are constitutes to all of the studied extracts. As seen from Table 1, the flowers and leaves extract contains the broad spectrum of antioxidant components in comparison with the two fruit extracts. They contain more monoterpenoids, steroids and phenol components compared to the fruits sample. Triterpenoids are detected mostly in green fruits extracts. On the grounds of these data we can suggest that the bigger antioxidant effect of flowers and leaves samples is due not only to the bigger amount of procyanidins, but also to the presence of additional components with antioxidant properties. The observed differences in the composition, defining the differences in the investigated properties we ascribe to the fact that in the different parts of the plant, the sources and magnitude of oxidative damages are different. In order to sustain the balance between oxidants and antioxidants, more antioxidant components are necessary on the sites with increase possibility of formation of reactive oxygen and nitrogen species. Our survey shows that the three investigated extracts have well manifested antioxidant properties in the used systems. The strongest effect is observed for the extract from flowers and leaves. Ripe and green fruits show similar but statistically different antioxidant activity. Ripe fruits demonstrate stronger effect in the model system with UV irradiation and chemiluminescent HRP-H2O2 systems, but green fruits antioxidant potential in the spectrophotometric system of Fe induced LP is stronger. The GC-MS analysis we carried out indicates that the observed differences are due to the presence of a bigger number of components possessing antioxidant properties in flowers and leaves extract, compared with samples.

v3-fos

2016-05-04T20:20:58.661Z

2015-07-01T00:00:00.000Z

6463468

s2

Low doses of gamma radiation in the management of postharvest Lasiodiplodia theobromae in mangos The postharvest life of mango is limited by the development of pathogens, especially fungi that cause rot, among which stands out the Lasiodiplodia theobromae. Several control methods have been employed to minimize the damages caused by this fungus, chemical control can leave residues to man and nature; physical control by the use of gamma radiation in combination with modified atmosphere and cold storage. The use of gamma radiation helps to reduce the severity of the pathogen assist in the ripening process of fruits, even at low doses (0.25, 0.35 and 0.45 kGy) chemical properties such as pH, soluble solids, acid ascorbic, titratable acidity and also the quality parameters of the pulp showed no damage that are ideal for trade and consumption of mangoes. This treatment can be extended for use in the management of diseases such as natural infections for penducular rot complex that has as one of L. theobroma pathogens involved. Introduction A survey of irrigated fruit farming in the semi-arid region of northeastern Brazil reveals that the pathogen Lasiodiplodia theobromae (Pat.) Griffon and Maubl accounts for major plant health problems in São Francisco Valley. The high degree of disease severity is due to climatic conditions that are favorable to the development of this fungus, with little variation throughout the year (Tavares et al., 1991). Based on scientific data, it is believed that the pathogenicity of L. theobromae increased as a result of environmental pressure, particularly in the region in question (Tavares, 2002). Moreover, the susceptibility of the mangos to the rot increases after harvest and storage as a result of physiological changes in fruit, disqualifying them for marketing due to involvement of the peel and pulp (Tavares et al., 1991). The postharvest life of mango is limited by the rapid ripening of fruits and the development of pathogens causing rot (Almeida et al., 2005). However, this conservation can be extended with the use of refrigeration and modified atmosphere (Jerônimo and Kanesiro, 2000). The recommended temperature for mangos is approximately 12°C (Alves et al., 1998). According to Calore and Vieites (2003), the use of a low storage temperature in combination with a modified atmosphere can slow the development of any microorganisms that may be present. Adequate postharvest handling in combination with food preservation methods should be employed to prolong the shelf life of fruits and vegetables, thereby extending the commercialization period (Jerônimo and Kanesiro, 2000). Advances in technological processes of food preservation over the last 50 years show that the gamma radiation of cobalt60 and cesium137 or even accelerated electrons is capable of inhibiting the proliferation of microorganisms (Kaferstein and Moy, 1993). According to Chitarra and Chitarra (2005), the irradiation of fruits and vegetables postharvest's has a main interest in the reduction or delay the damage caused by disease, presenting antifungal effect. However, it is also used as a preservation method, by prolonging the storage by delayed ripening and sprouting of some products. However, its use has some disadvantages, such as browning, softening, the appearance of surface depressions, abnormal maturation and loss of aroma and flavor of the products (Nagajata, 2007). The nature and extent of these changes depends on the type, composition and variety of the fruit, as well as the radiation dose applied and the environmental conditions during and after the radiation process. The application of ionizing radiation may alter the structural components of some fruits, giving them a better appearance and enhancing their firmness. Low doses of radiation can lead to the hydrolysis of some components, resulting in a longer shelf life and the conversion from starch to sugar (Lima et al., 2001). Irradiated fruits are often sweeter than non-irradiated ones (Thomas, 1986). During the ripening of mangos, the contents of organic acid diminishes and the soluble sugar increases, resulting in a predominance of sweetness in the ripe fruit (Bernardes-Silva et al., 2003). Monitoring these changes serves as a basis to evaluate if the ionizing radiation committed the ripening of fruits. This study aimed to evaluate the effect of gamma radiation (0.25, 0.35 and 0.45 kGy) associated with modified atmosphere in controlling of rot caused by L. theobromae and its impact on shelf life and the physicochemical characteristics of fruits. Materials and Methods The experiment was conducted at the Postharvest Pathology Laboratory at UFRPE and at the Department of Nuclear Energy of the Universidade Federal de Pernambuco (Brazil) in cooperation with the Postharvest of Fruit Laboratory of the Centro Regional de Ciências Nucleares (Brazil). Pathogen The phytopathogenic fungus used in the present study was obtained from the collection of the Empresa Brasileira de Pesquisa Agropecuária -Embrapa Semiárido (Brazil). The isolate was cultured on potato -dextrose -agar (PDA) and, inoculated into healthy fruits. Effects of gamma radiation on development of L. theobromae in mango Tommy Atkins mangos picked at mature-green or at maturity stages 2 and 3 (Deutsche Gesellschaft für Technische Zusammenarbeit -GTZ (1992)) from an orchard in Petrolina (Pernambuco State, Brazil) were immediately and carefully transported to the laboratory, where they were selected, washed and dried at 26 ± 2°C, relative humidity (RH) of 70 ± 5%. After that, fruits were wounded with a sterilized perforator (8 needles, 5 mm in diameter and 2 mm deep) and on each wound, was deposited 10 mL with of a L. theobromae spore suspension at a concentration of 10 6 conidia mL -1 . The Control were injured by the same way, but inoculum was switched for 10 mL of sterilized distilled water. Mangoes were packed in pairs, in expanded polystyrene trays (15 cm x 10 cm) coated whit PVC film and maintained at 26 ± 2°C. The trays were maintained in the laboratory conditions (26 ± 2°C; 70 ± 5% RH) for 24 h and 70 ± 5% RH for another 24 h. After this period, the trays containing the mango were treated whit radiation doses previously predetermined (0.25, 0.35 and 0.45 kGy) using a Gammacell ® 220Excel radiator (MDS Nordion, Canada), whose rate at time of application was cobalt60 source and 7.303 kGy/h. The positive control was composed of mangos inoculated with the phytopathogen but not irradiated and the negative control was composed of mangoes without inoculation or irradiation. After the application doses, irradiated and non-irradiated trays were kept under cold storage at 13°C for 15 days and then transferred to the incubation room (26 ± 2°C and 70% RH) without the PVC for 6 days. The experiment was conducted with an entirely randomized design consisting of 4 treatments and 4 replicates, with each replicate containing 5 mangos. The same design was used for evaluation of the physicochemical characteristics. The evaluation of disease severity consisted of the measurement of the diameter of the lesion using a caliper rule (Mitutoyo, Kaeasaki, Japão) first after the trays were removed from the cold chamber and every 2 days until the degeneration of the fruit occurred 6 days after leaving the cold chamber. The data were subjected to analysis of variance and regression analysis, the model was defined by the coefficient of determination, using the SAS Software (SAS Institute, 2000). Evaluation of physicochemical characteristics The fruits were evaluated at four different times: at the entrance of the cold chamber (Day 1); 15 days after the cold storage or at the end of the cold storage (Day 15), two days after leaving the cold chamber (Day 17); and four days after leaving the cold chamber (Day 19). The characteristics evaluated were: pulp firmness, pulp color, hydrogen potential (pH), solid soluble (SS), titratable acidity (TA), ascorbic acid (Vitamin C) and maturation index (MI) determined by SS/TA ratio. Pulp firmness was determined on opposite peeled sides of the fruit using a penetrometer model FT 327 (0-13 Lbs.) (Wagner Instruments, Greenwich, London) and the results were expressed in kgf. This analysis was performed at the end of the cold storage and at two and four days after leaving the cold chamber (Day 17 and Day 19). The pulp color was determined using a Minolta CR-300 colorimeter with three readings in different parts of the pulp of each fruit. The results were expressed in L*, a* and b*. L* values range from 0 (white) to 100 (black), represent the brightness of the pulp; a* values indicate chromaticity ranging from green (-) to red (+); and b* values indicate chromaticity ranging from blue (-) to yellow (+). The chemical characteristics were determined after the disintegration of the pulp, domestic centrifuge. The pH was checked at 10 g of pulp in potentiometer Quimis Model 400A. The levels of ascorbic acid in pulp were determined according to the method described by Carvalho et al. (1990) The solid soluble contents (SS) were quantified by direct reading on refractometer, Model Rez (0 -32ºBrix), and the results expressed in ºBrix, while the titratable acidity (TA) was determined according to the methodology described by Ohlweiler (1980). The maturation index (MI) was also determined by SS/TA ratio. In all of these analyzes were utilized three replicates for each fruit. The data were subjected to analysis of variance and regression analysis, the model was defined by the coefficient of determination, using the SAS Software (SAS Institute, 2000). Graphs were plotted using Sigma Plot (2008). Firmness data were analyzed using Tukey's test, with the level of significance set to 5% (p < 0.05), using the SAS Software (SAS Institute, 2000). Results and Discussion Effects of gamma radiation on development of L. theobromae in mango The doses of radiation used and the presence of PVC film during the cold storage of mangos did not cause the appearance of peel spots or caused other changes in fruits, differently the results reported by Calore and Vieites (2003) in peaches treated with 0.5 kGy and by Pfaffenbach et al. (2003) in mangoes packed in plastic film and kept under re-frigeration. In the first evaluation of the effects of radiation on rot development by L. theobromae made at the end of the cold storage it was observed that fruits had no symptoms of decay resulting from inoculation with the pathogen. In the second evaluation held two days after leaving the cold chamber it was checked disease incidence in all treatments, with significant differences in severity. Fruits treated with the lowest dose (0.25 kGy) showed similar disease development to Control (Figure 1). However, the highest dose (0.45 kGy) resulted in the greatest reduction in disease development. According to Morais et al. (2003) the cold storage slows the ripening of fruits, providing longer shelf-life beyond make feasible their exportation. The quality of the mangoes after their removal from the cold chamber is extremely important for the successful of the conservation process (Neves et al., 2002). The association between gamma irradiation and modified atmosphere may contribute to the maintenance of fruit quality as observed in this study. Evaluation of physicochemical characteristics The results of this study demonstrate that gamma radiation associated with cold storage reduces loss firmness mangos that received the highest dose (0.45 kGy) had the best values of firmness (Table 1) at all stages of evaluation. A loss of firmness hampers the commercialization of fruits and this factor can be attenuated by proper handling and preservation methods (Lima et al., 2009). The dose 0.45 kGy, was effective in preserving the firmness of the mangoes. These findings are similar to those reported by Miller and McDonald (1999), who found a positive interac-Gamma radiation postharvest 843 tion between gamma radiation and the maintenance of postharvest firmness in papaya (Sunrise Solo variety). The pulp color (Figure 2), no significant differences between gamma radiation doses were found in L*, a* or b* values. Regression analysis revealed reductions in all 3 variables throughout the evaluation period owing to the ripening of the fruits. The L* value ranges from 0 (black) to 100 (white). The mean L* value in the present study was 67.04 in the beginning of refrigeration and 52.03 at 19 days after placement in the cold chamber, indicating the ripening of the fruit. More positive a* values correspond to the ripening of the fruit. In the present study, the mean a* value was -13 before placement in the cold chamber and -1.23 at 4 days after removal from the chamber. Positive b* values represent the presence of the yellow color and negative values represent the presence of the blue color. In the present study, there was little variation in these values throughout the evaluation, from 44.61 in the control sample upon placement in the cold chamber to 22.3 at 4 days after removal from the chamber. In coloring of the pulp Allong et al. (2000) evaluated Kent mangoes, and reported reductions in these parameters considering this to be a common process in the ripening of the fruit. The statistical analysis demonstrated that only the soluble solids and pH were correlated with time and doses. Compared to the control, treated fruits showed a decrease in the values of soluble solids ( Figure 3A). Increasing amounts of soluble solids indicates the point of physiological maturity and hence the sampling point (Assis, 2004). In 844 Santos et al. a study of the behavior of different mango varieties, Ornelas-Paz et al. (2007) reported that the soluble solids content tends to increase during the ripening process. Thus, the low doses of gamma radiation in the present study did not alter the natural behavior of the mangos during the development. The pH showed similar behavior between the control and the doses 0.25 and 0.35 kGy only 0.45 kGy dose demonstrated a significant reduction between dose and slightly increased with respect to the valuation days ( Figure 3B). The results of pH were similar to those reported by Thomas et al. (1996), who irradiated mangos with doses of 0.3-1.0 kGy and found no significant changes in pH values, even when the ripening of the fruit was slowed. Lima et al. (2001) stressed the importance of evaluating pH as an intrinsic factor that exerts the greatest selective effect on the microflora likely to develop on fruits and vegetables. On regard to the ascorbic acid content ( Figure 4A), titratable acidity ( Figure 4B) or Ratio ( Figure 4C), we found no interaction between dose and valuation day and between the different gamma radiation doses. These findings differ from those reported by Youssef et al. (2002), who observed that gamma irradiation of mangos at doses of 0.5 and 2.0 kGy led to an increase in the ascorbic acid content, which may have been influenced by the combination of the radiation and quality of the fruit. In the present study, the ascorbic acid curve demonstrated that the mangos exhibited a reduction in this substance during the ripening process, which is a common finding in most fruits. On regard to titratable acidity, an increase in pulp acidity occurred during the ripening process. With few exceptions, the organic acid content is diminished during maturation owing to either respiration or the conservation of sugars (Chitarra and Chitarra, 2005). Gamma radiation had no effect on the acidity of the mangos. This finding is similar to that reported by Calore and Vieites (2003), who studied the effect of gamma radiation on the preservation of peaches and found that a dose of 0.1 kGy did not slow the ripening process with regard to acidity. The results of the present study demonstrate that higher doses (0.24, 0.35 and 0.45 kGy) were also not effective. No difference in Ratio was observed among the different gamma radiation doses, whereas difference was observed during the ripening of the fruit. This index indicates sweetness vs. acidity and commercially determines the maturity and quality of the fruit. Camargo et al. (2007) reported a tendency toward an increase in this index after Day 14 with the increase in the temperature, indicating that gamma radiation has little influence over Ratio. In Conclusion, the use of gamma radiation, even at low doses, can cause a positive effect on disease management during postharvest mango, with little or no influence on fruit quality. IT IS, recommended 0.45 kGy dose by pro-viding a decrease in the development of the pathogen without causing damage properties evaluated for physical and chemical sleeves. The results can be extended to natural infections can predict how the control behaves in the development of the fungus in this study was artificially inoculated.

v3-fos

2016-03-01T03:19:46.873Z

2015-04-01T00:00:00.000Z

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Emissions of Escherichia coli Carrying Extended-Spectrum β-Lactamase Resistance from Pig Farms to the Surrounding Environment The dissemination of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (E. coli) from food-producing animals to the surrounding environment has attracted much attention. To determine the emissions of ESBL-producing E. coli from pig farms to the surrounding environment, fecal and environmental samples from six pig farms were collected. In total, 119 ESBL-producing E. coli were isolated from feces, air samples, water, sludge and soil samples. Antibiotic susceptibility testing showed that the ESBL-producing isolates were resistant to multiple antibiotics and isolates of different origin within the same farm showed similar resistance phenotypes. Both CTX-M and TEM ESBL-encoding genes were detected in these isolates. CTX-M-14 and CTX-M-15 were the predominant ESBL genes identified. ESBL producers from feces and environmental samples within the same farm carried similar CTX-M types. The results indicated that the ESBL-producing E. coli carrying multidrug resistance could readily disseminate to the surrounding environment. Introduction The increasing prevalence of extended-spectrum β-lactamases (ESBLs) in the World has attracted wide attention [1]. ESBLs are enzymes that can destroy β-lactam antibiotics, including penicillins, first, second and third-generation cephalosporins and aztreonam which are susceptible to β-lactamase inhibitors [2]. The ability of ESBLs to confer bacterial resistance can dramatically decrease therapeutic options in disease control and treatment [3,4]. The ESBL enzymes mainly include three types, TEM, SHV and CTX-M [5]. Since first discovered in 1989 [6], CTX-M gene has become the most common ESBL enzyme and has spread quickly throughout the World taking the place of TEM and SHV types that were prevalent in the early 1990s [5]. Genes encoding these various ESBL genes are located on mobile genetic elements and could disseminate through horizontal gene transfer between bacteria, and even between different species [7]. Food-producing animals were considered reservoirs of zoonotic pathogens and resistant bacteria [8]. Escherichia coli can survive in the gastrointestinal tract of food-producing animals as a commensal bacterium and can also cause infections [9]. Extended-spectrum cephalosporins are effective drugs against such infections in veterinary clinical use; which creates a selective pressure for ESBL-producing E. coli. Animals colonized with ESBL-producing E. coli have been considered as potential sources of resistant E. coli infections in the community, which has attracted wide concern [10]. Furthermore; the ESBL-producing E. coli in animal farms could influence public health through environment pollution and contaminated animal products [11]. The dissemination of these resistant bacteria from animal houses through various routes exerts pressure on the surrounding environment and even influences the living environment of human beings. Aerosol transmission is an important route for virus and bacteria [12]. E. coli has been identified to transmit through air by aerosol formation [13]. Aerosol transmission of ESBL-producing E. coli with air flow contributes to its dissemination. The discharge of waste products and farmland application of effluents and feces could also promote the entry of drug-resistant bacteria into the environment [14]. To date, ESBL-producing bacteria have been found in various environments, where they may be a reservoir contributing to the spread of resistant bacteria [14,15]. In this study, to estimate the transmission of ESBL-producing E. coli originated from pig farms to the surrounding environment, ESBL-producing E. coli was collected from fecal and environmental samples from pig farms in China. Pig Farms Six pig farms located in different regions of Shandong Province, China and their surrounding environments were selected to collect samples to investigate the transmission of ESBL-producing E. coli from food animal-producing houses to the surrounding environment. ESBL-producing E. coli has been found in six (A, B, C, D, E and F) out of ten pig farms in our primary research. These farms are far away from villages. Negative pressure ventilation was used in these farms. Sampling Fecal and environmental samples were collected from these farms between April 2013 and June 2013 to evaluate the spread of resistant bacteria produced in pig farms to the surrounding environment. Air samples were collected using a six-stage Anderson sampler [16] at an airflow rate of 28.3 L/min placed at a height of 1.0 m indoors and outdoors in the down-and upwind positions as previously reported [13]. Each time, six MacConkey agar plates with 2 µg/mL cefotaxime were used as medium placed in an Anderson six-stage sampler for air sample collection. Inside and outside air samples were collected at the same time. In each house, inside air samples were collected at three locations along the passage with a time of 20-30 min. Outside air samples were collected at different distances including 10 m and 50 m upwind, and 10 m, 50 m, and 100 m downwind. No air samples were collected from farms E and F. At the same time, environmental samples were collected. Water and sludge were collected in the vicinity of pig farms A, B, C and D. River water samples were collected at 10 m upstream, and 10 m, 50 m and 100 m downstream away from the drain outlet. Sludge samples were collected at the outlet of the effluent. Soil samples were collected at different directions outside of the animal house. These samples were transferred to an ice box and then processed immediately upon arrival at the lab. Cefotaxime-Resistant E. coli Isolation After collection, the MacConkey agar plates with 2 µg/mL cefotaxime used for air samples were incubated at 37 °C overnight directly. Fecal samples were serially diluted twice with sterile phosphate buffered saline solution and then 100 µL was cultured on MacConkey agar plates with 2 µg/mL cefotaxime and incubated overnight. The river water samples were filtered using a nitrocellulose membrane filter and then the filter was placed on agar plates. Soil and sludge samples (2 g) were transferred into 50 mL Luria-Bertani (LB) broth for enrichment. Following bacteria enrichment, overnight cultures were streaked on MacConkey agar plates with 2 µg/mL cefotaxime at 37 °C overnight. One or two colonies with typical E. coli morphology were selected and further streaked on LB agar plates for purification. Presumptive pure cultures were identified by classical biochemical methods and the API 20E system [17]. Resistance Genes TEM-, SHV-, and CTX-M-encoding ESBL genes were identified using multiplex polymerase chain reactions (PCR) to determine the ESBL types of the ESBL producing E. coli from different samples, as previously described [19]. TEM-encoding genes were further amplified as described previously [20] and the amplicons were sequenced. The blaCTX-M genes were further amplified and analyzed using group primers CTX-M-1, CTX-M-2, CTX-M-8 and CTX-M-9, as described previously [21,22]. The PCR products were purified and cloned in pMD-18T for sequencing. The obtained DNA sequences were compared and blasted (http://www.ncbi.nlm.nih.gov/) to confirm the β-lactamase gene subtype. Statistical Analysis Pearson's chi-square test was used to compare the continuous data. The association between resistance phenotype of isolates from fecal and environmental samples were evaluated. Correlation coefficients (r values) and the levels of significance (p values) were used to interpret the results of correlation analyses. Two-tailed p values of 0.05 were considered statistically significant. The statistical analyses were conducted using the statistics software, SPSS, version 19.0 (IBM SPSS, Chicago, IL, USA). Samples Positive for ESBL-Producing E. coli from Feces and Environments One hundred and twenty samples positive for ESBL-producing E. coli were detected from the fecal samples from the six farms. Water and sludge samples were collected from A, B, C and D farms. In the vicinity of E and F farm, no river was found. Samples positive for ESBL producers were detected in air samples in three out of four pig farms. Isolation and Identification of ESBL-Producing E. coli A total of 120 cefotaxime-resistant E. coli strains were isolated from fecal samples and environmental samples collected from the six pig farms. One hundred and nineteen E. coli isolates from feces, indoor air samples and outdoor air samples, water and sludge samples and soil samples were confirmed to be ESBL-producing E. coli after the phenotypic confirmatory test. In three out of six pig farms (B, C and D), ESBL-producing E. coli were detected in air samples (six, one and two, respectively). Seven ESBL-producing E. coli were obtained from water samples outside the four farms, and five from sludge samples. A total of 12 ESBL-producers were isolated from soil samples. From farm A, five isolates were obtained from feces, one came from water (10 m downstream), and two were isolated from sludge samples. Among the 20 ESBL-producing E. coli isolated from farm B, 10 isolates were from feces, six were from air samples including three indoor air isolates and three outdoor air isolates from 10 m and 100 m downwind, and four were from water (10 m downstream) and sludge samples. In farm C, two water isolates from 10 m downstream and one from 50 m downstream, and one ESBL-producing isolates from an indoor air sample, outdoor air sample (10 m downwind), water and sludge sample respectively were obtained ( Table 2). Discussion With the use of antibiotics, more and more resistant bacteria occur in food-producing animals, including ESBL-producing E. coli. The spread of these bacteria through various routes to the environment creates a threat to public health. In this study, ESBL-producing E. coli was isolated from feces and environment samples including indoor air, outdoor air, water and sludge samples and soil samples from six pig farms in rural regions of Shandong, China. From the six farms, ESBL-producing E. coli was all detected in feces and different kinds of environmental samples, which indicated the possible transmission routes of ESBL-producers from food-producing animal farms. All 119 of the ESBL-producing isolates from fecal and environmental samples showed high rates of resistance to multiple antimicrobial agents. Isolates showing resistance to two or more classes of drugs were treated as multi-drug resistant (MDR). The resistance profiles varied between different farms, but were highly related between isolates from feces and environmental samples within the same farm. These results suggested that the ESBL-producers in the environment might originate from the pig farm. The CTX-M gene was the predominant ESBL gene in this region, consistent with previous reports [23]. BlaCTX-M-14 and blaCTX-M-15 were the most common CTX-M type, similar to what has been reported in pigs, cattle, and chickens [11,24]. In food-producing animal production, high concentrations of airborne microorganisms are often found in indoor environments [25]. These microbes in such an environment can survive in the form of aerosols for a long time in the air and transmit with air flow [13]. In this study, ESBL-producing E. coli was obtained from the indoor air and outdoor air samples. Isolates from indoor, outdoor and fecal samples showed high similarity, which indicated the airborne transmission of the ESBL-producing E. coli in pig farms. Previous studies had demonstrated the dissemination of ESBL-producing E. coli originated from chicken houses into the air [26,27]. The concentrations of microorganisms were closely related with the air quality. A poor air environment could benefit the spread of ESBL-producing E. coli. ESBL-producing bacteria have been increasingly reported in water and sludge [28][29][30]. Agricultural use of contaminated water or sludge could be a possible route for ESBL-producing E. coli to enter into the food chain [31,32]. In this study, ESBL-producing E. coli were also isolated from river water and sludge samples, which shared similar resistance profiles and ESBL genes with fecal isolates within the same farm. These results suggested the potential influence of pig farms on the surrounding water environments. In conclusion, the high similarities of isolates from environmental and fecal samples suggest a possible dissemination of resistant bacteria from pig feces into the surrounding environment. These results indicated the emissions of resistant E. coli isolates from pig houses to the surrounding environment, which constitutes a major threat to public health. As the origin of resistant bacteria, thus the rational use and antibiotics and the establishment of effective management of food-producing animal farms are necessary. Conclusions Comparison of isolation rates, resistance profiles and β-lactamase genes showed that fecal isolates and environmental isolates shared similar characteristics, which suggested the possible emissions of the ESBL-producing E. coli from feces to the environment. Author Contributions Tongjie Chai Zengmin Miao and Lili Gao designed this study. Lili Gao, Xiaodan Zhang and Jiaqing Hu took samples. Lili Gao and Xiaodan Zhang performed the bacterial isolation, microbiological experiments, and analyses. Liangmeng Wei and Tongjie Chai revised this manuscript.

v3-fos

2019-04-25T13:05:49.343Z

2015-12-07T00:00:00.000Z

3878728

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Mediterranean Diet Food: Strategies to Preserve a Healthy Tradition The traditional Mediterranean diet refers to a dietary pattern found in olive growing areas of the Mediterranean region. It’s essential characteristic is the consumption of virgin olive oil, vegetables, fresh fruits, grains, pasta, bread, olives, pulses, nuts and seeds. Moderate amounts of fish, poultry, dairy products and eggs are consumed with small amounts of red meat and wine. Over the past few decades there has been a growing interest in the role of the Mediterranean diet in preventing the development of certain diseases, especially cardiovascular disease. Mediterranean food products are now re-evaluated for the beneficial health effects in relation to the presence of bioactive compounds. The body of science unraveling the role of bioactives such as phenolic acids, various polyphenols, flavonoids, lignans, hydroxyl-isochromans, olive oil secoiridoids, triterpene acids and triterpene alcohols, squalene, αlpha-tocopherol and many others is growing rapidly. A challenge for future research is the magnitude of the contribution of each active compound to the overall positive health effect. Strategies to preserve and disseminate the healthy Mediterranean diet should focus on: the implementation of the claim recently approved by EFSA for the level of biophenols in olive oil and the protection of LDL oxidation; technological improvements based on the increased awareness about the role of minor constituents of Mediterranean foods; products that are innovative but also traditional. The Traditional Mediterranean Diet The traditional Mediterranean diet refers to dietary patterns typical of specific regions of the Mediterranean region in the early 1960s. It is characterized by an abundance of plant foods such as vegetables, fresh fruits, grains, pasta, bread, pulses, nuts and seeds, and a high level of monounsaturated fatty acids. A common feature of the Mediterranean diet is a high consumption of olives and olive oil as the primary sources of dietary fat and moderate amounts of fish, poultry, dairy products and eggs consumed along with small amounts of red meat and wine. Over the past few decades there has been a growing interest in the role of the Mediterranean diet in preventing the development of certain diseases, especially cardiovascular disease. The discovery of the cardioprotective properties of the diet is one of the great successes of epidemiology. Today there are many biochemical and other studies in the field of biosciences related to Mediterranean food products and their ingredients. These studies confirm the findings of epidemiology related to vascular health and hypertension and indicate also that a high intake of foods typical of the Mediterranean dietary pattern and a good adherence to it provides protection against coronary heart disease and may be inversely associated with the development of various types of cancer, arthritis, diabetes and neurodegenerative diseases. They may also be effective in improving health status and reduce mortality [1-3].Thus, the pattern has been garnering interest throughout the world and more people are interested in the health benefits it confers. Mediterranean diet pyramids The healthy traditional Mediterranean pattern has been represented by various pyramids indicating graphically the foods to be consumed on a daily basis or weekly. Mediterranean diet pyramids have been continuously re-designed and completed. The last 2010 version (Eighth International Congress on The Mediterranean Diet, Barcelona), incorporates lifestyle and cultural elements such as moderation, conviviality, seasonality and eco-friendliness. In other words, the new revised modern Mediterranean diet takes into consideration contemporary lifestyles and environmental constraints and is compatible with the development of a sustainable diet model for present and future generations [2]. Mediterranean Food Products The great progress made in the areas of natural products chemistry, food analysis, nutrition, biochemistry and other biosciences during the last two or three decades has demonstrated that some commonly encountered food constituents have hitherto unknown health promoting and disease preventive properties. Mediterranean food products are now re-evaluated for the beneficial health effects in relation to the presence of bioactive compounds. A lot of information is now available. A good example is biophenols. Phenolic compounds widely distributed in the plant kingdom and abundant in our diet are today among the most talked about classes of phytochemicals. This is indicated by the accumulated scientific work that focuses on: • Oxidation mechanisms and contribution of phenols and generally natural antioxidants in preventing free radical damage and oxidative stress • The dietary intake of phenolic compounds and its effect on lipoprotein metabolism, oxidative damage, inflammation, endothelial dysfunction, and blood pressure • The potential of certain phenols in oncology, chemoprevention, cell-specific cytotoxic and apoptotic effects Journal of Experimental Food Chemistry Boskou, J Exp Food Chem 2016, 1:1 http://dx.doi.org/10.4172/2472-0542.1000104 Review Article open access J Exp Food Chem ISSN:2472-0542 JEFC, an open access journal Volume 1 • Issue 1 • 1000104 • The clarification of molecular mechanisms accounting for the antioxidant, anti-inflammatory, and anticancer properties through gene transcription profiling • The extraction of phenols and other bioactive compounds from fruits processing residues The Traditional Mediterranean Diet The traditional Mediterranean diet refers to dietary patterns typical of specific regions of the Mediterranean region in the early 1960s. It is characterized by an abundance of plant foods such as vegetables, fresh fruits, grains, pasta, bread, pulses, nuts and seeds, and a high level of monounsaturated fatty acids. A common feature of the Mediterranean diet is a high consumption of olives and olive oil as the primary sources of dietary fat and moderate amounts of fish, poultry, dairy products and eggs consumed along with small amounts of red meat and wine. Over the past few decades there has been a growing interest in the role of the Mediterranean diet in preventing the development of certain diseases, especially cardiovascular disease. The discovery of the cardioprotective properties of the diet is one of the great successes of epidemiology. Today there are many biochemical and other studies in the field of biosciences related to Mediterranean food products and their ingredients. These studies confirm the findings of epidemiology related to vascular health and hypertension and indicate also that a high intake of foods typical of the Mediterranean dietary pattern and a good adherence to it provides protection against coronary heart disease and may be inversely associated with the development of various types of cancer, arthritis, diabetes and neurodegenerative diseases. They may also be effective in improving health status and reduce mortality [1][2][3].Thus, the pattern has been garnering interest throughout the world and more people are interested in the health benefits it confers. Mediterranean diet pyramids The healthy traditional Mediterranean pattern has been represented by various pyramids indicating graphically the foods to be consumed on a daily basis or weekly. Mediterranean diet pyramids have been continuously re-designed and completed. The last 2010 version (Eighth International Congress on The Mediterranean Diet, Barcelona), incorporates lifestyle and cultural elements such as moderation, conviviality, seasonality and eco-friendliness. In other words, the new revised modern Mediterranean diet takes into consideration contemporary lifestyles and environmental constraints and is compatible with the development of a sustainable diet model for present and future generations [2]. Mediterranean Food Products The great progress made in the areas of natural products chemistry, food analysis, nutrition, biochemistry and other biosciences during the last two or three decades has demonstrated that some commonly encountered food constituents have hitherto unknown health promoting and disease preventive properties. Mediterranean food products are now re-evaluated for the beneficial health effects in relation to the presence of bioactive compounds. A lot of information is now available. A good example is biophenols. Phenolic compounds widely distributed in the plant kingdom and abundant in our diet are today among the most talked about classes of phytochemicals. This is indicated by the accumulated scientific work that focuses on: • Oxidation mechanisms and contribution of phenols and generally natural antioxidants in preventing free radical damage and oxidative stress • The dietary intake of phenolic compounds and its effect on lipoprotein metabolism, oxidative damage, inflammation, endothelial dysfunction, and blood pressure • The potential of certain phenols in oncology, chemoprevention, cell-specific cytotoxic and apoptotic effects • The clarification of molecular mechanisms accounting for the antioxidant, anti-inflammatory, and anticancer properties through gene transcription profiling • The extraction of phenols and other bioactive compounds from fruits processing residues Virgin olive oil Virgin olive oil is a fundamental Mediterranean diet component and contributes substantially to the health benefits of this diet. It is rich in mono-unsaturated fatty acids and contains bioactive phenols such as the dialdehydic forms of elenolic acid linked to tyrosol and hydroxytyrosol, oleuropein and ligstroside aglycons, tyrosol and hydroxytyrosol. Other polar phenolics present in virgin olive oil are lignans, phenolic acids, flavonoids, derivatives of phenolic alcohols, hydroxy-isochromans and traces of glycosides (oleuropein, ligstroside). Non polar bioactive constituents and other nutritionally important compounds are alpha-tocopherol and non-phenolic compounds, mainly squalene hydroxyterpenic acids, triterpene dialcohols, phytosterols and carotenoids [4]. The present knowledge of olive oil's complex composition is due to the application of advanced techniques for the preparation of the samples (solid phase extraction techniques, semi-preperative high pressure chromatography, ultrasound-assisted emulsificationmicroextraction), and the identification and quantification of phenolic molecules (gas chromatography-mass spectrometry, (GC-MS), high pressure liquid chromatography-diode array detector/mass spectrometry (HPLC-DAD/MS), liquid chromatography diode array detector-electrospray time-of-flight mass spectrometry (HPLC-ESI-TOF/MS), other hyphenated techniques and high resolution mass spectrometry (HPLC-DAD-SPE-NMR/MS, ORBITRAP platform analyzers), as well as nuclear magnetic resonance techniques) [5,6] Published research work and ongoing studies are extended and their outcome may ultimately be used to integrate the results of experiments in various disciplines, including food chemistry, biochemistry, pharmacology and other biological sciences. Table olives Table olives that are green, turning color, or black are good sources of bioactive compounds but they have not yet been fully appreciated as a valuable functional food The phenols reported to be present in commercial samples of table olives, depending on the method of debittering, are verbascoside, hydroxytyrosol, tyrosol, luteolin, and apigenin 7-O-glycosides, and phenolic acids. Traditionally prepared table olives have been also reported that are rich sources of oleuropein. Other bioactive constituents in processed olives are maslinic and oleanolic acids, which are found in abundance in olive fruits. Other mediterranean diet constituents The enzyme Paraoxonase 1 (PON1) has been implicated in the prevention of cardiovascular diseases development of those conditions, especially atherosclerosis [7]. Extra virgin olive oil, the main source of fat, has been particularly effective in increasing PON1 activity. Other Mediterranean diet constituents such as nuts, fruits and vegetables, have been effective in modulating the activity of the enzyme. Pomegranate has attracted much research interest as a source of some potent phenolic antioxidants involved in the protection of LDL and HDL from lipid oxidation The enhancement of PON1 activity by pomegranate was demonstrated in animal models and correlated to the presence of phenolics such as punicalagin, gallic acid and ellagic acid. Nuts in the diet (walnuts, hazelnuts, almonds) may also boost memory in the elderly. A Mediterranean style diet supplemented by olive oil and nuts was found to counteract age-related cognitive decline, as indicated in a recent study [8]. Dried fruits Traditional dried fruit such as raisins, figs, dates, apricots and many others have been a staple of Mediterranean diets for millennia. Modern research focuses on polyphenols. Dates contain quercetin, apigenin and luteolin; prunes have a very high chlorogenic acid content. Dried apricots and peaches are also important sources of carotenoids, compounds which are precursors of vitamin A and antioxidants. Sesame seeds and sesame seeds paste (tahini) Sesame seed is a reservoir of biologically important compounds and its health promotion properties in humans are well known [10,11].The bioactive components present in the seed include vitamins, phytosterols, minerals, polyunsaturated fatty acids, tocopherols and lignans such as .sesamin and sesamolin, These lignans have been shown to have a cholesterol-lowering effect in humans, to prevent high blood pressure, and to increase vitamin E supplies in animals [10,11]. Health claims The body of science unravelling why olive bioactives such as polar phenols, phenolic acids, lignans, flavonoids triterpene acids and triterpene alcohols, squalene, αlpha-tocopherol, hydroxyl-isochromans and many others is growing rapidly. Yet, the magnitude of the contribution of each active compound to the overall positive health effect cannot be easily estimated. Besides, several of the health benefits assigned to many dietary constituents are still under controversy; this can be deduced from the large number of applications rejected by the European Food Safety Authority about health claims of new foods and ingredients. Olive oil is probably a unique case since there are two health claims for this natural product. In 2004 the Food and Drug Administration (FDA) announced the availability of a qualified health claim for monounsaturated fat from olive oil and reduced risk of coronary heart disease (http://www.fda.gov/-dms/qhcolive/htlm). These claim however, does not seem to be complete. The protective effects could be ascribed to the fatty acid composition of the oil but minor constituents, as indicated by recent studies, may be equally or even more important. The European Food Safety Authority has approved a claim for phenols in olive oil (listed in the European Community Council Regulation No. 432/2012), concluding that a cause and effect relationship can be considered established between the phenolic and protection of LDL (low density lipoproteins) This is a claim for "Virgin olive oil with a high level of naturally present polar phenols". Authorization of the health claim aroused enthusiasm and was considered by the SMEs in the producing countries as a means to convey more benefits from virgin olive oil consumption to consumers. In its implementation, however, this claim seems to have some problems that derive from a lack of clarity in terminology and mainly due to the absence of a suitable and generally accepted analytical protocol for the determination of the bioactive compounds hosted under the claim [12]. If such a procedure is soon available, then analysis of a great number of samples will indicate if the required minimum amount of bioactive phenols in the health claim (5 mg of "polyphenols": /20 g oil) is realistic. Quality and authentication Good quality is protected by proper regulations and specifications. Still authenticity and safety are not always guaranteed. Rules set by various organisations and the European Union can now be supported by DNA or chemistry based fingerprinting. Improved protocols to extract DNA and amplify DNA sequences are continuously proposed for the identification of origin, genuineness and typicality of the products, while new effective and sensitive analytical methods to detect traces of adulterants are also developed These are based on methodologies such as nuclear magnetic resonance spectroscopy, gas chromatography, high-performance liquid chromatography and hyphenated methods (HPLC-ESI-MS, MALDI-TOF MS, LC-NMR) combined with principal components or other correlation analyses [13,14]. The interplay of science, innovation and tradition Innovative food science is based on the increased awareness about the role of minor constituents. Therefore, major technological developments that aim at improving quality and preservation focus on an amelioration of the composition and retention of bioactive ingredients in the final product. People give value to the cultural identity of food but also to the findings in the area of chemistry, nutrition and biosciences related to Mediterranean foods. Producers should identify innovative products that are also traditional. Technology and tradition, these two seemingly contradictory factors, can interplay successfully. Non cultivated vegetables A common feature of the Mediterranean diet, the Mediterranean aliment culture, is a high consumption of vegetable. The scientific literature concerning the Mediterranean diet and its constituents is very extensive. Still systematic ethnobotanical studies focusing on wild vegetables traditionally gathered and consumed in many areas are rather scant and sporadic [15]. Wild vegetables of the Mediterranean diet have been the subject of a series of recent phytochemical and phytopharmacological studies that indicated a reduced risk of thrombosis and related conditions due to the consumption of specific boiled plants [16]. An efficacy as anti-obesity agents was also observed [17] Still, the usual picture of the Mediterranean diet underlines mainly cultivated food plants. The wild vegetables remain yet a largely unknown section. Strategies to preserve and evaluate further this type of food from non-cultivated plants should promote nutritional, biochemical, and other studies in the field of biosciences but also the traditional culinary heritage of a territory and sustainable gastronomy. Substitutes and imitation products Diet is now associated with hundreds of "healthier" products. Among them many patented preparations based on the incorporation of Mediterranean food into other food or various substitutes and imitation products promoted with the claim that they "simulate" the Mediterranean diet. These products are loosely controlled; more documentation and updated legislation is needed. Strategies are also necessary to promote consumer awareness in the countries of the Mediterranean basin but also in other countries where consumers are now seriously interested in the benefits of the real diet and the dietary patterns and lifestyle found in olive-growing areas of the Mediterranean basin. Conclusion The traditional Mediterranean diet is characterized by a high intake of plant products, virgin olive oil, fruits, olives, grains, bread, pulses, nuts and seeds. Moderate amounts of fish, poultry, dairy products and eggs are consumed with small amounts of red meat and wine .It emerged in the last three decades and it was introduced by the classic cross-cultural epidemiological "Seven countries study". The healthy traditional Mediterranean pattern is represented by various pyramids indicating graphically the foods to be consumed on a daily basis or weekly. Data on the association between this diet and cardiovascular disease, cancer and other chronic diseases are continuously accumulating. Mediterranean food products are now being reevaluated for the beneficial health effects they offer in relation to the bioactive compounds they contain. Olive oil constitutes a major component of the "Mediterranean diet. " The chief bioactive components of olive oil include oleic acid, phenolic constituents such as the dialdehydic forms of elenolic acid linked to tyrosol and hydroxytyrosol, oleuropein and ligstroside aglycons, as well as lignans, phenolic acids, alpha-tocopherol and squalene. The European Food Safety Authority has approved a claim for phenols in olive oil recognizing that a cause and effect relationship can be considered established between the phenolics and protection of LDL (low density lipoproteins). This claim needs some clarity in the terminology of phenols and should be accompanied by standard analytical protocols for the quantitation of bioactive compounds hosted under it. Table olives are also rich sources of bioactive compounds such as oleuropein, hydroxytyrosol, lutein and apigenin glycosides and triterpenic acids. Other Mediterranean diet constituents such as nuts, pomegranate, dried fruits, sesame seeds and sesame seeds paste (tahini) have been also evaluated and found good reservoirs of bioactive compounds, mainly unsaturated fatty acids, phenolic acids, carotenoids, tocopherols, lignans and others. Strategies to preserve and disseminate the healthy Mediterranean diet should focus on: education of the consumers who are poorly adhered to their traditional diet; implementation of the claim recently approved by EFSA for the level of biophenols in olive oil and the protection of LDL oxidation; technological improvements based on the increased awareness about the role of minor constituents of Mediterranean foods; products that are innovative but also traditional; edible non-cultivated plants and territorial culinary heritage.

v3-fos

2019-04-01T13:12:30.160Z

2015-06-30T00:00:00.000Z

88739088

s2

Effect of biocide addition on plantlet growth and contamination occurrence during the in vitro culture of blueberry Interest and great demand for blueberry ( Vaccinium corymbosum ) have increased, as V. corymbosum is now one of the most economically important crops in Korea. It is expected that blueberry production and the area planted for cultivation will increase consistently in the years ahead because of high profitability and the consumer's demand for healthy ingredients. Effective mass production of blueberry is urgently needed for commercial cultivation establishment, but a main limitation is lack of a propagation system that produces a disease-free plant material for commercial plantation. A large amount of research has focused entirely on developing tissue culture techniques for blueberry propagation. However, controlling fungal and bacterial contamination of woody plant material is extremely difficult. Our study was conducted to investigate the effect of biocide addition during the in vitro culture of blueberry on plantlet growth and contamination occurrence. Four biocides, including Plant Preservative Mixture (PPM TM ), vancomycin, nystatin and penicillin G, were used in varying concentrations during the in vitro propagation of blueberry. When nystatin was added into the medium at low concentrations, the overall growth of blueberry plantlets was retarded. Addition of vancomycin and penicillin G in high concentrations decreased contamination but induced plantlet mortality. On the other hand, when 1ml/L PPM TM was added, the growth characteristics of blueberry plantlets did not significantly differ from non-treatment (control), and the contamination occurrence rate was very low. From these results, we found that the addition of the appropriate biocide could provide an effective method to reduce contamination in the culture process, thereby raising in vitro production efficiency. Abstract Interest and great demand for blueberry (Vaccinium corymbosum) have increased, as V. corymbosum is now one of the most economically important crops in Korea. It is expected that blueberry production and the area planted for cultivation will increase consistently in the years ahead because of high profitability and the consumer's demand for healthy ingredients. Effective mass production of blueberry is urgently needed for commercial cultivation establishment, but a main limitation is lack of a propagation system that produces a disease-free plant material for commercial plantation. A large amount of research has focused entirely on developing tissue culture techniques for blueberry propagation. However, controlling fungal and bacterial contamination of woody plant material is extremely difficult. Our study was conducted to investigate the effect of biocide addition during the in vitro culture of blueberry on plantlet growth and contamination occurrence. Four biocides, including Plant Preservative Mixture (PPM TM ), vancomycin, nystatin and penicillin G, were used in varying concentrations during the in vitro propagation of blueberry. When nystatin was added into the medium at low concentrations, the overall growth of blueberry plantlets was retarded. Addition of vancomycin and penicillin G in high concentrations decreased contamination but induced plantlet mortality. On the other hand, when 1ml/L PPM TM was added, the growth characteristics of blueberry plantlets did not significantly differ from non-treatment (control), and the contamination occurrence rate was very low. From these results, we found that the addition of the appropriate biocide Introduction Vaccinium fruits are now considered as a health food because they exhibit relatively high antioxidant and anti-inflammatory properties (Prior et al. 1998;Ehlenfeldt and Prior 2001;Zheng and Wang 2003). Blueberry (Vaccinium corymbosum) is by far the most important commercial crop worldwide, as well as in Korea. Cultivars of these crops are vegetatively propagated by stem cuttings to maintain their hereditary characteristics (Douglas 1966). This propagation method is slow and many genotypes do not properly respond to root-inducing growth regulators. As an effective alternative, tissue culture techniques have been used for rapid mass propagation of superior genotypes (regardless of season) and the production of virus-free plants. In addition, these in vitro culture techniques are broadly applied in conventional breeding programs (Meiners et al. 2007). However, microbial contamination usually occurs during the plant tissue culture process, causing many serious problems. Contamination by different sources, such as bacteria and fungi, reduces productivity and prevents successful culture procedures. Therefore, various methods are used to eliminate bacterial and fungal contamination, including the application of sterilizing agents and antibiotics and fungicides, and inactivation by heat and light (Kneifel and Leonhardt 1992;Leifert et al. 1992;Salehi et al. 1997;Haldeman et al. 1987;Reed and Tanprasert 1995;Seckinger 1995). After surface sterilization, explants are either submerged in an antibiotic-antimycotic solution, or these agents are added to the culture medium to hinder the growth of microorganisms on the explants (Colgecen et al. 2011). Contaminants are often difficult to detect because they predominantly remain within the plant tissue (Viss et al. 1991). Contaminated plants may exhibit no visible symptoms, reduced multiplication and rooting rates, or may die (Leifert et al. 1989). Introduction of microorganisms results from poor aseptic technique or inadequately sterilized equipment, but can be overcome with improvements in training or equipment handling. However, elimination of internal contaminants is much more serious and problematic (Buckley et al. 1995). Ideal antimicrobial agents should be soluble, stable, unaffected by pH and media, lacking in side effects, broadly bactericidal and fungicidal, suitable in combinations, nonresistanceinducing, inexpensive and nontoxic to human health (Falkiner 1990). Therefore, many biocides have been evaluated on the bacterial and fungal contaminants of various plants. Plant Preservative Mixture (PPM TM ) is a combination of two broad-spectrum industrial isothiazolone biocides, chloromethylisothiazolone and methylisothiazolone. The active ingredients in PPM TM also include magnesium chloride, magnesium nitrate, potassium sorbate and sodium benzoate. This agent is heat stable and can be autoclaved with culture medium (Lunghusen 1998). Mutants resistant to PPM TM rarely develop because it targets specific multiple enzymatic sites in the Krebs cycle and electron transport chain of microorganisms (Chapman and Diehl 1995). Niedz (1998) tested PPM TM with many types of citrus tissue culture and demonstrated that it could be used with culture media to prevent bacterial and fungal contamination. The positive effects of PPM TM have been reported in a number of plants, including the non-embryogenic callus of sweet orange, shoot regeneration of rough lemon, adventitious melon, petunia, tobacco and pepper (Compton and Koch 2001;Guri and Patel 1998). Nystatin is a fungistatic and fungicidal polyene antibiotic that increases the permeability of the cell membrane of sensitive fungi by binding to sterols, chiefly ergosterol (Brezis et al. 1984). Its main action is against Candida species. It is also effective against Aspergillus, Coccidioides immitis, Cruptococcus neoformans, Histoplasma capsulatum, Blastomyces dermatidis and other yeasts and fungi (Garrod et al. 1981;Medoff and Kobayashi 1980). Vancomycin is an amphoteric glycopeptide antibiotic produced by Streptomyces orientalis. It inhibits bacterial cell wall synthesis by binding to peptidoglycans. This antibiotic has a relatively narrow spectrum of activity and prevents the growth of Gram-positive bacteria. All Gram-negative bacteria are known to be resistant (Pollock et al. 1983). Penicillin G acts by inhibiting cell wall synthesis through binding to penicillin binding proteins (PBPs), inhibiting peptidoglycan chain cross-linking. This antibiotic is active against Gram-positive bacteria but much less effective against Gram-negative organisms (Pollock et al. 1983). Nowadays various biocides are industrially used as bactericidal and fungicidal compounds, but careful checks need to be performed to confirm that they do not inhibit or alter plant growth during in vitro culture procedures. Therefore, in this study we describe contamination inhibition using different biocides incorporated into the culture media and verify their effects on plantlet growth during the in vitro culture of blueberries. Materials and Methods In vitro culture of blueberry plantlets Three blueberry cultivars ('Spartan', 'Northland' and 'Woodard'), which were cultured for in vitro shoot proliferation in woody plant medium (WPM) and Murashige and Skoog medium adjusted to pH 5 and supplemented with growth regulators were used in our experiments. These plantlets were individually obtained from meristem cultures and incubated in 450㎖ glass vessels containing 90ml of culture medium (medium composition depended on the cultivar) at 23±1°C with a 16h photoperiod (40 μmol･m -2 ･s -1 light intensity). Biocide addition into the culture medium Four biocides were used: Plant Preservative Mixture (PPM TM ), vancomycin, nystatin and penicillin G. For culturing, 0, 0.5, 1, 2 and 4 ml/L PPM TM were added into the blueberry culture medium before autoclaving. Stock solutions of other biocides were made up fresh, filter-sterilized and added to the medium after autoclaving. Vancomycin (0, 1, 2.5, 5, and 10 mg/L), nystatin (0, 10, 25, 50, and 100 mg/L) and penicillin G (0, 0.5, 1, 2, and 4 mg/L) were tested at various concentrations. Blueberry plantlets cultured in proliferation medium were transferred into a separate biocide-containing medium, and each treatment was replicated 5 times. After three months, contamination occurrence, as well as growth characteristics, such as shoot number, shoot length and death rate, were calculated. Statistical analysis Data were subjected to a two-way analysis of variance (ANOVA) and the differences among means were compared using Duncan's multiple range test (Duncan 1955). Results and Discussion Effect of biocide addition on plantlet growth and contamination occurrence in blueberry cultivar 'Northland' Addition with higher concentrations of biocides decreased in vitro contamination, increased death rate, and prevented the overall plantlets growth of blueberry cultivar 'Northland' (Table 1). In particular, nystatin treatment at a low concentration induced plantlet death, as well as growth retardation. Nystatin, a fungicide known to be mainly active against Candida species and yeast, increases cell membrane permeability of sensitive fungi by binding to ergosterol and hinders cell wall synthesis. Therefore, it was suggested that this inhibitory action results in blueberry plantlet growth inhibition and death. When vancomycin and penicillin G were added at relatively higher concentrations, contamination rates decreased by 40~48%, but phytotoxicity, as indicated by plantlet death, was observed. Although both vancomycin and penicillin G have been used as popular antibiotics to prevent the growth of various bacteria, these agents showing phytotoxicity to plants could not be recommended in our experiment. Other studies also reported different effects of these antibacterial agents. Martina (1999) evaluated the effect of vancomycin on the growth of in vitro Primula vulgaris; a 40~80 mg/L vancomycin application had no effect on plant growth in comparison to control groups grown on the same medium without vancomycin. Gholamhoseinpour et al. (2012) reported that 200 mg/L vancomycin resulted in the least amount of bacterial infection without having any deleterious effect on the growth and proliferation of peach x almond hybrids. Penicillin G was effective in eliminating bacterial contamination on ginseng, but caused abnormal and depressed callus formation and an inhibition of somatic embryogenesis (Teng and Nicholson 1997). In another study, a penicillin agent had a positive effect on the growth of Oryza sativa seedlings (Mukherji and Biswas 1985). Such stimulatory effects have not yet been fully explained, but these antibiotics are not likely to act as growth regulator substitutes. At 1 ml/L PPM TM , shoot proliferation and death rate were not shown to be appreciably different from non-treatment (control), as the contamination rate significantly declined by 52%. On the other hand, death rates doubled at 2 and 4 ml/L PPM TM , as these levels were seen to induce phytotoxicity. PPM TM is a relatively new, broad-spectrum agent developed by Plant Cell Technology, Inc. (USA) and has been increasingly used in the tissue culture of many species, including salad burnet, melon, petunia, tobacco, chrysanthemum, European birch and rhododendron at concentrations ranging from 0.5 to 10 ml/L (Babaoglu and Yorgancilar 2000;Crompton and Koch 2001;George and Tripepi 2001). PPM TM is designed to kill cells of bacteria and fungi and inhibits spore germination (Plant Cell Technology 2006). It is either used for surface sterilization or included in the culture medium to remove the internal contaminants that may be present in explants. Babaoglu and Yorgancilar (2000) reported that the use of PPM TM was very effective in controlling in vitro contamination without impairing shoot regeneration from petiole and hypocotyl explants of salad burnet (Poterium Effect of biocide addition on plantlet growth and contamination occurrence in blueberry cultivar 'Spartan' and 'Woodard' Biocide addition at relatively higher concentrations also decreased in vitro contamination, increased death rate and prevented plantlet elongation of the blueberry cultivar 'Spartan' ( Table 2). Nystatin was found to be effective in reducing the contamination rate by half, but had a phytotoxic effect on plantlets in vitro at low concentrations. Vancomycin and penicillin G treatments at higher concentrations decreased contamination efficaciously, but phytotoxicity, as indicated by plantlet death, increased severely. When PPM TM was incorporated into medium at 1 ml/L, the negative effect impairing shoot growth was rarely observed, as contamination considerably declined by 58%. Table 3 shows that 1 ml/L PPM TM was consistently very effective in controlling contamination while being gentle to the shoot growth and proliferation of the blueberry cultivar 'Woodard'. Our results from these experiments demonstrated that the industrial biocide PPM TM can be used effectively to control the growth of bacteria and fungi in blueberry tissue cultures. The isothiazolones present in PPM TM exhibited little phytotoxicity at the recommended levels. PPM TM appears to be most effective in inhibiting the growth of air-and waterborne bacteria and fungi. Since it is heat stable and can be autoclaved with the plant growth medium, it may be best suited for use as a preservative agent in culture medium to control contamination.

v3-fos

2018-12-22T15:27:46.624Z

2015-07-01T00:00:00.000Z

59358818

s2

Bromatological and mycotoxin analysis on soybean meal before and after the industrial process of micronization Aflatoxins, fumonisins and zearalenone take part of the most studied mycotoxin groups due to their toxic effects on animal and human health. This research evaluated samples of soybeans meal used in animal food industry. A hundred and twenty one soybean meal samples were analyzed, so that 66 were analyzed before the industrial processing of micronization and 55 after it. The bromatological average of samples before micronization showed the following answers: 12.4% moisture; 46.4% protein; 79.5% protein solubility; 5.9% ash content; 2.2% fat; 4.3% fiber and 0.02 (ΔpH) of urease activity. The samples of micronization soybean meal showed 7.0% average values for moisture and 48.6% for crude protein. The mycotoxin levels were low in natura soybean meal; therefore, average values were 0.5μg kg-1, 29.6μg kg-1 and 56.8μg kg-1 for aflatoxin, zearelenone and fumonisin, respectively. After micronization, the average values for the studied samples were 1.3μg kg-1, 67.5μg kg-1 and 89.1μg kg-1, respectively for the same mycotoxins. The results for bromatological and mycotoxin analyses indicate similarity with the established patterns according to the Brazilian Compendium for Animal feed and reference literature. However, at least one of the three studied mycotoxin was detected in all of the analyzed samples and there was greater contamination of soybeans meal after the micronization process. INTRODUCTION Companies responsible for the animal feed have worked hard to produce adequate and free of contaminants food in order to improve a great nutrient absorption. The adequate offer of nutrients which will meet the animals' needs allows better food conversion and greater production (TININI et al., 2012). The essential raw matters to produce animal feed are corn and soybean meal, which when associated to minerals, vitamins, amino acids and additives meet the nutrients need for vital functions of the animal (BELLAVER & SNIZEK JR, 2012). Micronization is a grinding process that transforms soybean meal into very fine particles (microns). Due to the increase of the superficial area of a raw matter generated through this process, micronization enables an increase in solubility and bioavailability, which improves powder uniformity and mixture quality (CAMPOS & SILVA, 1986). The presence of mycotoxin in animal feed also affects the product quality. This factor may cause problems to the industry and farming since it may increase the incidence of diseases and lessen yield efficiency (SASSAHARA et al., 2003). There are over four hundred types of different toxins that are produced by over 350 types of fungi. The well-known mycotoxins in grains and processed products in soybeans are: Aflatoxins (B1, B2, G1 and G2), deoxynivalenol, nivalenol, ochratoxin A and zearalenone. The aflatoxins and ochratoxins are produced by fungi of Aspergillus genus, while deoxynivalenol, nivalenol and zearalenone are produced by fungi of Fusarium genus (SALINAS, 2006) The fumonisins are produced by Fusarim verticillioides and F. proliferatum under high humidity conditions and high temperature. These fungi are widely distributed in a global scale and found in soil and on plants surface and they contaminate grains in both field and storage process. Fumonisin B1 (FB1) prevails in nature and it is the most toxic fungus. It represents almost 70% of total contamination on food and on animal food naturally contaminated (KOBASHIGAWA, 2010). Legal mechanisms have been adopted in many countries in the attempt to avoid the harmful effects caused by mycotoxins in ingredients that compose animals' feedstuffs. The most well-known laws are the ones that regulate aflatoxin levels this also happens to the one established by MERCOSUL and the other one adopted by countries in America which establish laws for mycotoxins in such products. Nevertheless, laws for other mycotoxins are under implementation in the same way they already are for different animal species in European Union (FREIRE et al., 2007). Although there are legal standards for aflatoxins or, in some countries, for other mycotoxins, these micotoxins rarely appear isolated. There is usually simultaneous interaction among different mycotoxins, and this may decrease the safety limits established by law. In Brazil, research on food quality for animal consumption has shown increasing problems caused by mycotoxins. The tropical and subtropical climates in specific Brazilian regions are appropriate fungi development and consequently mycotoxins. The demands of the consumer market plus this climate feature and the excessive control employed by the national and international governmental agencies point to the need of implementing programs to monitor food quality (SASSAHARA et al., 2003). Due to the importance of quality control in the production of animal feed in all steps of this process, this study aimed at evaluating soybean meal samples before and after the industrial process of micronization according to bromatological and mycotoxin analyses. Soybean meal sampling collection Samples of almost 1,000 grams were weekly collected and analyzed in a manufacturing unit in an industry for animal nutrition from Santa Catarina state, from September, 2010 to November, 2011. A total of 121 soybean meal samples were analyzed and 66 of them were obtained before the industrial micronization process while 55 were collected after it. The soybean meal samples were collected in the arrival of trucks and trailer with the aid of a depth probe. After sampling, were sent to the laboratory in identified plastic bags. Then, the samples were grinded and homogenized in a Retsch ® ZM 200 mill (Retsch, Inc.-Germany) at 18,000 rpm speed in order to obtain particles smaller than 0.5mm. The samples of micronized soybean meal were collected by a scoop in the storage silo or during the product weighting. Since the particles of micronized soybean meal samples were less than 212 µm the end of the processing, were analyzed without the milling step. The analyses were performed in triplicate Bromatological analyses The bromatological analyses of soybean meal samples before micronization were: moisture (MC), soluble protein in KOH (SP), ashes content (AC), lipids (L), crude fiber (CF) and urease activity according to Analytical Methods for Animal Feed Control (BRASIL, 1991 The analyses for micronized soybean meal were moisture (MC) and crude protein (CP), according to the above mentioned methods. Each toxin was extracted using 5 grams of sample in 25mL methanol 70% that was stirred for 3 minutes. Later, they were filtered in a filter paper Quanty ® JP 41black label and used for the immunoassay. Quantitative determination was obtained by a Stat Fax 321 Plus (Awareness technology, Inc.-EUA) plate reader at a 650nm wavelength. Calibration curve preparation Neogen Veratox ® patterns were inserted to prepare the calibration curve of each mycotoxin. For the aflatoxins, the used patterns varied from 0 to 50μg kg -1 concentrations; while for fumonisin, this range was from 0 to 6,000μg kg -1 and for zearalenone, from 0 to 500μg kg -1 . The absorbance graph versus wavelength of each toxin analyzed was drawn and the linearity of calibration curve was in r 2 ≥0.99. The limit of detection (LD) and quantification (LQ) of the method used for aflatoxins, zearalenone and fumonisin was 1,4μg kg -1 and 5,0μg kg -1 ; 200μg kg -1 and 500μg kg -1 ; 10μg kg -1 and 25μg kg -1 , respectively. Based on the calibration curve, the amount of aflatoxins, fumonisins and zearalenone was calculated by the line equation through the linear regression. RESULTS AND DISCUSSION The bromatological averages of the studied 66 soybean meal samples before micronization are shown on table 1. This study recorded an average result of 0.02 (ΔpH) for urease activity in soybean meal samples (Table 1), which it is under the minimum answer established by the Brazilian Compendium of Animal Feed, whose reference values for soybean meal with protein vary from 44% to 48% and for maximum urease activity is 0.15 (ΔpH) (CBAA, 2009). The urease activity of crude grain varies from 2.0 to 2.5 (ΔpH) (BUTOLO, 2002). Soybean meal is obtained from the soybeans grains milling to extract oil, which is used for human consumption as it is an important ingredient for animal feed (BUTOLO, 2002). Due to the presence of anti-nutritional factors in soybeans, it's in natura use in diets formulation is not recommended since there may show some deleterious effects to health, especially in swine and poultry. Soybeans grains have some inhibitors of trypsin, chymotrypsin, lectins, among others (BELLAVER & SNIZEK Jr., 2012). The determination of urease activity aims at evaluating whether soybean meal has received the right thermal processing to inactivate the anti-nutritional factors present in soybeans (LIMA et al., 2011). Thus, the results obtained in this study have indicated an appropriate process as well as the studied presented soybean meal with excellent classification pattern concerning urea activity, between 0.01 to 0.05, according to LIMA et al. (2011). Protein solubility is recognized as one of the best methods to evaluate sub or over-processing (BELLAVER & SNIZEK JR., 2012) and indicates the available protein percentage to be absorbed by the animal. According to MENDES et al. (2004), the ideal range for solubility concerning animal feed is 73-85%. Values below 70% have indicated overheating and over 85%, they are associated to the sub-processed soybeans. Thus, the results of protein solubility to 79.5% soybean meal samples tested in this work, showed that are in accordance to the allowed variation, so, there was no sub or overheating in the processing. The lipid content variable is influenced by the oil extraction process (OST et al., 2005). The values obtained for soybean meal samples before micronization showed a 2.2% average (Table 1). Similar results were observed by HENZ et al. (2009), who recorded the same average (2.2%). On the other (OST et al., 2005). The average result for crude fiber analysis was 4.3%. So, when these data were compared with GENEROSO et al. (2008) results (5.2%), it was possible to correlate it with the centesimal of crude protein. According to OST et al. (2005), variation can occur as addition of the soybean hulls derived from soybean grain processing, thereby reducing the percentage protein. After micronization process, it could be observed a moisture decrease on soybean meal from 12.4% to 7.0%. Micronization is a heat treatment of soybean meal, thus, there is greater loss of water available as well as greater concentration of nutrients occurs. This can also be evidenced by the averaged values of crude protein (CP) that accounted for 46.4% before and 48.6% after processing step. The results of the studied mycotoxins analyses are represented in Figures 1A and 1B. Among the analyzed samples of soybean meal before micronization, 92% of them (n=61) were contaminated with zearalenone, 55% (n=36) were contaminated with aflatoxins and 30% (n=20) with fumonisin ( Figure 1A). The maximum aflatoxin contamination was 3.8μg kg -1 , fumonisin 500μg kg -1 and 204μg kg -1 zearalenone. Besides the values variability, it is possible to observe the contamination of the same sample with different mycotoxins. Mycotoxins determination in soybean meal before micronization reported the following average level of contamination in samples of aflatoxins, Zearalenone and Fumonisins of 0.5μg kg -1 , 29.6μg kg -1 and 56.8μg kg -1 , respectively. Among micronized soybean meal samples, it was observed that 21 samples (38%) were contaminated with fumonisin, 45 (82%) had aflatoxin and 55 (100%) contained zearalenone toxin ( Figure 1A). The maximum aflatoxin contamination was 4.7μg kg -1 , 1000μg kg -1 fumonisin and 159.2 μg kg -1 zearalenone. The contamination average level of micronized soybean meal samples of Aflatoxin, Zearalenone and Fumonisin was 1.3μg kg -1 , 67.5μg kg-1 and 89.1μg kg -1 , respectively ( Figure 1B). Similar results to the obtained ones in this study have been described by NETTO et al. (2002), who researched various sources of mycotoxins in animal feed. Among the 21 soybeans samples analyzed by these researchers, aflatoxin was detected with 14.3% and zearalenone with 42.9% in the same samples. Moreover, TININI et al. (2012) have analyzed 34 soybean meal samples and observed the presence of mycotoxins in 44.1% of them (n=15), but, the average values reported for aflatoxin were 7.4μg kg -1 and 2.58μg kg -1 for zearalenone, which was different from the average of the values obtained in this trial. Although the fumonisin produced by Fusarium moliniforme strains is often found in maize (MAZIERO & BERSOT, 2010), in the present study, this mycotoxin was present in 30% in natura samples and in 38% micronized soybean meal ( Figure 1A). Different results were reported by MALLMANN et al. (2001), Who evaluated fumonisin B1 levels in 407 samples of several cereal (maize, Rice, wheat, barley, oats and soybean meal) in Southern Brazil and did identify mycotoxin in soybean meal samples. Average values observed for the studied mycotoxins are shown in figure 1B and there is a higher level of the studied three toxins after micronization manufacturing process. The micronization process may represent an increase in mycotoxins levels, since the external environment variations due to temperature change or humidity in silos, stores or even during grinding stage, can quicken secondary metabolites production due the stress fungus will be submitted (LAZZARI, 1997). According to the incidence percentage, zearalenone was the most present toxin regarding a greater number of samples. It was identified in 92% samples before micronization and in 100% postprocessing samples. Zearalenone is an analog of estrogen and may cause hyperestrogenism. There are also several incidents of advance puberty in children because of its presence (IAMANANKA et al., 2010). In this research, fumonisin and aflatoxin presented lower incidence ( Figure 1A). TININI et al. (2012) researched about mycotoxin presence in dairy cattle diet on family farms in western Paraná and identified that 44.1% soybean meal samples were positive for aflatoxin and zearalenone. The presence of aflatoxin can be associated to the storage conditions of soybeans or soybean meal. Fumonisins and zearalenone are produced by field fungi, from Fusarium gender. The possibility of the presence of these mycotoxins in products analyzed may be due to contamination of soybeans by fungi. When they find the ideal conditions of temperature and humidity for their development during storage of these grains, they just produced them. Another likely origin may be associated with the presence of impurities (OLIVEIRA et al., 2010) that have not been properly removed during the pre-cleaning step that is performed soon after harvest. Although the obtained contamination levels are below the standard or found ones in other studies, it is of paramount importance to consider all the components on animal feed formulation because each raw matter can present different values of mycotoxins that can contribute to increased toxic levels as well as may cause acute or chronic effects. CONCLUSION The results of bromatological and mycotoxin analyses have pointed out some compliance with the standards established by the Brazilian Compendium of Animal Nutrition and reference literature. However, it has been observed at least one of the three mycotoxins in all studied samples. Furthermore, the micronization process has contributed to the increased levels of all studied mycotoxins.

v3-fos

2016-03-22T00:56:01.885Z

2015-12-25T00:00:00.000Z

7749928

s2

Bioactive Compound Content and Cytotoxic Effect on Human Cancer Cells of Fresh and Processed Yellow Tomatoes Tomato, as a fresh or processed product, has a high nutritional value due to its content of bioactive components such as phenolic compounds. Few studies describe the effect of processing on antioxidant content and the cancer cell growth inhibition activity. In this study we determined the phenolic and ascorbic acid content of three yellow tomato varieties, before and after thermal processing. Moreover, we determined the antioxidative power and tested the effects of tomato extracts on three human cancer cell lines. We found that the amount of phenolic acids (chlorogenic acid and caffeic acid) decreased in all the samples after processing, whereas the flavonoid content increased after the heat treatment in two samples. A cytotoxic effect of tomato extracts was observed only after processing. This result well correlates with the flavonoid content after processing and clearly indicates that processed yellow tomatoes have a high content of bioactive compounds endowed with cytotoxicity towards cancer cells, thus opening the way to obtain tomato-based functional foods. Introduction Among crops, tomato (Solanum lycopersicum), with a total production of around 160 million tons per year, is the second most important source of nourishment (after potatoes) for the World's population [1]. Its consumption has increased in the last few years with the commercialization of several processed products such as sauces, juices, soups and purees. It has been estimated that 35% of raw tomatoes are consumed as sauces, 18% as tomato paste, 17% as canned tomatoes, 15% are transformed into juices, and 15% into catsup [2,3]. Tomato fruits show a high nutritional value, due to their content of several important micronutrients such as carotenoids, vitamins (C and E) and phenolic compounds [4,5]. Recent studies have demonstrated that the regular intake of tomato, either fresh or processed, is associated with a reduced risk of inflammation, cancer, cardiovascular diseases, diabetes and obesity and can increase cell protection from DNA damage by oxidant species [3,6]. In particular, phenolic compounds, including phenolic acids (chlorogenic, caffeic, ferulic, gallic, p-hydroxybenzoic, protocatechuic and p-coumaric acids) and flavonoids (rutin, quercetin, naringenin, kampferol and derived), are of particular interest since they are effective free radical scavengers through the presence of para-hydroxyl groups. These compounds are able to regulate cellular signaling processes during inflammation or may operate as signaling agents themselves thus reducing the risk of neurodegenerative diseases, cancer and aging [7,8]. Flavonoids are the main group of phenolics in tomatoes and include flavonols (such as quercetin), flavanols (such as catechins), flavanones (such as naringenin), anthocyanidins and stilbenes (such as resveratrol). These compounds are usually located in the skin and contribute to the aroma, fragrance and color [9,10]. It has been demonstrated that rutin, quercetin, glycosides of quercetin, resveratrol and catechin are active in rheumatoid arthritis and exert intestinal anti-inflammatory activity. Their mechanisms of action comprise the inhibition of differentiation and function of osteoclast/macrophage and the modulation of estrogen [11][12][13][14]. Diet-derived flavonoids have also been demonstrated to possess antiallergic, antiulcer, antioxidant, antiradical, antidiabetic, cardioprotective, antiviral, antibacterial, antifungal, antiproliferative and anticarcinogenic activities [15]. Food-derived quercetin is able to inhibit the growth of various cancer cell lines, although the exact mechanism of this action is not thoroughly understood [15]. To date, only a few studies have focused on the impact of processing on the general nutritional quality and antioxidant activities of tomato fruits. In addition, little is known regarding the biological activities of tomato polyphenolic antioxidants [16,17]. Since during tomato pasteurization several changes in bioactive molecules composition can occur [18,19], the present study was aimed at investigating the effect of a classical processing procedure on the antioxidant content and in particular on the phenolic profiles of yellow tomatoes. These varieties have been selected on the basis of their high amount of bioactive molecules and, in particular, of phenolic compounds in the fresh fruit [20]. We characterized their nutritional value (including carotenoids, ascorbic acid and phenolics content) before and after processing. In addition, we evaluated their effect, before and after processing, on three human cancer cell lines by using MTT assays. Content of Antioxidant Compounds Herein we evaluated the content of bioactive compounds in yellow tomato genotypes before and after processing. These genotypes are one Italian (GiàGiù, coded E40) and two Bolivian landraces (M-4 and 284, coded E87 and E92, respectively) and all are cherry-type tomatoes. They were previously selected in our laboratory for their high levels of ascorbic acid (AsA) and/or phenolics [20]. Before chemical extraction, the moisture content both in the processed and un-processed samples was measured and found to be around 93% for all the genotypes under study. The solid content for each sample is reported in Table 1. Table 1. Solid content (g/100 g FW), total carotenoids (mg/100 g FW), lycopene (mg/100 g FW) and beta-carotene (mg/100 g FW) content in yellow tomato lines before (NP) and after (P) processing. Total values are presented as means˘SD (n = 9). NP: Not processed. P: Processed. Asterisks indicate values that are significantly different from the non-processed samples (** p < 0.01). Carotenoids The levels of total carotenoids, lycopene and β-carotene before and after processing are reported in Table 1. Total carotenoids in the genotypes E40 and E87 did not reveal a significant change after heat treatment, whereas a significant mean increase was observed in E92 fruits (63.1%). As expected, the analyzed yellow varieties did not show the presence of lycopene in either unprocessed or processed samples as also reported in earlier studies [20,21]. β-Carotene, the major found carotenoid, decreased significantly (p < 0.01) after the heat treatment only in the E40 genotype. Conflicting data on tomato carotenoid stability during thermal processing can be found in the literature. Many authors have reported that carotenoid content was stable in tomatoes submitted to different thermal treatments [22,23]. Georgé et al. [21] found that in yellow tomato genotypes β-carotene content was greatly reduced in processed yellow tomatoes compared to fresh tomatoes, while the thermal treatment did not significantly affect the amount of β-carotene and lycopene in red tomatoes. On the contrary, Capanoglu et al. [18] showed a significant decrease in both lycopene (32%) and β-carotene (36%) content in processed red tomatoes, while other authors reported an increase in lycopene amount in tomato products. In particular, Re et al. [24] found a general increase in the lycopene level in several processed products, with up to 30% in a tomato paste. These contrasting results may be due to the temperatures and durations adopted for the different processing methods. Ascorbic Acid and Total Phenolics The levels of ascorbic acid (AsA) in both fresh fruit and processed samples are reported in Table 2. In the three analyzed varieties (E40, E87 and E92), a significant decrease (p < 0.001) was observed in the processed samples with respect to the mean value in fresh fruits (59.8%, 54.8% and 48.4%, respectively). Several examples of AsA loss during thermal processing of tomato products have been reported. In accordance with our data, Abushita et al. [16] and Capanoglu et al. [18] observed a loss of about 50% after thermal processing, whereas Pérez-Conesa et al. [25] reported a mean AsA degradation value of 90% after pasteurization of tomato purées. Gahler et al. [26] correlated a reduction in AsA levels in different tomato products with an increase in the heating time and the number of processing steps. Dewanto et al. [27] found a loss of AsA in heat-processed tomatoes at 88˝C with an estimated D 88˝C value (the time taken for 90% reduction of the initial vitamin C content at 88˝C) of 276 min. The loss of ascorbic acid during processing could be decreased by using mild treatments and lower temperatures [28]. In this study, spectrophometric methods were used to study the effects of processing on phenolic compounds content. In accord with the behavior observed for AsA, total phenolics levels exhibited a general reduction after thermal treatment ( Table 2). Mean decreases of 12%, 16.6% and 26% were found in E40, E87 and E92 from the initial mean values of 50.9˘1.7, 52.9˘1.8 and 53.5˘1.2 mg/100 g FW, respectively. The changes in the content of phenolic compounds we observed in this work could be due to the oxidation of the molecules due to the presence of oxidative and hydrolytic enzymes released during the process. Total flavonoids amount did not reveal a significant variation after processing in E40, while a significant increase of 20.5% and 76.9% (p < 0.001) was observed in the genotypes E87 and E92, respectively ( Table 2). We observed a general increase of the ratio flavonoids/total phenolics in the three analyzed genotypes after processing. In particular, the increase of this parameter after the treatment ranged from 1.15 fold in E40 to 2.45 fold in E92. Marinova et al. [29] found a ratio of around 0.17 in red tomato fruits. Our data are consistent with evidence from the literature. Indeed, Georgé et al. [21] found a significant (28%) decrease of total polyphenol content after thermal processing in yellow tomatoes. Pérez-Conesa et al. [25] also observed a decrease of these compounds attributed to the pasteurization step. On the contrary, Gahler et al. [26] found an increase in the total phenolic content during the processing of tomato juice and tomato sauce. Dewanto et al. [27] reported no significant variations in total phenolic compounds after heat treatment. The differences observed in these studies can be due to the different processing methods adopted, together with the different analyzed matrixes and their composition. However, it has to be taken into consideration that total phenolic values are only indicative of the amount of polyphenols in tomatoes, since no single analytical procedure is able to accurately measure the total polyphenol amount. This is due to the structural diversity found among phenolic molecules and the high variation in content depending on the tested matrix [30,31]. Table 2. Ascorbic acid, total phenolics, total flavonoids content and flavonoids/phenolics ratio in yellow tomato lines before (NP) and after (P) processing. Values are presented as means˘SD (n = 9). Data are expressed as mg/100 g FW. NP: Not processed. P: Processed. Asterisks indicate values that are significantly different from the non-processed sample (*** p < 0.001). Identification of Individual Phenolic Compounds In order to evaluate changes in the composition of individual phenolic compounds after processing, we carried out HPLC analyses (Tables 3 and 4 Figure 1). Chlorogenic acid, its isomer 5-caffeolylquinic acid (5-p-CQA) and rutin were the most abundant compounds in the three analyzed varieties, in both unprocessed and processed forms (Tables 3 and 4), in accordance with data reported by Garcìa-Valverde et al. [6] and Slimestad and Verheul [32]. Table 2. Ascorbic acid, total phenolics, total flavonoids content and flavonoids/phenolics ratio in yellow tomato lines before (NP) and after (P) processing. Values are presented as means ± SD (n = 9). Data are expressed as mg/100 g FW. NP: Not processed. P: Processed. Asterisks indicate values that are significantly different from the non-processed sample (*** p < 0.001). Identification of Individual Phenolic Compounds In order to evaluate changes in the composition of individual phenolic compounds after processing, we carried out HPLC analyses (Tables 3 and 4, Figure 1). Chlorogenic acid, its isomer 5-caffeolylquinic acid (5-p-CQA) and rutin were the most abundant compounds in the three analyzed varieties, in both unprocessed and processed forms (Tables 3 and 4), in accordance with data reported by Garcìa-Valverde et al. [6] and Slimestad and Verheul [32]. A general significant decrease of phenolic acids was found in the three genotypes after the heat treatment (Table 3). In particular, in the E40 genotype we found that chlorogenic acid, caffeic acid and 5-p-CQA were reduced by 56.4%, 21.7% and 38.1%, respectively. For the same compounds, in E87 we found a decrease of 48.3%, 35.1% and 28.1%, respectively. Finally, in E92 decreases of 58.5%, 25% and 42.1%, were found for the three detected phenolic acids. The reduction of chlorogenic acid was also reported by Vallverdú-Queralt et al. [19] and Bugianesi et al. [33]. Chlorogenic acid and caffeic acid may be oxidized to reactive o-quinones through the catalytic oxidation process and so heat treatment could result in their degradation. In addition to the loss of total phenolic acids, we found a significant decrease (26.9%) in the content of total flavonoids in E40 (Table 4). However, despite the decrease of phenolic compounds observed, the E40 genotype maintained significantly higher levels of phenolic acids and flavonoids, compared to the other two varieties, even after the thermal treatment (p < 0.05). No significant differences were observed in the flavonoid content of E87 extracts, whereas a significant increase in E92 extracts (about 41%) was observed after processing. These last results relate well with those obtained using spectrophometric methods. The increase of flavonoids content in E92 could be also due to the fact that homogenization and thermal processing disrupt cell membranes and cell walls, thus facilitating the release of some nutrients from the tissues, making them more easily extractable from the matrix [34]. In all the analyzed genotypes variations in the distribution of flavonoids were detected after processing. For example, in E40 and in E92 the amount of rutin decreased significantly by about 25.4% and 20%, respectively, whereas in E87 the amount of rutin did not change significantly from the initial value of 10.7˘0.02 mg/100 g FW. In accordance with our data, Capanoglu et al. [18] found that rutin decreased after treating the samples in a three-effect evaporator unit. Interestingly, we also observed a significant increase in quercetin levels, of about 1.3-, 1.5-and 3-fold in E40, E87 and E92, respectively, and an increase of about 6-folds of quercetin3-β-D-glucoside in E92 from the mean initial value of 1.2˘0.17 mg/100 g FW. By contrast, a decrease of quercetin3-β-D-glucoside of 58.9% and 29.5% was found in E40 and E87, respectively. The difference in stability of some phenolics in the three analyzed genotypes could be related to different factors linked to the composition of the analyzed fruits. Finally, naringenin chalcone was converted into naringenin after heat treatment. This is probably due to unforeseen cyclization of the chalcone to the corresponding flavanone during the processing [35,36]. Though naringenin and naringenin chalcone have analogous chromatographic properties using the HPLC method reported in the Experimental Section, they were easily distinguished due to their different wavelength of their maximum absorptions (280 nm for naringenin and 330 nm for naringenin chalcone). Antioxidant Activity In the processed E40 genotype LAA showed a mean increase of 13.2%, from the mean initial value of 39.5˘2.5 µmol TE/100g FW in fresh fruit (Table 5). In contrast, E87 revealed a significant decrease after processing (p < 0.001). No significant changes were on the other hand observed in the E92 samples. Before processing the genotype E87 showed a significantly higher value of LAA compared with the other two analyzed cultivars (p < 0.001), while after processing a significantly higher LAA value was observed in the genotype E40 (p < 0.001). These results do not correlate with the amount of total carotenoids and β-carotene calculated in the different samples, and so a significant contribute to the LAA could be attributed to other molecules, such as vitamin E. Indeed, Seybold et al. [37] investigated the content of carotenoids and vitamin E in samples of tomato sauce, soup, baked slices, and juice taken after different heating times. The authors demonstrated that carotene amount decreased or was stable, while tocopherol content significantly rose during short-term heating. HAA was evaluated by both ABTS and FRAP assays. In accordance with the AsA and phenolic content, HAA revealed a general decrease in processed samples. In particular, by ABTS test, decreases of 19.4%, and 17.8% were recorded for E40 and E92, respectively, whereas in E87 an increase of 13.4% was observed. A reduction of 27.8%, 12.7% and 45.4%, was observed by FRAP test for E40, E87 and E92 respectively. The antioxidant potential of tomatoes varied with the assay method used, confirming that the two methods measure different antioxidative effects [38,39]. The discrepancy of the results here obtained by the two different methods was found also in other studies [5,38,39]. In accordance with our results, Graziani et al. [22] found a loss of HAA of the samples after heating treatment. Vallverdú-Queralt et al. [19] reported an increase in HAA when fresh tomatoes were processed into puree and juices as a consequence of tomato cream addition, whereas the antioxidant level did not increase when processing was performed in the absence of cream. To determine if the measured contents of the analysed samples can be related to antioxidative effects, we calculated the Pearson product-moment correlation coefficients (r). The analyses show that antioxidative data determined by FRAP well correlate with ascorbic acid levels (r = 0.852) and total phenolics (r = 0.885). Effects of Tomato Extracts on Cell Viability To test the anticarcinogenic potential of E40, E87 and E92, before and after processing, we used three human cancer cells derived from: liver (HepG2), kidney (Hek293) and cervix (HeLa). Cells were treated for 48h with 20% of tomato extracts, corresponding to a concentration of phenolics ranging between 156 and 212 µg/mL, and cell viability was tested by the MTT reduction assay, as an indicator of metabolically active cells ( Figure 2) and by trypan blue dye exclusion assay ( Figure A1). By using the MTT reduction assay we observed after processing (grey bars, Figure 2) a cytotoxic effect of E87 and E92 extracts on the analyzed human cancer cells, whereas E40 extract was highly toxic only on renal cancer cells. No significant effect on cell viability was observed when cells were exposed to the same extracts before processing (black bars, Figure 2). Noteworthy, we observed a general proliferative effect of non-processed tomato extracts on the cell lines analyzed. Since it is known that cancer cells are actively proliferating, the presence of nutrients in combination with low levels of flavonoids may have a positive effect on cell growth [5]. Previous studies demonstrated that processed tomato products inhibited the growth on cancer cells more than the fresh tomatoes [38,39]. The cytotoxic effect of tomato extracts after processing could be due to the increased levels of compounds with reported antiproliferative activity, such as certain flavonoids, which are known to induce apoptosis on different cell lines [40]. In particular, quercetin3-β-D-glucoside has been reported to possess antiproliferative activity on different cell lines [41,42]. The sugar moieties confer to quercetin glycosides a higher stability in water with respect to quercetin. In addition, it has been suggested that the conjugation with glucose enhances quercetin absorption mainly in the small intestine, as this compound can be better absorbed than quercetin and rutin [41]. In addition, it has been demonstrated that quercetin3-β-D-glucoside exerts a more potent antiproliferative effect than quercetin and rutin on various cancer cell lines [41]. You et al. [42] demonstrated that the growth-inhibitory effect of the quercetin 3-β-D-glucoside on colon (HT-29 and HCT 116), breast (MCF-7), hepatocellular (HepG2) and lung cancer (A549) cells was higher than that exerted by quercetin and rutin, with rutin being the least cytotoxic. Therefore, the observed E92 cytotoxic effect could be due to the increased quercetin 3-β-D-glucoside content detected after processing only in this genotype (Table 4). It has been reported that quercetin3-β-D-glucoside exhibits a significant antiproliferative activity at doses ranging between 20 and 60 µM, in a dose dependent manner [42,43]. In our study the amount of quercetin 3-β-D-glucoside in the tested tomato extracts was between 8.3 µM and 65 µM. However, we hypothesize that differences in the cytotoxicity of the extracts is due to a "balance" between quercetin 3-β-D-glucoside content and the total phenolic acids levels. Indeed, after thermal treatment, E92 showed the highest flavonoids/phenolics ratio ( Table 2). The different antiproliferative activity of E40 extracts compared to the other ones could be also due to the higher level of phenolic acids, in particular chlorogenic acid, recorded in this genotype. According to this hypothesis, it has been reported that in HepG2 cells chlorogenic acid has no cytotoxic effect and improves cellular tolerance against oxidative factors, by activating survival/proliferation pathways [44]. Finally, the higher cell growth inhibition observed with E87 and E92 tomato extracts after processing, in particular on Hek293 cells, could be due to the presence of higher naringenin levels in these samples. This flavanone is known to induce cytotoxicity and apoptosis in various human cancer cells [45][46][47]. These results have been confirmed by trypan blue dye exclusion assay ( Figure A1). apoptosis in various human cancer cells [45][46][47]. These results have been confirmed by trypan blue dye exclusion assay ( Figure A1). Chemicals and Reagents Standards and reagents were purchased from Sigma (St. Louis, MO, USA), whereas solvents were from Fluka (Buchs, Switzerland). Chromatographic solutions were degassed for 20 min by a Branson 5200 ultrasonic bath (Branson Ultrasonic Corp., Phoenix, AZ, USA). Plant Material and Processing Material Plant material consisted of three tomato genotypes (GiàGiù, 284, M-4) used for fresh consumption. They belong to a wide tomato germplasm collection available at the Department of Agricultural Sciences, University of Naples Federico II, and are referred as E40, E87, E92, respectively. Additional details on the genotypes, including those on their source and distribution, are deposited on the Lab Archives repository hosted http://dx.doi.org/10.6070/H4TT4NXN. Plants were cultivated according to a randomized design with three replicates (10 plants/replicate), in an experimental field located in Acerra (Naples, Italy) in the year 2014. Each sample consisted of 20-pooled fruits per plot. The samples were harvested at full yellow ripe stage, as used in the industry. The fruits were chopped, ground in liquid nitrogen a FRI150 blender (Fimar, Rimini, Italy) to a fine powder, and kept at -80˝C until analyses. Tomatoes were also processed according to a classical thermal treatment. Briefly, after washing for 5 min with water, tomatoes were treated for 10 min at 92˝C. A part of treated tomatoes was passed through a pulper in order to obtain a puree. Glass cans were filled with 60% of treated whole tomatoes and 40% of puree and were successively vacuum sealed. The filled jars were pasteurized at 100˝C for 60 min and cooled by water. The processed samples were homogenized by using the Fimar FRI150 blender and kept at -80˝C until analyses. Three cans for each genotype were collected and analyzed. The dry matter contents of all the samples was determined by vacuum-drying the samples for at least 12 h at 60˝C to constant weight. The moisture content, both in the processed and un-processed samples, was around 93% for all the genotypes under study. Solid content for each sample is reported in Table 1. Chemical Extractions Hydrophilic and lipophilic fractions were obtained according to Rigano et al. [5]. Relative to hydrophilic extract, 70% methanol (30 mL) was added to samples of unprocessed or processed tomatoes (3 g) and the mixture was put in an ultrasonic bath for 60 min at 30˝C. Then the mixture was centrifuged at 3500ˆg using a Rotina 420R Hettich 84 Zentrifugen centrifuge (Tuttlingen, Germany) for 10 min at 4˝C, and the supernatant was kept at´20˝C until evaluation of total phenolic compounds, HPLC analysis and hydrophilic antioxidant activity (HAA). The pellet was extracted three successive times with 16 ml of solution acetone/hexane (40/60, v/v) using an Ultraturrax 115VAC IKA T 25 High Speed Homogenizer (Cole-Parmer, Vernon Hills, IL, USA) in order to obtain the lipophilic extract. The mixture was centrifuged at 3500ˆg for 5 min at 4˝C according to a modified procedure reported by Zouari et al. [48]. The supernatants were collected and stored at -20˝C until the determination of lycopene, β-carotene, total carotenoids and lipophilic antioxidant activity (LAA). Carotenoid Determination For carotenoids determination, absorbance of lipophilic extracts was read at 663, 645, 505 and 453 nm. Amounts of β-carotene and lycopene in extracts were calculated according to equations reported by Zouari et al. [48]. Total carotenoids were calculated by reading the absorbance at 480, 648, 666 nm according to the formula reported by Wellburn [49]. Results were then converted into mg/100 g FW. Ascorbic Acid Determination Ascorbic acid (AsA) determination was performed by a colorimetric method [50] with modifications as reported by Rigano et al. [5]. Briefly, TCA 6% (300 µL) was added to sample (500 mg). The mixture was vortexed, incubated for 15 min on ice, and centrifuged at 16,000ˆg for 20 min at 4˝C (Eppendorf Centrifuge 5415R, Hamburg, Germany). Successively, 0.4 M phosphate buffer (20 µL, pH 7.4) and double distilled (dd) H 2 O (10 µL) were added. Eighty milliliters of color reagent prepared as reported by Stevens et al. [50] were added. The mixture was incubated at 37˝C for 40 min and the absorbance was read at 525 nm. Three biological replicates were analyzed for each sample. The standard curve was obtained in the range of 0-70 nmol and the values were transformed into mg/100 g FW. Total Phenolics and Total Flavonoids Determination Total phenolics were determined by the Folin-Ciocalteu assay [51] with modifications reported by Rigano et al. [5]. Briefly, Folin-Ciocalteu's phenol reagent (62.5 µL) and dd H 2 O (250 µL) were added to supernatant (62.5 µL) obtained from the hydrophilic extract obtained as described in Section 3.3. After 6 min, 7% Na 2 CO 3 solution (625 µL), and dd H 2 O (500 µL) were added to the mixture, which was incubated for 90 min and the absorbance was read at 760 nm. The standard curve was obtained in the range of 0-70 µg/mL gallic acid. Total phenolic content of tomato fruits was expressed as mg gallic acid equivalents (GAE)/100 g FW. Three biological replicates and three technical assays for each biological repetition were analyzed. Total flavonoids were estimated by the aluminum chloride colorimetric assay reported by Marinova et al. [29] with slight modifications. An aliquot (500 µL) of methanolic extract (se Section 3.3) was added to 5% NaNO 2 (30 µL) and, after an incubation of 5 min, 10% AlCl 3 (30 µL) was added. After 6 min 1 M NaOH (200 µL) and H 2 O (240 µL) were added and the absorbance of the resulting solution was measured at 510 nm. The standard curve was obtained in the range of 0-100 µg/mL of quercetin. Total flavonoids content was expressed as mg quercetin equivalents (QE)/100 g FW. Three biological replicates and three technical assays for each biological repetition were analyzed. Individual Phenolics Compounds Determination Twenty-five millilitres of methanolic extract (Section 3.3) were dried by a rotary evaporator (Buchi R-210, Milan, Italy) and resuspended in 70% methanol (500 µL) containing around 0.175 g of solid weight. The extract was passed through a 0.45 µm Millipore nylon filter (Merck Millipore, Bedford, MA, USA). Flavonoids and phenolic acids were identified and quantified by using a HPLC Spectra System SCM 1000 (Thermo Electron Corporation, San Jose, CA, USA) equipped with a Gemini column (3 µm C18, 110 A, 250ˆ4.6 mm; Phenomenex, Torrance, CA, USA) and UV-visible detector (Shimadzu, Riverwood Drive, Columbia, MD, USA) according to the procedure reported by Rigano et al. [5]. Chromatograms were recorded at 256 nm for rutin, quercetin and derived, 280 nm for naringenin, 330 nm for chlorogenic acid and derivate, caffeic acid, kampferol-rutinoside, naringenin chalcone and derived. For quantification, integrated peak areas from the tested extracts were compared to the peak areas of known amounts of standard phenolic compounds. The results were expressed as mg/100 g FW. The ABTS assay is based on the ability of the antioxidants present in the samples to reduce the ABTS + radical action capacity. The FRAP assay displays the reduction of a ferric ion complex to the ferrous form. After the addition of antioxidants, the production of the oxidation products is reduced and the solution changes its color [6,38]. An ABTS ‚`s olution was prepared and diluted as described by Miller and Rice-Evans [53]. One hundred microliters of supernatant obtained from the methanolic extraction (Section 3.3) were added to diluted ABTS ‚`( 1 mL) and then the mixture was incubated for 2.5 min. The absorbance was read at 734 nm. The FRAP test was performed by adding acetate buffer (2.5 mL, pH 3.6), TPTZ solution (0.25 mL, 10 mM in 40 mM HCl), FeCl 3¨6 H 2 O solution (0.25 mL, 12 mM), and supernatant (150 µL) obtained from the above extraction. After an incubation of 30 min at room temperature, the absorbance of the complex was read at 593 nm. The standard curve resulted linear between 20 and 800 µM Trolox. Results were expressed as micromoles of Trolox equivalents (TE) per 100 g FW. Cell viability assays Human adenocarcinoma cells (HeLa), human renal epithelial cells (Hek 293), and human liver hepatocellular cells (HepG2) were from ATCC (American Type Culture Collection, Manassas, VA, USA) and were cultured in Dulbecco's modified Eagle's medium (Sigma-Aldrich), supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA), 2 mM L-glutamine, and antibiotics. Cells were grown in a 5% CO 2 humidified atmosphere at 37˝C [54]. Cells were seeded in 96-well plates (100 µL/well) at a density of 5ˆ10 3 /well. Methanolic tomato extracts, obtained as reported above, were dried by rotovapor (R-210, Buchi, New Castle, DE, USA) at 30˝C, redissolved in dimethyl sulfoxide (DMSO) 5% in PBS, and then added to the cells 24 h after seeding for cytotoxicity assays (20% v/v). Cell viability was assessed by the MTT assay after 48 h incubation. The MTT reagent, dissolved in DMEM in the absence of phenol red (Sigma-Aldrich), was added to the cells (100 µL/well) to a final concentration of 0.5 mg/mL. Following an incubation of 4 h at 37˝C, the culture medium was removed, and the resulting formazan salts were dissolved by adding isopropanol containing 0.1 N HCl (100 µL/well). Absorbance values of blue formazan were determined at 570 nm using an automatic plate reader (Microbeta Wallac 1420, PerkinElmer, Shelton, CT, USA). The decrease in absorbance in the assay measures the extent of decrease in the number of viable cells following exposure to the test substances calculated by using the following formula: % inhibition of cells = (A control -A test substance)/A controlˆ100. Three separate analyses were carried out with each extract. Control experiments were performed either by growing cells in the absence of the extract and by supplementing the cell cultures with identical volumes of extract buffer (5% DMSO in PBS). The method used avoids any possibility of a DMSO effect on the results. In parallel experiments, cell viability was evaluated by trypan blue dye exclusion assay. After incubation for 48 h at 37˝C with tomato extracts, cells were detached by trypsin, centrifuged at 1000ˆg for 5 min at r.t. and the cell pellet was resuspended in 0.4% trypan blue buffer (Sigma-Aldrich) and counted in the hemocytometric chamber (Burker chamber, Sigma-Aldrich). Statistical Analyses The biological replicates of samples were analysed in triplicate. Quantitative parameters were expressed as the mean value˘SD. Differences among not processed and processed samples were determined by using SPSS (Statistical Package for Social Sciences) Package 6, version 15.0 (SSPS Inc., Chicago, IL, USA). Significance was determined by Student's t-test at a significance level of 0.05. SPSS Package 6, version 15.0 was also used to calculate Pearson's correlation between measured parameters. The percentage of the variations of quantitative parameters before and after the processing was calculated by using the following formula: Increase or Decrease p%q " value after processing-value before processing value before processingˆ1 00 (1) Conclusions In all the samples analyzed, a decrease in the amount of ascorbic acid, total phenolics, phenolic acids and hydrophilic antioxidant activity was observed after heat treatment. Interestingly, processing was able to enrich tomato fruits in bioactive compounds, such as certain flavonoids. In particular, in the two Bolivian genotypes (E87 and E92), heat treatment led to a significant increase in the level of naringenin and quercetin glycoside, which correlates well with the observed antiproliferative activity of these tomato extracts against some human cancer cells. It is worth saying that the anticarcinogenic potential of foods containing phenolics cannot be based only on the effects of individual compounds but may involve a synergistic effect among phytochemicals [43]. In the Italian E40 genotype, high levels of phenolic acids and flavonoids were detected both in the fresh and processed fruits, and the content of these bioactive compounds indicates that it has a potential use for the production of tomato-based functional food. Nowadays, there is an increasing demand of natural products, which are expected to be safe and health-promoting. In this context, our data could have a significant impact on consumer's food selection, increasing their awareness of the health benefits of fresh and processed tomato fruits. Figure A1. Cell viability assay of E40, E87 and E92 on human cancer cells. HepG2 (A); HeLa (B) and Hek293 (C) were treated for 48 h at 37˝C with 20% (corresponding to a concentration of phenolics ranging between 156 and 212 µg/mL) extracts from E40, E87 and E92 before (black bars) and after (grey bars) heat treatment. Cell number was evaluated by trypan blue dye exclusion assay. All values are given as means˘SD.

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2017-05-29T15:22:17.078Z

2015-09-24T00:00:00.000Z

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Screening Genetic Resources of Capsicum Peppers in Their Primary Center of Diversity in Bolivia and Peru For most crops, like Capsicum, their diversity remains under-researched for traits of interest for food, nutrition and other purposes. A small investment in screening this diversity for a wide range of traits is likely to reveal many traditional varieties with distinguished values. One objective of this study was to demonstrate, with Capsicum as model crop, the application of indicators of phenotypic and geographic diversity as effective criteria for selecting promising genebank accessions for multiple uses from crop centers of diversity. A second objective was to evaluate the expression of biochemical and agromorphological properties of the selected Capsicum accessions in different conditions. Four steps were involved: 1) Develop the necessary diversity by expanding genebank collections in Bolivia and Peru; 2) Establish representative subsets of ~100 accessions for biochemical screening of Capsicum fruits; 3) Select promising accessions for different uses after screening; and 4) Examine how these promising accessions express biochemical and agromorphological properties when grown in different environmental conditions. The Peruvian Capsicum collection now contains 712 accessions encompassing all five domesticated species (C. annuum, C. chinense, C. frutescens, C. baccatum, and C. pubescens). The collection in Bolivia now contains 487 accessions, representing all five domesticates plus four wild taxa (C. baccatum var. baccatum, C. caballeroi, C. cardenasii, and C. eximium). Following the biochemical screening, 44 Bolivian and 39 Peruvian accessions were selected as promising, representing wide variation in levels of antioxidant capacity, capsaicinoids, fat, flavonoids, polyphenols, quercetins, tocopherols, and color. In Peru, 23 promising accessions performed well in different environments, while each of the promising Bolivian accessions only performed well in a certain environment. Differences in Capsicum diversity and local contexts led to distinct outcomes in each country. In Peru, mild landraces with high values in health-related attributes were of interest to entrepreneurs. In Bolivia, wild Capsicum have high commercial demand. (http://link.springer.com/article/10.1007/s00217-014-2325-6). The biochemical characterization data of the CIFP core collection in Bolivia are within the paper and its supporting information files, as well as the Peruvian agromorphological evaluation data in three locations of the 23 promissory accessions that were also biochemically analysed. Finally, the paper encloses a third supporting file with the agromorphological data of all Peruvian promising accessions in a fourth evaluation site. most important vegetables and spices in international trade [15]. In several countries, native Capsicum diversity is gaining importance to develop processed food products for niche markets [16], [17]. Capsicum is also of interest for a wide range of other applications such as a natural colorant for food, an ingredient in pharmaceuticals, and a highly pungent extract used for self-defense sprays, animal repellents and insecticides [18]. Domesticated Capsicum species show wide morphological variability, including traits which are useful for discriminating among the species such as plant pubescence, number of flowers per node, calyx constriction, corolla color and spots, and seed color [19] (Fig 1), as well as variability in other, commercially valuable traits such as fruit color, shape, flavor, and biochemical components, including health and taste-related attributes like antioxidant capacity and capsaicinoids, the latter being responsible for Capsicum's pungency [9], [20][21][22]. The high degree of genetic diversity found within the genus provides both opportunities and challenges for plant breeders as the full extent and structure of this diversity remains underutilized and imperfectly understood. Crossing experiments and molecular marker studies have shown that the five domesticated species of Capsicum pertain to three distinct primary gene pools (sensu [23]), [24], [25], which are centered around C. annuum, C. baccatum, and C. pubescens, respectively, and are based on the degree of genetic proximity and reproductive compatibility with the target cultigen [26]. The three gene pools have little reproductive compatibility between them, although successful crosses have been reported between plants from the C. annuum and C. baccatum complexes [24], [27], [28]. Together, they include all of the cultivated landraces and local varieties pertaining to the five domesticates, as well as at least eight wild Capsicum taxa and conspecific wild populations (Fig 3). Given the rich variety of Capsicum in its primary center of diversity, we expected that a wealth of commercially valuable properties for product development could be found in the cultivated and wild pepper species present in Peru and Bolivia, The specific objectives of this study were to: 1) demonstrate, with Capsicum as a model crop, the application of phenotypic and geographic indicators of diversity as effective criteria to select a set of promising accessions for multiple uses from crop centers of diversity; and 2) evaluate the expression of biochemical and agromorphological properties of the selected Capsicum accessions in different environmental conditions. Methods We followed four steps in the selection of promising accessions (Fig 4): 1) Strengthening and expansion of existing genebank collections; 2) biochemical characterization of Capsicum fruits from representative genebank subsets; 3) selection of promising accessions for multiple uses from the subsets; and 4) evaluation of promising accessions in different environments. All activities were conducted between 2010 and 2013 in Peru and Bolivia. 1) Strengthening and Expansion of Existing Genebank Collections At the outset of this study, the Instituto Nacional de Innovación Agraria (INIA) from Peru maintained a collection of 106 accessions from only three regions of this country. During the Cultivated Capsicum gene pools. Capsicum annuum L., C. chinense L. and C. frutescens L., while morphologically distinguishable, are largely interfertile with one another and are commonly regarded forming a species complex [24]. These three sister cultigens, together with their conspecific wild populations, including C. annuum var. glabriusculum (Dunal) Heiser & Pickersgill, constitute the Capsicum annuum primary gene pool. The Capsicum baccatum primary gene pool is formed by the cultivated C. baccatum L. or more specifically C. baccatum var. pendulum (Willd.) Eshbaugh; its conspecific wild relative C. baccatum var. baccatum; and the more distantly related species, C. baccatum var. praetermissum (Heiser & P. G. Sm.) Hunz. Capsicum baccatum's secondary gene pool consists of C. chacoense Hunz. The Capsicum pubescens primary gene pool is made up of the cultivated species (C. pubescens Ruiz & Pav.), together with the closely related purple-flowered wild species C. cardenasii Heiser & P. G. Sm. and C. eximium Hunz. [24], [25]. Several other wild edible species, C. eshbaughii Barboza and C. caballeroi Nee are recently discovered in Bolivia and resemble C. eximium and C. cardenasii in certain fruit and flower aspects, but their relativeness to C. pubescens still needs to be clarified [42], [54]. And so there are other wild species that require further analysis. The little-studied wild species Capsicum galapagoense Hunz. and C. tovarii Eshbaugh et al. have been suggested by some authors to form part of the C. annuum complex and C. baccatum gene pools, respectively, but are intentionally left out of this diagram until more conclusive evidence supporting their inclusion becomes available. study, INIA carried out eight collecting trips which combined the following criteria for determining the collection routes in Peru: 1) available knowledge on Capsicum diversity in Peru [17]; 2) geographic origin of existing genebank and herbarium accessions (see www.gbif.org); and 3) knowledge of INIA experts on Capsicum production areas. The pre-existing Bolivian germplasm collection of 396 accessions held at the Centro de Investigaciones Fitoecogenéticas de Pairumani (CIFP) in Bolivia already included a wide range of native Capsicum diversity. Complementary collecting activities in search of landraces and wild Capsicum species in cloud forests and seasonally dry woodlands in the departments of La Paz, Cochabamba, Sucre and Chuquisaca were carried out. Taxonomic identification of the Peruvian accessions was carried out by David E. Williams, while accessions of the Bolivian collection were botanically identified by Gloria Barbosa, Margoth Atahuachi and Ximena Reyes. Both collections were regenerated as part of this study following exclusion protocols to avoid cross pollination and to assure genetic integrity of the accessions. INIA and CIFP did not require specific permission for the collection locations as they were national authorities in conservation of plant genetic resources at the time of field collection. 2) Biochemical Characterization of Capsicum Fruits from Representative Genebank Subsets Subsets of about 100 accessions per country, representing the overall diversity of each collection, were selected for biochemical characterization of Capsicum fruits (see Fig 4). Diversity was maximized by using available data on botanical classification and passport data of geographic origins as a proxy for genetic variation [29]. Fruit sampling was done during the seed regeneration of the accessions. Early-fruiting accessions had greater representation in the subsets compared to late-fruiting accessions because we only selected from accessions with mature fruits at the time of fruit collection. Twelve seedlings were planted per accession, and of these up to six healthy plants were selected randomly for collection of bulk samples of fruits to be used in a first biochemical screening. In Peru, the accessions were all grown in a common plot in Huaral, Lima (Table 1). In Bolivia, the accessions were grown in five separate environments recognizing that some materials were highly adapted to specific climate zones, particularly the wild and semi-domesticated peppers like 'arivivi' (C. baccatum var. baccatum) and 'ulupica' (C. eximium) ( Table 1). Fruit samples were shipped to Germany, as dried and crushed material after complying with Peruvian, Bolivian and German legal and phytosanitary regulations, for biochemical analysis. 3) Selecting Sets of Promising Accessions for Multiple Uses About 40 promising accessions were selected in each country from the subsets based on the biochemical characterization of Capsicum fruits designed to capture a wide range of options for product development. Variation in pungency, differences in color, and accessions with high values for the other biochemical attributes were important selection criteria. We also included as many Capsicum species as possible since phenotypic differences may occur both within and between species. 4) Evaluation of Promising Accessions in Different Environments Once promising accessions had been identified, they were grown in four evaluation sites each in Bolivia and Peru in different climate zones (Table 1). At each site, local management practices were followed (For Peru see S1 Table; for Bolivia see S2 Table). Three repetitions were established within each site. For each accession, 12 seedlings from its regenerated seed material were planted in a row with a distance of 70 cm between individuals. The distance between rows of accessions was 80 cm. The two outer seedlings of each accession were not considered in further studies to avoid border effects. A row of seedlings was planted as a buffer alongside the outer accessions to avoid border effects for these materials. Seedlings that died within two weeks were replaced. In the evaluation sites, the promising accessions were sampled for a second round of biochemical analyses in Germany following the same methodology as explained above (i.e. fruit bulk samples from up to six healthy plants selected at random). These accessions were also morphologically characterized for a number of highly discriminating traits of agronomic and commercial interest according to the Capsicum descriptors developed by IPGRI et al. (1995) [19]. These traits were mean plant height (cm) per accession, flowering time (number of days to 50% plants flowering), mean fruit length (cm), weight (g) and width (cm). We applied a two-way ANOVA to examine differences in biochemical and morphological attributes between species and locations. We identified these two factors as fixed following Annicchiarico (2002) [32], considering species as a group factor of promising accessions and locations as specific combinations of management and environment representing certain production areas. In case of differences, post-hoc Tukey`s Honest Significant Difference (HSD) analyses allowed significantly different groups to be identified. Since data for several traits did not follow a normal distribution and in order to process data of all traits similarly, values of each trait were transformed into ranked data. These transformed data were then used in F analyses, t tests, HSD and multivariate analyses (see [33]). To reduce the false positive rate in the multiple trait tests, we adjusted the p values obtained with a False Discovery Rate (FDR) correction. To facilitate further use of germplasm for different purposes, we classified promising accessions with similar trait-value combinations in separate groups. Trait values of each accession across locations were averaged and then ranked as explained above. We applied hierarchical clustering, calculating Euclidian distances between accessions with the rank scores and applying Ward´s method to group them. The final number of clusters was defined with the Kelley-Gardner-Sutcliffe penalty function [34]. A Principal Component Analysis (PCA) was done with these rank scores to visualize how traits related to each other, between species and among cluster groups. We examined the phenotypic stability of the promising accessions for each trait by calculating environmental variance following Annicchiarico (2002) [32]: In which S 2 is the environmental variance for a specific accession; R ij is the average value for a particular trait in a certain location; m i is the average value of an accession for a particular trait across locations; and e is the number of locations. We compared differences in phenotypic stability between the accessions with an ANOVA, in which the environmental variances measured per trait were considered as a repeated measure. To compare the environmental variances obtained for the different traits, we standardized the S 2 values for each trait between 0 and 1. The environmental variance for an accession is usually large when its m i value is high [35]. To identify traits for which accessions with high values are stable across environments, we correlated for each trait, the m i values with the corresponding S 2 values. Finally, we examined how consistent the plants of promising materials were in the expression of biochemical properties compared to the mother plants that were used for seed regeneration of each accession. Therefore, we compared for each trait, the m i values from the genotype by environment (G x E) analysis with the results from the first biochemical screening. Pairwise t tests were applied to compare the rank scores. Most analyses were carried out with basic statistical functions in the R statistical environment [36]; HSD analyses were done with the Agricolae package [37]; the PCA with help of the Vegan package [38]; the Kelley-Gardner-Sutcliffe penalty function was applied with the Maptree package [39]. As this work progressed and results of the germplasm screening became available, we shared the results with several actors in the Capsicum value chain: entrepreneurs, farmers and others convened at three meetings in Peru and two meetings in Bolivia in 2011 and 2012 [40], [41]. 1) Strengthening and Expansion of Existing Genebank Collections In Peru, we established one of the most diverse national collections of native Capsicum varieties in the world consisting of 712 accessions encompassing the five domesticated species and dozens of locally recognized landraces [31] (Fig 5; Table 3), and a representative subset of 90 accessions from which 39 promising accessions were selected for evaluation in different environments. The CIFP collection in Bolivia now contains 487 accessions encompassing the five domesticated species and at least four wild taxa consumed by humans, including the recently discovered wild species C. caballeroi [42] (Fig 5; Table 3), which is unique to this collection. This collection conserves an important amount of C. baccatum variability compared to international Capsicum collections because Bolivia is a primary center of diversity of cultivated C. baccatum var. pendulum [6], [43] (S3 Table). Over 80% of the accessions are duplicated in the national Bolivian collection held by Instituto Nacional de Innovación Agropecuaria y Forestal (INIAF). A representative subset of 96 accessions was established for Bolivia from which 44 promising materials were selected. 2) Biochemical Characterization of Capsicum Fruits from Representative Genebank Subsets Table 4 presents the mean values and ranges of biochemical attributes of the representative subsets in each country. For individual passport and biochemical characterization data please refer to S1 Dataset. The 90 accessions of the Peruvian subset included four of the five domesticated species (C. annuum, C. baccatum, C. chinense and C. frutescens) originating from ten regions. The biochemical results from Peruvian C. pubescens accessions are reported separately because they have a longer growing season compared to the other domesticates and were characterized separately [44]. In Bolivia, the subset of 96 accessions included the five domesticated species and two wild taxa, C. eximium and C. baccatum var. baccatum, from seven departments. The two wild taxa were of special interest to Bolivian entrepreneurs because of their potential for high-value product differentiation [41]. Accessions from other Bolivian wild pepper species like C. caballeroi, did not produce fruits in time for the biochemical screening and further selection. 3) Selecting sets of Promising Accessions for Multiple Uses The 44 promising accessions from Bolivia included all species from the subset and covered a range from low to high pungency. Sixteen had exceptionally high values in one or more of the attributes of ASTA extractable color, antioxidant capacity, polyphenols, and fat content compared to the other accessions of the Bolivian subset. We did not find any differences in biochemical properties between the representative subset and the promising accessions according to posterior t tests (S4 Table). In Peru, at the time of selecting promising materials, results of the biochemical analyses were only available for 24 of the subset accessions. From these, we selected eight promising materials which had high or low values in capsaicinoids and ASTA extractable color and/or high levels in antioxidant capacity, polyphenols or fat compared to the other Peruvian accessions that had been analyzed at that moment. Other promising accessions were selected later during a field visit to the regeneration site when most accessions were fruiting, with a special emphasis on variation in fruit form and color, to complete a total of 39 promising accessions representing the four species from the subset (see S1 Dataset). The variation in biochemical traits in the set of promising accessions did not differ compared to the representative subset (S4 Table). 4) Evaluation of Promising Accessions in Different Environments In Bolivia, no promising accession produced fruits in time in two or more sites for the second round of biochemical analyses. Therefore, here we report only on the results of the Peruvian G x E evaluation experiment. In Peru, fruits from 23 of the 39 accessions, in three of the four evaluation sites, were mature at the time planned for the second biochemical screening. In the fourth Peruvian site, Huaral, the accessions did not produce fruits in time for this round of biochemical analyses. Of the two promising C. frutescens accessions, only one produced fruits in the three sites. On its own it would not constitute a representative group, so we analyzed it together with the accessions of the genetically close C. chinense species (see [12], [25]). For most accessions, sufficient fruits were only available for one single bulked sample per site for biochemical screening, while agromorphological evaluation was completed in all three Table 4. Biochemical fruit screening of the Capsicum accessions from the representative subsets. Mean, minimum and maximum values are presented per species. In Peru, all accessions were grown in a common plot. In Bolivia, we anticipated that accessions were highly adapted to specific environments and were therefore grown in different locations. Screening Capsicum Pepper Diversity repetitions in each site (S2 Dataset). Differences between repetitions were found for plant height between and within sites and for flowering within but not between sites (S5 Table). For one high-yielding accession, results of three repetitions in all sites were compared to confirm that biochemical stability within sites was higher than between sites [30]. To be able to compare the agromorphological performance of the 23 accessions with the biochemical expressions in their fruits, we averaged within each site agromorphological trait values from the three repetitions (S3 Dataset). Eventually the species and location factors of our two-way ANOVA consisted of three germplasm groups and three evaluation sites respectively. The ANOVA revealed highly significant differences in performance between species for several traits and to a lower degree between different locations (Table 5). We observed most of these differences for the biochemical traits rather than the agromorphological features ( Table 5). The four C. annuum accessions flowered earlier than the materials of the other species. Their fruits also contained significantly higher fat content and concentrations of tocopherols compared to the other species and expressed an intense red color as indicated by the high values for ASTA extractable color (Fig 6). Capsicum baccatum accessions weighed more and contained on average higher flavonoid and quercetin concentrations compared to most C. chinense-C. frutescens accessions, but not compared to the C. annuum accessions. A few C. chinense-C. frutescens accessions had exceptionally high flavonoid and quercetin content compared to the other promising accessions (Fig 6). In the Chiclayo location, the accessions contained higher concentrations of flavonoids and quercetin, and higher values for ASTA extractable color compared to the other sites (Table 5; Fig 6). In Pucallpa, plants flowered earlier in contrast to the other locations. In Chiclayo, higher polyphenol levels were observed compared to Tambo Grande. Between individual accessions, no differences in environmental variance were detected (F (Table 6). Accession scores for ASTA extractable color, fat, flavonoids, polyphenols and quercetin were consistent with the first biochemical characterization of these accessions (S6 Table). Antioxidant capacity and capsaicinoid scores were not consistent between the two screenings. We don´t report here on tocopherols because these phytonutrients were only measured in the second screening. Most variation between promising accessions was seen in capsaicinoid levels and fruit shape and weight as well as the antioxidant capacity and the concentration of polyphenols (Fig 7). The cluster analysis classified the 23 accessions into six groups (Fig 7; Table 7). These are: Discussion In this study we have shown how using kinds of phenotypic and geographic diversity as selection criteria is an effective approach to find accessions with a diverse set of features of interest, in a relatively short time span of three years including field collecting and seed regeneration to obtain the necessary diversity for selection. This is particularly useful when screening germplasm from centers of crop diversity because of the high variation found here. In our case, it has allowed in both Peru and Bolivia for the identification of a set of promising Capsicum genebank materials for multiple uses including the development of a wide range of fresh and processed food products [31], [45]. Our approach is similar to the development of mini-core collections [46], and differs from more traditional germplasm selection for breeding, which usually focuses on one or two specific traits of interest such as fruit size or pungency in the case of Capsicum. The extensive biochemical analyses allowed us to detect accessions with interesting properties for novel products. A similar approach of using diversity in biochemical properties to select promising accessions has been applied successfully in other Capsicum studies [21], [47]. In our study, we were able to take this a step further and evaluate biochemical and agromorphological properties of promising materials in different environments after these accessions were selected from a first biochemical characterization. Most advances were made with this approach in Peru where we were able to evaluate promising accessions from the C. annuum and C. baccatum gene pools in different environments to select elite material. The differences in agromorphological and biochemical performance observed between these accessions are largely the result of the divergent processes of human selection as different species and landraces were domesticated and used in different areas. It is less likely that these differences are a result of natural evolutionary processes. The genetically Although most trait combinations that we observed and the differences between species and accessions can be explained as a result of domestication, some biochemical traits correlate because they functionally relate to each other. Quercetin is a specific flavonoid [30]. The levels of polyphenols and capsaicinoids determine partly the antioxidant capacity of a fruit [48]. The biochemical traits were more influenced by environmental factors than agromorphological attributes, even if we just consider uncorrelated phytochemical traits such as ASTA extractable color, polyphenols, and flavonoids. These results are in line with other studies [49], [50]. Yet, our G x E experiment also revealed that for accessions with high antioxidant capacity and high concentrations of fat and tocopherols, and different levels of capsaicinoids, these traits remained stable across environments. Fat content in fruits of the promising accessions appeared to be consistent over the two biochemical screenings carried out during the selection and evaluation process. Accessions with high fat content are therefore interesting for further evaluation and genetic improvement. On the other hand, although, antioxidant capacity and capsaicinoid levels were stable traits across environments in the G x E evaluation, the values of these traits expressed by the promising accessions were not consistent over the two biochemical analyses carried out. The reasons for these contrasting results are not clear and require further evaluation between generations and across different environments for these traits. Some accessions displayed exceptionally high values of flavonoid and quercetin concentrations and ASTA extractable color in specific locations as demonstrated by their high environmental variance related to high average values across locations (see Table 6). It would be interesting to investigate whether these trait expressions can be linked to the environmental conditions of specific locations. The differences in concentrations of these biochemical compounds and in the level of polyphenols between the peppers in the Chiclayo and Pucallpa locations could possibly be explained by differences in water access. Peppers growing in the desert in Chiclayo were irrigated while in the humid rainforest conditions of Pucallpa they were rain- fed. The content of capsaicinoids is reported to increase when plants suffer from drought, and other phytonutrients may behave similarly [51]. The evaluation sites in both Chiclayo and Tambo Grande were located in the tropical desert but fruits were harvested later in Chiclayo (Table 1; S1 Table). This extra time in Chiclayo may have allowed the plants to develop high concentrations of phytonutrients, as has been reported in other studies [49]. It is difficult to estimate how differences in soil conditions among sites influenced plant performance and biochemical composition of Capsicum fruits. More controlled experiments are necessary to understand the interaction between drought, maturation and agromorphological and biochemical properties. Of all the agronomic traits, flowering time differed mostly between species and among locations. Timing of flowering and fruit set within a season is largely determined by responses to temperature and photoperiod [52]. This explains early flowering in Pucallpa, given the high annual mean temperature compared to the other locations, and the delayed flowering in Huaral, given the low annual mean temperature compared to the other sites, and which led to the retarded fruiting in the latter site (Table 1). Yet, it´s unclear why the Capsicum annuum landraces flowered earlier than accessions from the other species in all three sites where accessions produced fruits on time for biochemical evaluation. Our study focused only on the characterization and evaluation of promising accessions for agronomic and biochemical properties and did not include breeding. It is encouraging, however, that INIA has included all 39 promising Capsicum materials identified in this study in their horticultural improvement program. Several promising Peruvian accessions, such as 'ayuyo' (C. baccatum) and 'ají cerezo' (C. annuum) landraces, are already of interest for direct production because they match the interests of national entrepreneurs who participated in project information-sharing workshops. These materials are non-pungent, mild or semipiquant, have high values for several health-related attributes, and are highly productive compared to other accessions [31]. Other commercially and agronomically important characteristics of the fruits and plants still need to be explored, such as taste profiles and resistance to pests and diseases. Genomic advances in Capsicum hold promise to complement selection, evaluation and breeding activities, for example in selecting promising accessions and elite material, which have genes that are related to certain traits of interest [28], [53]. Some promising Bolivian accessions, including several wild C. eximium and C. baccatum var. baccatum landraces, produced fruits with exceptionally high fat content in specific evaluation sites compared to other Bolivian accessions (Table 4), yet they performed poorly across different environments, which suggests that these landraces only respond well to the local conditions under which they were developed. Further research on landrace-environment interactions and crop improvement is required to develop varieties that perform well in a broader environmental range. The wild Capsicum species of Bolivia deserve special attention. Bolivia is probably the country in the world where humans consume the highest diversity of wild Capsicum. Human consumption of C. cardenasii, the recently identified C. eshbaughii and C. caballeroi is exclusive to Bolivia according to current knowledge [6], [42], [54]. Capsicum eximium is consumed in Bolivia and Northern Argentina [6]. Capsicum baccatum var. baccatum and C. chacoense are commonly harvested and consumed in Bolivia and also in Argentina, Paraguay and Brazil (D. E. Williams, pers. comm.). In many traditional communities throughout Latin America, wild and semi-wild Capsicum species are often found growing in close proximity to cultivated peppers, in home gardens or at the edges of cultivated fields, where introgressive hybridization takes place between the cultigen and closely related wild species or sub-species, spontaneously giving rise to novel or intermediate forms which are then selected and propagated by observant farmers in an ongoing process of on-farm crop evolution [55]. Many wild Capsicum species are already recognized by plant breeders as useful material for the genetic improvement of cultivated peppers [5], [56]. These wild genetic resources also present an opportunity to domesticate, breed, and cultivate novel pepper varieties with high market potential [41]. As a result of the taxonomic identification of the Bolivian C. baccatum collection, several accessions of supposedly wild 'arivivi' have been revealed to be in fact small-fruited landraces of the cultivated C. baccatum var. pendulum. These landraces are promising candidates for commercial cultivation to reduce the uncontrolled extraction pressure on wild populations of C. baccatum and C. chacoense (this latter species is also known as 'arivivi'). Sustaining 'ulupica' (C. caballeroi, C. cardenasii, C. eshbaughii, and C. eximium) production is a greater challenge because these species have not yet been domesticated. Further work is required to develop commercially and ecologically sustainable extraction and cultivation systems for wild Capsicum species. Conclusions With the use of phenotypic and geographic diversity as selection criteria, we were able to identify promising Capsicum accessions in Peru and Bolivia for a potentially wide range of highvalue products within a short time span of three years. The strengthened genebank collections in the center of cultivated Capsicum diversity provided the necessary variation for selection. Biochemical analysis allowed us to identify accessions with promising traits beyond the basic distinction between sweet and hot peppers. These accessions are relevant for novel food, health and other products. Although we used the same approach for germplasm screening in Bolivia and Peru, the differences in Capsicum diversity between the two countries, distinct local environmental conditions, and culture and market opportunities led to different outcomes from the selection process. The promising accessions resulting from this process are the basis for national and local genetic improvement programs, which ideally should be developed in collaboration with entrepreneurs and farmers to ensure that the developed varieties match both market and farmers' needs. In Peru, some accessions are already being used for commercial production because of their high productivity and the interest shown by entrepreneurs. These include several sweet, mild and semi-piquant peppers with high concentrations of flavonoids and quercetin or high levels of tocopherols. These accessions produced their highest concentrations of these compounds in an irrigation-controlled system in the tropical desert in Chiclayo. Rain-fed systems in the Amazon provide a good alternative as a low-input system. In Bolivia, the selected cultivated and wild pepper landraces only performed well in specific environments, suggesting that they are strongly adapted to local conditions. Wild Capsicum harvesting requires further research for sustainable management. Our study highlights the limited knowledge about the breeding potential and potentially useful traits present in crop gene pools, even for globally important crops like Capsicum peppers. Many opportunities remain to further screen and evaluate genetic resources from Capsi-cum´s center of diversity. Similar opportunities are awaiting for the under-researched genetic resources of other crops in their respective centers of diversity. Supporting Information S1 Dataset. Biochemical characterization and passport data of the Bolivian and the Peruvian representative subsets. Table. One-way ANOVAs with nested design to verify differences among the three repetitions between and within the Peruvian evaluation sites for the agromorphological attributes. (DOCX) S6 Table. Probability values (p values) that the order in values for biochemical attributes of the promising materials in the genotype by environment evaluation and the initial biochemical characterization are consistent with each other; t tests were applied separately for each attribute. (DOCX) (CIDRA) for the use of photos in Fig 1, and finally three anonymous reviewers for their constructive comments on an earlier version of this paper.

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Bile acid mediated effects on gut integrity and performance of early-weaned piglets Background Early weaning (EW) results in a transient period of impaired integrity of the intestinal mucosa that may be associated with reduced plasma concentration of glucagon-like peptide-(GLP) 2. We have previously shown that intragastric infusion of chenodeoxycholic acid (CDC) increases circulating GLP-2 in early-weaned piglets. The aim of this study was to expand previous work to establish whether feeding piglets a cereal-based diet supplemented with CDC can improve gut integrity and animal performance immediately after EW. A cohort of 36 piglets weaned at 20 days of age, 6.2 ± 0.34 kg of body weight (BW) were randomly assigned (n = 18) to receive a standard prestarter diet or the same diet supplemented with 60 mg of CDC per kg of initial BW for ad libitum intake until day 14 postweaning. Thereafter, all pigs were fed the same untreated starter diet for 21 days until the end of the study on day 35. On days 1, 7 and 14 blood samples were collected from 6 pigs per treatment to measure plasma GLP-2. On day 15, 6 pigs per treatment were euthanized to obtain intestinal tissue samples for later histological and gene expression analyses. Results Supplementing the diet with CDC tended to increase plasma GLP-2 (P < 0.07; 39 %) and the weight of the large intestine (P < 0.10; 11 %), and increased ileal crypt depth (P < 0.04; 15 %) after 14 days of treatment exposure. Although feed intake and BW gain were not affected by treatments, feeding CDC induced the expression of the cytokines TNF-α (P < 0.02; 1.9 fold), IL-6 (P < 0.01; 2.4 fold), and IL-10 (P < 0.006; 2.2 fold) and the tight junctional protein ZON-1 (P < 0.02; 1.5 fold) in the distal small intestine. Conclusions This study showed that the oral administration of CDC to early-weaned pigs has the potential to improve the protection of the intestinal mucosa independently of relevant changes in gut growth. Background Early weaning (EW) is a widespread practice in modern settings of pig production. At that time, piglets are exposed to a variety of stressors including abrupt separation from sow and changes in diet and environment, which jointly result in a period of transient anorexia, gut mucosal atrophy, and intestinal dysfunction [1][2][3]. The weaning-induced deterioration of gut integrity could be partly related to the marked reduction in circulating glucagon-like peptide (GLP)-2 that typically accompanies EW in pigs [4,5]. GLP-2 is an intestinotrophic peptide released by the enteroendocrine L cells mainly in response to luminal nutrients [6,7]. Of interest, exogenous GLP-2 restores mucosal growth, transcellular transport, and the expression of tight junction (TJ) proteins that control paracellular permeability in a number of animal models of intestinal atrophy or dysfunction [8][9][10]. Recently, it has been found that the chronic administration of GLP-2 at supraphysiological levels to neonatal pigs for 42 days increased villus height and crypt depth in the small intestine and colon 21 days after EW [11]. More important, the administration of a long-acting analog of GLP-2 at pharmacological doses to 25-days-old suckling piglets increased intestinal weight and enzyme activity 5 days after weaning [12]. Although available evidence suggests that GLP-2 treatment can contribute to improve intestinal adaptation to weaning, it is reasonable to expect that strategies capable of enhancing secretion and (or) stability of endogenous GLP-2 might be equally effective but easier to implement under commercial schemes of pig production. In recent years bile acids have emerged as potent hormonal regulators capable of stimulating the secretion of GLP-1 (a co-product of proglucagon, released in parallel with GLP-2) from the intestine. This action is mediated by the G-protein-coupled bile acid receptor 1 (GPBAR1, also known as TGR5), which is a bile acid sensor expressed on the luminal surface of intestinal L cells [13,14]. Interestingly, the continuous enteral administration of chenodeoxycholic acid (CDC), a primary bile acid known to activate TGR5, to newborn piglets fed parenterally increased the plasma concentration of GLP-2 and prevented gut atrophy otherwise resulting from the lack of enteral nutrition [15]. In a later study, we investigated whether CDC could induce a similar response in weanling pigs. In this study, piglets weaned at 21 days of age, fed a cereal-based diet, and infused intragastrically with a single dose of CDC had increased circulating GLP-2 and tended to have a longer and heavier intestine than their control counterparts [16]. As proposed in that report, it is plausible that the dose of CDC and administration procedure used in our study might have limited the impact of increased GLP-2 secretion on intestinal adaptation to EW. It is important to note, however, that bile acids may also control the integrity of the intestinal barrier by regulating the expression or cellular distribution of TJ proteins through mechanisms unrelated to GLP-2 [17,18]. In summary, available evidence indicates that activating intestinal signaling pathways controlled by bile acids allows stimulating the release of endogenous GLP-2 and thereby improving gut integrity in experimental models of intestinal atrophy and dysfunction. Therefore, the aim of this study was to expand previous work to establish whether the inclusion of CDC in the diet of earlyweaned piglets fed according to current standards of pig production can improve gut integrity and animal performance immediately after weaning. Animals and housing All experimental procedures were approved by the Laboratory Animal Care Advisory Committee of the Faculty of Veterinary Sciences of the Universitat Autónoma de Barcelona, Spain. A total of 36 pigs (Large White x Landrace x Pietrain; 18 of each sex) weaned at 20 ± 0.9 days of age and 6.2 ± 0.34 kg of body weight (BW) were used in a study conducted at the Swine Experimental Unit of Lucta S.A. (Girona, Spain). At arrival, piglets were distributed into 36 individual pens (0.35 m 2 /pen) thoroughly cleaned and equipped with fully-slatted plastic floor plus a nipple drinker and a feeder. Animals were randomly assigned to receive a standard prestarter diet (CONd; n = 18; 50:50 male to female ratio) or the same diet supplemented with 60 mg of CDC (Sigma-Aldrich) per kg of initial BW (CDCd). Animals were fed the solid diets from weaning until day 14; thereafter, all pigs were fed the same (untreated) starter diet for 21 days until the end of experiment on day 35 (Table 1). During the study, all pigs had ad libitum access to feed and water. Starting at weaning BW was measured weekly, whereas feed intake was recorded daily until day 13 and weekly from day 15 to 35. Plasma collection and analysis Blood samples were obtained from six randomly-chosen pigs per treatment via jugular venipuncture on day 1, 7, and 14 after 12 h of feed deprivation. Samples were collected into tubes containing EDTA and aprotinin (BD Vacutainer®), held in ice-cold water for 30 min, centrifuged at 2000 × g for 10 min, stored at −80°C, and analyzed later on for bioactive GLP-2 by radioimmunoassay as described previously [19]. Tissue collection On day 15 after 3 h of feed deprivation, 6 pigs per treatment were euthanized with an intravenous injection of sodium pentobarbital (200 mg per kg of BW; Fatro Ibérica, Spain). The abdomen was opened and the intestines were removed and dissected into sections arbitrary designated as jejunum (from the pyloric sphincter to the first Peyer's patch), ileum (from the first Peyer's patch to the ileocecal valve) and large intestine (from the ileocecal valve to the rectum). Intestinal sections were measured, flushed with saline, and weighted. A 10-cm segment was removed from the midsection of the jejunum and ileum, divided into 5-cm halves, and opened longitudinally. Half of these samples were fixed in 10 % buffered formalin for subsequent histological examination, whereas mucosal scrapings were taken from the other half and stored in RNAlater® (Ambion, USA) at −80°C until analysis of gene expression. Morphometric analysis Samples of jejunum and ileum were dehydrated and embedded in paraffin, sectioned (~4 μm), and stained with hematoxylin and eosin. Villus height, crypt depth, number of intraepithelial lymphocytes in villi, and number of goblet cells in crypts were measured in 10 well-oriented villi and crypts using a light microscope (BHS, Olympus) and a linear ocular micrometer (Olympus, Microplanet). All determinations were done by the same person, who was blinded to treatments, at 400× magnification as described previously [20]. Statistical analysis Analyses were performed using the mixed-model procedure of SAS (release 9.2, SAS Institute Inc.). Performance data (BW, average daily gain, feed intake and feed conversion) for animals that were slaughtered on day 15 (n = 6) and those that completed the study on d 35 (n = 12) were analyzed separately. These results and GLP-2 data were analyzed using a mixed-effect model with repeated measures in which pig within treatment was used as random variable whereas treatment, time (day or week), and the interaction treatment by time were considered fixed. The smallest value for the Akaike's information criterion was used to identify the most appropriate covariance structure. The same model but without repeated measures was used to analyze intestinal weight, length, and morphology. To achieve normality, data for GLP-2 were transformed prior to analysis. Least squares means were separated into significant effects using the Fisher adjustment option of SAS. Differences in gene expression resulting from the comparison of the CDCd group with the CONd group were determined using a linear mixed-model in which treatment was included as fixed effect and the sample as random [24]. Gene specific residual variance (heterogeneous residual) was fitted to the gene effect [25]. For genes displaying efficiencies different from 2 (E ≠ 2), Ct values were adjusted according to the model described by Steibel et al. [24]. The geometric mean of the reference genes TBP and ACTB was used to correct Ct values of target genes [26]. Differences among treatments were considered to be significant when P < 0.05, whereas when P > 0.05 but < 0.10 differences were considered to indicate a trend towards a significant effect. Animal performance The onset of feed consumption after weaning and its time course during both the first 13 days of exposure to treatments (Fig. 1) and the 5 weeks of study (Fig. 2) were similar between treatment groups. Likewise, supplementing the CONd prestarter diet with CDC did not alter weight gain of piglets that ended the study either on days 15 or 35 (Table 2). In both treatment groups the incidence of diarrhea was low and did not differ between them ( Table 2). In addition, animals exhibited normal behavior and signs of adverse treatment effects were not observed during the study. Intestinal growth and morphology The inclusion of CDC in the prestarter diet tended to enhance the weight of the large intestine (P < 0.10) but did not modify the size (weight and length) of the small intestine (Table 3). In addition, the morphology of the mucosa from the jejunum and ileum was similar between treatments, with the exemption of the ileal crypts that were deeper (P < 0.04) in pigs fed CDC (Table 4). Intestinal gene expression The feeding of CDCd during the 14 days that followed EW did not modify the relative concentration of mRNA transcripts from genes examined in the jejunal mucosa (data not shown). Although expression of OCLN, GLP-2R, ASBT, EGFR and GCG was similar between groups (data not shown), the expression of ZON-1 (P < 0.02), TNF-α (P < 0.02), IL-10 (P < 0.006), and IL-6 (P < 0.01) increased 1.5, 1.9, 2.2 and 2.4 folds, respectively, in the ileum of CDCd-fed piglets relative to their CONd counterparts (Fig. 4). Discussion Feeding piglets a cereal-based diet supplemented with CDC for 2 weeks after EW induced genes involved in the barrier function and protection of the intestinal mucosa with marginal effects on the concentration of circulating GLP-2 and mass of the intestine. The enteroprotective action of CDC was not associated with changes in food intake and BW gain either during the period of CDC supplementation or after withdrawal of CDC from the diet. The low incidence of diarrhea observed in both treatment groups suggests that keeping pigs in individual pens under high sanitary conditions might have hindered improvements in animal performance otherwise mediated by CDC. Taken together, data indicate that the oral administration of bile acids to weaned pigs has the potential to improve the protection of the intestinal mucosa independently of relevant changes in gut growth. Weaning-induced intestinal atrophy and dysfunction are associated with a transient decrease in circulating GLP-2 [4,5]. In a previous study, we have showed that the intragastric administration of a single dose of CDC to piglets during the first 6 days after EW remarkably increased the plasma concentration of endogenous GLP-2 but that this response tended to enhance only the length and weight of the ileum [16]. Thus, we speculated that supplementing the postweaning diet with CDC with the aim of distributing its enteral supply throughout the day may augment the nutrient-dependent secretion of GLP-2 and improve its efficacy to preserve gut integrity immediately after EW. We observed that plasma GLP-2 tended to increase at the end of the period of exposure to CDC on day 14, when food intake increased on average by 160 g/d (178 %) relative to the first postweaning week. Coincidentally, in our previous study piglets consumed during the first 6 days after EW about 66 % more feed (+48 g/d) than in the present study [16]. It seems therefore that there is a minimum of enteral nutrition required for CDC to potentiate the release of GLP-2 in animals fed solid diets. Although this effect was paralleled by a deepening of the ileal crypts, which is a distinctive trophic action of GLP-2 [5], only the mass of the Data are least squares means, n = 6. CONd, prestarter diet; CDCd, prestarter diet supplemented with 60 mg chenodeoxycholic acid per kg of initial body weight large intestine was marginally increased. Paradoxically, enteral administration of CDC prevented gut atrophy in newborn piglets fed via parenteral [15]. In line with our findings, however, recent studies also observed modest enlargements of the epithelium of the intestine of enterally-fed weanling pigs in response to prolonged treatment with exogenous GLP-2 [11,12]. Collectively, data indicate that the intestine of pigs remains responsive to the trophic effect of bile acids after weaning, an action presumably mediated by GLP-2, but that the magnitude of this effect is rather small and likely irrelevant from a developmental standpoint. However, one cannot rule out that the deepening of crypts induced by CDC might accelerate the recovery of the intestinal mucosa function after weaning and contribute to maintain TJ and adequate enterocyte turnover. A critical function of the intestinal epithelium is to form a dynamic physical barrier to luminal contents to protect the host from infection and chronic exposure to inflammatory stimuli. Adjacent mucosal cells accomplish this by interacting through TJ proteins that are connected to the actin cytoskeleton and regulate the intestinal paracellular permeability [27]. Importantly, mounting evidence links increased gut permeability with intestinal inflammation, systemic immune activation, and disease progression in humans and animals [28]. Because EW dysregulates intestinal permeability in pigs [3,29], targeting TJ proteins may illuminate ways to maintain the integrity of the intestinal barrier and thereby improve piglet health, growth, and welfare during the weaning period. Based on this prediction and the notion that bile acids [17,18] and GLP-2 [30] regulate the expression and (or) cellular distribution of TJ proteins, we decided to examine the impact of dietary supplementation with CDC on the expression of some genes involved in the control of the barrier function of the intestinal mucosa. We found that feeding CDCd resulted in proinflammatory (i.e., TNF-α and IL-6 expression) and antiinflammatory (i.e., IL-10 expression) responses that were associated with increased concentration of ZON-1 transcripts in the epithelium of the distal small intestine. Considering that during the development of intestinal inflammation TNF-α disrupts TJ [31] whereas IL-10 antagonizes its action [32], it seems reasonable to suggest that CDC triggered a homeostatic immune response that ultimately appeared to enhance the integrity of TJ of the intestinal epithelium. The question as to whether these effects were mediated directly by CDC via activation of the bile acid sensors TGR5 [18] and farnesoid X-activated receptor (FXR) [17] or indirectly via enhanced released of GLP-2 [30] cannot be addressed with data reported herein. However, the observation that the anti-inflammatory action of GLP-2 involves suppression of both crypt-cell proliferation and inflammatory cytokines through a mechanism unrelated to Th2 cytokines such as IL-10 [33], suggests that the tolerogenic response triggered by CDC was not associated with GLP-2. Yet, it is more important to note that in a recent study with early-weaned pigs weaning disrupted intestinal permeability partly by repressing the expression of TJ proteins, including ZON-1, and that this effect lasted for 14 days postweaning albeit the morphology of the intestinal mucosa was fully recovered by then [34]. Therefore, our findings support the proposal that the oral administration to pigs of bile acids, or compounds that mimic their action, holds potential for enhancing the integrity of the mucosal barrier at weaning and beyond this critical time. It is widely accepted that disorders caused by EW, including increased susceptibility to diarrhea and growth retardation, mainly result from the transient absence of feed consumption that follows EW [1]. Expectably, supplementing the postweaning diet with CDC comprehended the risk of aggravating EW-induced anorexia because of reduced diet acceptability and (or) enhanced satiety mediated centrally by GLP-2 [35]. We found, however, that feeding CDCd did not affect the onset of feed intake following EW nor the amount of feed consumed during the 5-weeks study. Likewise, feed intake of weanling pigs was not affected when the plasma concentration of GLP-2 was increased via the intragastric infusion of CDC [16] or the administration of exogenous GLP-2 at a supraphysiological dose [11]. Despite the absence of anorectic effects, the aforementioned enteroprotective impact of feeding CDCd did not translate into CONd, prestarter diet; CDCd, prestarter diet supplemented with 60 mg of chenodeoxycholic acid per kg of initial BW; ZON-1, zonula occludens 1; TNF-α, tumor necrosis factor alpha; IL-10, interleukin 10; IL-6, interleukin 6. *P < 0.05, **P < 0.01 improved animal performance (i.e., BW gain and incidence of diarrhea). As suggested before, the high sanitary conditions under which this study was conducted might have accounted for such results. Certainly, the efficacy of exogenous GLP-2 for improving gut integrity during EW was most evident when pigs developed severe diarrhea [12]. Furthermore, bile acid-mimicking compounds administered orally to mice suppressed intestinal inflammation and signs of diarrhea in models of chemically-induced colitis [18,36]. Thus, available data provide a rationale for exploring the value of bile acids and compounds that mimic their action as dietary supplements to improve performance of pigs under situations of increased incidence of enteric disorders. Conclusions Supplementing the diet of early-weaned pigs with CDC enhanced the expression of genes involved in the protection and barrier function of the mucosa of the distal small intestine. These effects, however, were only associated with a trend towards increased concentration of endogenous GLP-2 and intestinal growth. Even though dietary supplementation with CDC did not affect feed intake, the high sanitary conditions that prevailed in this study might have negated improvements in piglet performance resulting from the enteroprotective action of CDC. Results from this study warrant further research to examine the use of bile acids and compounds that mimic their action as dietary interventions to improve gut health and performance of pigs under situations of increased susceptibility to enteric inflammation and infection (e.g., poor environmental hygiene, increased microbial exposure, physical stress, etc.).

v3-fos

2018-05-21T21:28:04.260Z

2015-05-21T00:00:00.000Z

21698788

s2

Effects of dietary protein level on nutrients digestibility and reproductive performance of female mink (Neovison vison) during gestation The objective of this study was to determine whether nutrient digestibility and reproductive performance of pregnant mink (Neovison vison) were affected by different dietary protein levels. One hundred and twenty female mink were randomly assigned to four groups, receiving diets of fresh material with different protein levels. The dietary protein levels, expressed as percentage of dry matter (DM), were 32, 36, 40 and 44% respectively. These values corresponded to average 320, 360, 400 and 440 g protein/kg DM, respectively. Results were as follows. All of crude protein digestibility, nitrogen (N) intake, N retention increased along with dietary protein level increasing. Low protein level (32%) significantly reduced the above indicators (P < 0.05). DM digestibility and ether extract digestibility were not affected by dietary protein level. Results of mated females, barren females, kids per litter, live born kids per mated female, birth survival rate, and birth weight showed that mink achieved optimal reproductive performance when dietary protein level was 36%. In conclusion, dietary protein was anticipated to significantly influence some nutrients' utilization. Adopting the appropriate dietary protein level allow better reproduction performance. The most preferable reproductive performance was achieved when diet contained 275.5 g digestible protein per kg DM for female mink in gestation. Introduction Mink are widely farmed in China as economic carnivorous animals with a higher demand for protein than other domestic animals. Protein constitutes the most expensive part of mink feed. Consequently, possibilities to decrease the level of dietary protein in order to provide inexpensive feed but still support animal performance and health have been the subject of research for several years (Nutrition, 1982;Skrede, 1978aSkrede, , 1978bTy€ opp€ onen et al., 1986). Furthermore, there has been a general wish to minimize the emission of nitration via urine and feces to the environment (Bikker et al., 2010). Reproduction of mink has been intensively studied in many research projects. Dietary protein deficiency was associated with reduced body weight of the offspring (Vesterdorf et al., 2012). A diet with low dietary protein content, which is 14% of metabolizable energy (ME) during late gestation, affected reproductive performance (Matthiesen et al., 2010). And the progression in breeding result is poor when compared with other mammals like sows or cows. Research has been conducted to demonstrate the effect of different feeds on reproductive capacity (Crum et al., 1993;Dahlman et al., 2002;Fink and Børsting, 2002;Fink et al., 2006;Matthiesen et al., 2010;Skrede, 1978b;Travis and Schaible, 1961). However, no specific information was found about the protein requirement of female mink during gestation. Thus, it is necessary to carry out the study on dietary nutrient levels on female mink in breeding season. This present study investigates whether different dietary protein levels affect reproductive performance of female mink during gestation. The specific aims were to investigate the effect of gestational protein supply on feed intake, nutrients digestibility, nitrogen (N) metabolism and reproductive performance. Materials and methods The experiment was carried out at the Fur Animal Breeding Base of Institute of Special Economic Animal and Plant Science, Chinese Academy of Agricultural Sciences (44.02 N,126.15 E) in the northeast of China. The animals used in this work were managed according to the requirements of the national Experimental Animals Protection Law, and with bioethics and biosecurity committee approval. Animals, diets and management A total of 120 two-year-old female mink (Neovison vison) of the standard black genotype were housed in roofed standard sheds with open sides. The experimental period extended from 14 d before mating until parturition. All animals were weighed at start of experiment and distributed randomly into four experimental groups from A to D with dietary protein levels of 32, 36, 40 and 44%, respectively. These levels corresponded to average 320, 360, 400 and 440 g protein/kg dry matter (DM), respectively. Decreasing dietary protein levels (in the experiment from 317.9 to 453.9 g/kg DM) from groups A to D were compensated by increasing the level of dietary carbohydrates (from 413.8 to 246.7 g/kg DM). Ether extract (EE) content (g/kg DM) in the diets remained constant within the range from 166.5 to 167.0 g/kg DM, so the diets were isocaloric. Throughout the experimental period, mink were fed the experimental diets twice a day ad libitum at 0800 and 1600 (Beijing time), and drinking water was taken freely by mink. The ingredients of the four diets were listed in Table 1, chemical composition in Table 2, and amino acid content in Table 3. Mating and nitrogen balance experiment The female mink were mated starting from 5 March, 2010. After about 25 d, eight pregnant mink were randomly selected from each group and housed in individual cages used for digestibility measurements and N-balance experiments. The experiment consisted of a three-day collecting and recording period carried out on the eight pregnant mink from respective treatment groups. The animals were kept in metabolism cages constructed for separate collection of feces and urine. Feed residues, feces and urine were quantitatively collected, weighed and recorded daily and stored at À20 C until the end of the balance period. To avoid ammonia evaporation from the urine, 20 mL sulfuric acid (5% solution) was added to the urine collection bottles, and the urine collection trays were sprayed with citric acid (20% solution) daily. In the N-balance calculations, retained N was determined as ingested N-(fecal N þ urinary N). Chemical analysis Wet samples of diets and feces were analyzed for DM and N (AOAC and Helrich, 1990). The freeze dried samples of diets and feces were analyzed for crude protein (CP) and EE. Urine and citric acid rinse were analyzed for N content. DM, ash, CP (Kjeldahl-N  6.25) and EE after acid hydrolysis were determined according to standard procedures (AOAC and Helrich, 1990). Crude carbohydrate was calculated as the difference by subtracting ash, CP and EE from the DM content. Amino acids in diets were analyzed by amino acid analyzer (L-8900, HITACHI, Japan), as described by Ma et al. (2010). The apparent digestibility (AD) coefficient of nutrients was calculated as follows: AD ¼ (a À b)/a  100, where "a" is nutrient intake from feed, and "b" is nutrient excretion in feces. The calculation of ME content and the proportional composition of ME were based on the digestibility coefficients achieved and the following values of ME: (Clauss et al., 2010). Reproductive performance evaluation Mated female mink, barren female mink, kids per litter, and live born kids per mated female were recorded or calculated. Mink kids were weighted at birth. These were used as indexes to determine the reproductive performance of dams (Korhonen et al., 2002;Shaw et al., 1997;Tauson and Ald en, 1984;Vesterdorf et al., 2012). Statistical analysis The statistical analyses of data were carried out using the Chi-square statistics and one-way ANOVA procedure of SAS (version 8.2, SAS Institute, Inc., Cary, NC, USA). Data were represented as mean ± SD. Values of P less than 0.05 were considered statistically significant and those of P less than 0.01 were considered statistically highly significant. Feed intake and nutrients digestibility Effects of different dietary protein levels on performance and nutrient digestibility are showed in Table 4. DM intake reached the peak in group B and was significantly higher than group D (P < 0.05). No significant difference was observed among the four groups in DM output and DM digestibility (P > 0.05). CP digestibility linearly increased with increasing dietary protein levels. Group D had a significantly higher CP digestibility than group A (P < 0.05). Results also showed protein supply had no effect on EE digestibility in gestation (P > 0.05). Nitrogen-balance Effects of different dietary protein levels on N-balance are presented in Table 5. Dietary protein caused highly significant higher N intake (P < 0.01), while fecal and urine N were not affected (P > 0.05). Reproductive performance Data of reproductive performance is presented in Table 6. Assessed by using chi-square statistics, the numbers of mated females and barren females were not affected by dietary protein levels, but reduced number of barren females was observed in group B (P > 0.05). None of kids per litter, live born kids per mated female, birth survival rate or birth weight was significantly affected by dietary protein level (P > 0.05), but the birth survival rate of group A was significantly decreased (P < 0.05), and most of these indexes reached the highest in group B. Discussion Present studies suggested that protein intake may play a part in feed intake regulation, and high protein diets caused a depression in feed intake (Harper et al., 1970;Musten et al., 1974;Scharrer et al., 1970). In this study, DM intake increased initially, reaching the highest in the 36% dietary protein group and then decreased, indicating mink are regulating the feed consumption so as to balance their gains of protein (Mayntz et al., 2009). Less DM intake in the higher protein diet group was probably due to the higher energetic costs of using amino acids as glucose precursors, as demonstrated by Chwalibog et al. (2004). The apparent digestibility of DM was highly dependent on the dietary protein level. The lower the dietary protein level, the lower was the DM digestibility (Dahlman et al., 2002). Our results showed that dietary protein level had no significant effect on DM digestibility, probably because energy densities of all diets were within the range where mink could be expected to regulate their intake. As strict carnivores, mink have a higher demand for protein than other domestic animals. Previous studies found that decreasing dietary protein level leads to the decreasing values of apparent digestibility (Skrede, 1979;Szymeczko and Skrede, 1990). Our study demonstrated that the apparent protein digestibility decreased in group A with the lowest protein content, but not significantly affected at higher protein levels. During the breeding season, pregnant mink need protein not only for growth of the gravid uterus, but also for reserving protein as mobilizable amino acids available to meet the increased N requirement of early lactation, the Means in the same row with different lowercase of superscripts were significantly different at P < 0.05. A, B Means in the same row with different capital letters of superscripts were greatly and significantly different at P < 0.01. 1.54 ± 0.12 1.53 ± 0.12 1.68 ± 0.14 1.72 ± 0.16 N retention 0.81 ± 0.08 Bb 1.30 ± 0.08 ABab 1.41 ± 0.11 ABa 1.74 ± 0.13 Aa Groups A to D denoted dietary protein levels, 32, 36, 40 and 44%, respectively. N ¼ nitrogen. Means in the same row with different lowercase of superscripts were significantly different at P < 0.05. A, B Means in the same row with different capital letters of superscripts were greatly and significantly different at P < 0.01. Means in the same row with different lowercase of superscripts were significantly different at P < 0.05. development of the mammary gland, and a likely increase in protein requirement for the hypertrophy of visceral tissues, including gut and liver, in late pregnancy (Fell et al., 1972;Robinson et al., 1978) and in maternal tissue. In adequately nourished dams fed high protein diets (groups with dietary protein levels of 36, 40 and 44%), part of the protein digested for fetal growth could possibly be sustained by mobilization of a labile protein reserve that has accreted during early gestation . According to the present research, N excretion in fecal and urine were not significantly affected by protein provision during gestation, probably due to endogenous N secretion from the digestive tract. A previous study on pigs demonstrated that reduced dietary protein would lead to an enhanced relative amount of endogenous N secretion (Le Bellego and Noblet, 2002), which could explain the stable N excretion among groups. In the present study, CP intake had a significant effect on N retention as determined by collection of urine and feces during pregnancy, as previously found in mink (Matthiesen et al., 2010;Zhang et al., 2013). Although N retention increased along with protein level, one cannot make conclusion on the protein requirement of pregnant mink until the reproductive performance is investigated. Our data showed mink of all groups were successfully mated. The group with 36% dietary protein showed the lowest number of barren females, and significantly higher number of kids per litter, the number of live born kids per mated female, and birth survival rate. The fact that low protein diets impair the reproductive performance has been demonstrated by extensive studies (Bellinger et al., 2006;Galler and Tonkiss, 1991;Matthiesen et al., 2010;Pinheiro et al., 2008;Sherman et al., 1999). Hansen (1974) found that low levels of protein (25% of ME from protein) throughout gestation resulted in a tendency for suboptimal reproductive performance. A diet with lower dietary protein content resulted in 15% barrenness, 30.6% fewer kids per mated female and a lower number of live kids born per litter, and F1-generation kids with protein restriction during fetal life had a lower birth weight than F1generation kids with adequate protein (Matthiesen et al., 2010). Studies had shown that increasing dietary protein level increased protein oxidation rates and metabolic rates, which leaded to an acute increase in reactive oxygen species (ROS) generation (Fink and Børsting, 2002;Mohanty et al., 2002;Rouvinen-Watt, 2003). Agarwal et al. (2005) reported that, ROS, playing a role with regard to the female reproductive tract, can affect oocyte maturation and follicle development, fertilization and implantation, the development of the fetus, and pregnancy itself by causing abortions, birthing complications and defects in the offspring. Thus, excessive protein intake may stimulate ROS generation in high protein level groups, leading to poorer reproductive performance. Therefore, it could be concluded that birth rate of female mink could be adversely affected by both deficiency and excess of protein, and 36% dietary protein level (275.5 g digestible protein per kg DM) met the protein requirement of pregnant mink in this investigation. Conclusions Our findings suggested that dietary protein level during gestation significantly affected the nutrients digestibility and N-balance of mink. Optimal reproductive performance was observed with the dietary protein levels up to 36% (275.5 g digestible protein per kg DM), which we recommend to be adopted in mink farming business.

v3-fos

2015-09-18T23:22:04.000Z

2015-08-01T00:00:00.000Z

40241848

s2

Sources and Amounts of Animal, Dairy, and Plant Protein Intake of US Adults in 2007–2010 Dietary guidelines suggest consuming a mixed-protein diet, consisting of high-quality animal, dairy, and plant-based foods. However, current data on the distribution and the food sources of protein intake in a free-living, representative sample of US adults are not available. Data from the National Health and Nutrition Examination Survey (NHANES), 2007–2010, were used in these analyses (n = 10,977, age ≥ 19 years). Several US Department of Agriculture (USDA) databases were used to partition the composition of foods consumed into animal, dairy, or plant components. Mean ± SE animal, dairy, and plant protein intakes were determined and deciles of usual intakes were estimated. The percentages of total protein intake derived from animal, dairy, and plant protein were 46%, 16%, and 30%, respectively; 8% of intake could not be classified. Chicken and beef were the primary food sources of animal protein intake. Cheese, reduced-fat milk, and ice cream/dairy desserts were primary sources of dairy protein intake. Yeast breads, rolls/buns, and nuts/seeds were primary sources of plant protein intake. This study provides baseline data for assessing the effectiveness of public health interventions designed to alter the composition of protein foods consumed by the American public. Introduction Protein is, unquestionably, required in the human diet [1]. Dietary protein is the primary source of amino acids, particularly the essential amino acids, which cannot be synthesized from endogenous precursors, and are required for growth, development, and maintenance of human health. The recommended dietary allowance (RDA) for protein is 0.8 g protein per kilogram body weight (g/kg BW) and is considered adequate for nearly all healthy US adults [1], although consuming protein above the RDA has consistently been shown to be metabolically advantageous by promoting healthy blood lipids, weight management, satiety, and enhancing long-term bone mineralization [2]. We recently reported that habitually consuming a higher protein diet, regardless of body size, was associated with lower adiposity and higher HDL-cholesterol compared to consuming protein at levels consistent with the RDA [3]. The source of dietary protein is perhaps as important as the total quantity consumed. Animal, dairy, and some plant proteins are considered high-quality proteins that confer health and metabolic benefits based on the digestible levels of the essential amino acids they contain. Previous work has shown that many protein foods, regardless if classified as animal, dairy, or plant, are major contributors of other critical nutrients (e.g., zinc, vitamin B-12, iron, calcium, phosphorus, magnesium, vitamin E, and dietary fiber) [4]. The 2010 Dietary Guidelines for Americans recommends consuming a mixed-protein diet, consisting of a variety of high-quality animal, dairy, and plant-based foods [5]. Recently, the 2015 Scientific Report of the Dietary Guidelines Advisory Committee (DGAC) recommended increasing certain plant-based foods, including whole-grains, legumes, and nuts, as well as increasing the intake of low-fat dairy and certain animal-based foods, such as seafood. In contrast, the DGAC recommends consuming a diet lower in plant-based foods that contain refined grains and added sugars and certain animal-based foods, primarily red and processed meat [6]. Whether these recommendations are being met or to what extent dietary modification is required to comply with the DGAC recommendations is not known, largely because no studies have provided comprehensive data of habitual intakes of animal, dairy, and plant-based foods, particularly as they relate to total protein intake, in a representative sample of the free-living US adult population. The objective of the current study was to determine dietary intake level and food sources of animal, dairy, and plant protein among US adults using data from NHANES 2007-2010. Based on a 2005-2006 characterization of protein intake among older adults using NHANES data [7], we anticipated that animal protein intake, with or without including dairy, would be the predominant source of protein in the diet followed by plant protein. We expected dairy protein intake alone to contribute the lowest amount of total protein to the diet, and that milk, which may provide the highest-quality protein, would be the primary source of dairy protein in the diet. Participants The study sample consisted of 10,977 adults (age ě 19 years) who completed a 24-h dietary recall in What We Eat in America, the dietary interview component of the NHANES, 2007-2010. Analyses included only individuals with complete and reliable dietary records using the USDA automated multiple-pass method. Pregnant or lactating women were excluded. All participants or proxies provided written informed consent and the Research Ethics Review Board at the National Center for Health Statistics approved the survey protocol. Detailed description of the survey design and the data collection procedures are reported elsewhere [8]. Estimating Level and Source of Protein Intake USDA food composition databases were used to determine protein gram intake and protein type from foods consumed by NHANES participants. This process estimates nutrient content of reported foods, by linking the Food and Nutrient Database for Dietary Studies (FNDDS) with food composition data provided by the USDA Nutrient Database for Standard Reference (SR). The ingredients of disaggregated survey food recipes (coded using the SR food codes) were linked to the appropriate food composition databases using the SR-Link file of the FNDDS (versions 4.1 and 5.0 link SR releases 22 and 24 respectively) [9,10]. Protein gram amounts by type associated with an intake in the NHANES individual foods file was obtained via the FNDDS SR Links and SR nutrients files. Every SR code with protein was assigned via the SR code description to a source; animal, dairy, plant or mixed protein. Mixed protein was used to denote that the source for the SR code was from more than one of animal, dairy or plant proteins. For each food code, the SR weights and SR links were used to determine the percentage of protein of each of the types (animal, dairy, plant, mixed) that made up the protein in the food code. These percentages were then applied to the total protein for the food code if each food consumed by each subject. The calculations were done separately for each NHANES data release using the individual food files, FNDDS and SR files corresponding to that NHANES release. The nutrient profiles for some missing SR codes were obtained from addendums to FNDDS files for missing SR codes. There were only 13 remaining SR codes with missing nutrient profiles. These were obtained from the nearest SR version where it was available or from SR codes with similar descriptions. An analysis of all the protein intake in the NHANES files indicated these methods result in more than 90% of all protein gram intake categorized into animal, dairy or plant with only less than 10% in the mixed category. An example of protein in the mixed category was pizza. For example, the food code 58106220 "Pizza, cheese, from restaurant or fast food, thin crust" links to the single SR code 21301 denoting pizza. Using the SR description the protein is assigned to the mixed category since its source contains both dairy and plant-based protein and the individual amounts in the dairy and plant categories cannot be calculated from the SR data. Several food categories (such as mixed dishes, burritos and tacos, soups, cakes and pies, and eggs and omelets) were common for more than one source of protein. Additionally the USDA list of 150 total food categories [11], of which 24 food categories were identified as sources of animal proteins (providing at least 1% animal protein), 20 food categories as sources of dairy protein (providing at least 1% dairy protein), and 31 food categories as sources of plant protein (providing at least 1% of plant protein), was used to define sources of protein by type in the US diet. Statistical Analysis Data were analyzed using SAS 9.2 (SAS Institute) and SUDAAN release 11.0 (Research Triangle Institute). Appropriate weighting factors were used to adjust for oversampling of selected groups, survey non-responses of some individuals, and for day of the week the interview was conducted [12]. Mean and percentages˘standard errors (SE) of animal, dairy, and plant protein were determined using PROC DESCRIPT of SUDAAN using data from the first 24-h recall. To develop deciles of animal, dairy, and plant protein intake, individual usual intakes were estimated using the National Cancer Institute (NCI) method [13] similar to that we reported previously [3]. Briefly, usual intake of animal, dairy, and plant protein was estimated using both days of 24-h recall with a single component model since these dietary components are consumed by almost all subjects on most days. Results Overall protein intake (mean˘SE) was 82.3˘0.8 g/day (98.6˘1.1 g/day for men and 67.0˘0.7 g/day for women) regardless of protein source among this representative sample of US adults. The proportion of total protein intake attributable to animal protein was 46%, whereas dairy and plant protein accounted for 16% and 30% of total protein intake, respectively ( Figure 1). About 8% of the total protein intake (mainly from mixed foods) was undefined because its protein type could not be determined with confidence. t protein (providing at least 1% of plant protein), was used to define sources of protein by typ S diet. Statistical analysis ata were analyzed using SAS 9.2 (SAS Institute) and SUDAAN release 11.0 (Research Trian tute). Appropriate weighting factors were used to adjust for oversampling of selected grou ey non-responses of some individuals, and for day of the week the interview was conducted [1 n and percentages ± standard errors (SE) of animal, dairy, and plant protein were determined us C DESCRIPT of SUDAAN using data from the first 24-h recall. To develop deciles of anim , and plant protein intake, individual usual intakes were estimated using the National Can tute (NCI) method [13] similar to that we reported previously [3]. Briefly, usual intake of anim , and plant protein was estimated using both days of 24-h recall with a single component mo e these dietary components are consumed by almost all subjects on most days. esults verall protein intake (mean ± SE) was 82.3 ± 0.8 g/day (98.6 ± 1.1 g/day for men and 67.0 ± y for women) regardless of protein source among this representative sample of US adults. ortion of total protein intake attributable to animal protein was 46%, whereas dairy and p ein accounted for 16% and 30% of total protein intake, respectively ( Figure 1). About 8% of protein intake (mainly from mixed foods) was undefined because its protein type could not rmined with confidence. Figure 1. Percentage of animal, dairy, and plant protein intake among US adults combined and separated by sex using data from NHANES 2007-2010 (n = 10,977, ≥19 years). Total protein intake (mean ± SE) was 82.3 ± 0.8 g/day (98.6 ± 1.1 g/day for men and 67.0 ± 0.7 g/day for women). Total protein intake (mean˘SE) was 82.3˘0.8 g/day (98.6˘1.1 g/day for men and 67.0˘0.7 g/day for women). Animal, dairy, and plant protein intake more than doubled from population decile 1 to decile 10 ( Table 1). Animal protein intake was 2.6-fold and dairy protein intake four-fold higher in decile 10 compared to decile 1. Plant protein intake was 2.2-fold higher in decile 10 compared to decile 1. More than half of the women surveyed reported consuming levels of animal and plant protein in deciles 1 through 5, whereas the proportion of women consuming dairy protein was relatively constant across all deciles. The percentage of White individuals decreased and Hispanic individuals increased across deciles of protein intake from animal foods. However, the percentage of Whites reporting consuming more dairy protein increased across deciles, whereas the percentage of Hispanics and Blacks decreased across deciles. More Hispanics tended to consume higher levels of plant protein, while a greater percentage of Blacks reported consuming lower levels of plant protein (Table 1). Twenty-four food categories were identified as contributing at least 1% of total animal protein intake. Chicken and beef were the top two food categories for animal protein, providing 26% of total animal protein intake, 13% of total dietary protein intake, and 5% of total energy intake. The top 10 animal protein food categories, each of which contributed to more than 3% of total animal protein intake, provided approximately 67% of total animal protein intake but less than 16% of total energy intake ( Table 2). * These food categories also contribute as dairy and/or plant protein food sources and are included in Table 3 and/or Table 4. Twenty food categories were identified as providing at least 1% of dairy protein intake. Cheese and reduced fat milk were the top two food categories for dairy protein, providing approximately 35% of total dairy protein intake, 6% of total protein intake, and 4% of total energy intake (Table 3). Reduced fat, nonfat, whole, and low fat milk combined provided approximately 28% of total dairy protein intake, 6% of total protein intake, and 3% of total energy intake. The top 10 dairy protein food categories provided nearly 70% of total dairy protein intake and 11% of total energy intake. * These food categories also contribute as animal and/or plant protein food sources and are included in Table 2 and/or Table 4. Thirty-one food categories were identified as providing at least 1% plant protein intake. These 31 plant protein sources provided nearly 73% of plant protein in the diet. Yeast breads and rolls/buns were the top two plant protein food categories, providing nearly 18% of total plant protein intake, 6% total protein and total energy intake. The top 10 plant protein food categories provided approximately 40% of total plant protein intake and 20% of total energy intake (Table 4). * These food categories also contribute as animal and/or dairy protein food sources and are included in Table 2 and/or Table 3. The protein density (g/100 kcal) of animal protein food sources (providing at least 1% animal protein) was more than two times the density of plant protein food sources (providing at least 1% plant protein) and 50% more than dairy protein food sources (providing at least 1% dairy protein). The protein density of dairy protein food categories was also 50% more than the density of plant protein food categories ( Figure 2). Discussion The primary finding from this cross-sectional study confirms that Americans habitually consume protein that is predominately animal-based. Chicken, a lean source of high-quality protein, and beef were the primary source of animal protein intake. Plant protein did account for more than a third of total protein intake, although the major food sources of plant protein intake were breads and thus not plant foods that contain generally high-quality sources of protein. The proportion of total protein intake attributed to dairy (16%) was relatively low in comparison to animal (46%) and plant (30%) protein intake, although the primary sources of dairy protein intake in the American adult diet (e.g., cheese and milk) are considered to be among the highest-quality sources of protein. Although our estimates of total protein intake are consistent with our previous reports [3,14], few studies using national representative data have systematically characterized the level and also the food sources of animal, dairy, and plant protein intake in the American diet. Not surprisingly, those studies reported that animal-derived protein (including dairy) was the main source of protein intake. Analyses of data from NHANES [7,15] and European adults [16,17] show animal and dairy protein intake accounted for more than two thirds of total protein intake. Their findings are consistent with the combined total intake of protein from animal and dairy-based sources we found. Plant protein accounted for nearly one third of total dietary protein intake in our study, primarily from grains, which were also the primary source of plant protein intake among US adults nearly two [18] and three decades ago [15]. These data suggest American dietary habits are, essentially, unchanged, despite official national policy recommendations [19] promoting substantial changes and the popularity of various diets (e.g., the Paleo, Atkins, Gluten-free, South Beach, DASH, etc.). Food sources of animal protein were the most efficient source of dietary protein compared to food sources of dairy and plant-based protein when expressed as protein density (i.e., the amount of protein Discussion The primary finding from this cross-sectional study confirms that Americans habitually consume protein that is predominately animal-based. Chicken, a lean source of high-quality protein, and beef were the primary source of animal protein intake. Plant protein did account for more than a third of total protein intake, although the major food sources of plant protein intake were breads and thus not plant foods that contain generally high-quality sources of protein. The proportion of total protein intake attributed to dairy (16%) was relatively low in comparison to animal (46%) and plant (30%) protein intake, although the primary sources of dairy protein intake in the American adult diet (e.g., cheese and milk) are considered to be among the highest-quality sources of protein. Although our estimates of total protein intake are consistent with our previous reports [3,14], few studies using national representative data have systematically characterized the level and also the food sources of animal, dairy, and plant protein intake in the American diet. Not surprisingly, those studies reported that animal-derived protein (including dairy) was the main source of protein intake. Analyses of data from NHANES [7,15] and European adults [16,17] show animal and dairy protein intake accounted for more than two thirds of total protein intake. Their findings are consistent with the combined total intake of protein from animal and dairy-based sources we found. Plant protein accounted for nearly one third of total dietary protein intake in our study, primarily from grains, which were also the primary source of plant protein intake among US adults nearly two [18] and three decades ago [15]. These data suggest American dietary habits are, essentially, unchanged, despite official national policy recommendations [19] promoting substantial changes and the popularity of various diets (e.g., the Paleo, Atkins, Gluten-free, South Beach, DASH, etc.). Food sources of animal protein were the most efficient source of dietary protein compared to food sources of dairy and plant-based protein when expressed as protein density (i.e., the amount of protein per 100 kcal). Animal protein foods were twice as dense as plant protein foods. Dairy protein foods were also more protein-dense than plant protein foods. Animal and dairy proteins are considered high-quality proteins and excellent sources of other required nutrients, including iron, calcium, and vitamin D [20]. Plant protein foods are generally less protein-dense and, as a consequence, their consumption results in intake of more energy relative to protein. Grains, most of which were likely refined grains and foods with added sugar, were the largest contributor to total plant protein intake. Grains are often considered incomplete proteins because the essential amino acid content is low, with lysine as the most deficient [21]. Perhaps most importantly, the number of plant-based food categories contributing to total plant protein intake are, essentially, the same types of foods (e.g., doughnuts, cakes, pies, biscuits, candy, etc.) that the DGAC [6] recommends should be consumed less in order to achieve a healthier diet. Overconsumption of these foods likely diminishes the quality of total protein intake at the expense of simply consuming more energy. Our sample size (10,977), consistent approach to classifying protein type, and use of the NCI usual intake methodology to estimate usual intakes are strengths of the current study. However, there are some limitations, particularly reliance on self-report dietary recalls, which can under and overestimate actual intake data [22], even though NHANES uses sophisticated interview procedures. We acknowledge the timeframe assessed (NHANES 2007-2010) may not entirely reflect current intakes and that our analytical approach was unable to differentiate 100% of the protein in the diet, although we were able to confidently differentiate more than 90% of protein intake as animal, dairy, and plant. Conclusions This population-based, descriptive study provides a comprehensive, contemporary analysis of total level and food sources of animal, diary, and plant protein intake in a representative sample of US adults. Our analyses indicate that, in the American adult diet, animal protein is the predominant source of dietary protein followed by plant and dairy protein. These data demonstrate that in a representative sample of the American adult population about 30% of protein is coming from plant-based foods. However, most of these foods are relatively low in protein density and any dietary recommendation that recommends a diet higher in plant-based foods should consider their effects on energy intake and the quantity and quality of protein consumed, and the positive and negative impact on intake of nutrients associated with these protein-containing foods.

v3-fos

2016-05-18T09:40:07.010Z

2015-02-28T00:00:00.000Z

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Comparison of Bioactive Compounds and Quality Traits of Breast Meat from Korean Native Ducks and Commercial Ducks The aim of this research was to compare the bioactive compound content and quality traits of breast meat from male and female Korean native ducks (KND) and commercial ducks (CD, Cherry Valley). Meat from three 6-wk old birds of each sex from KND and CD were evaluated for carcass and breast weights, pH, color, cooking loss, shear force, and bioactive compound (creatine, carnosine, anserine, betaine, and L-carnitine) content. KND showed significantly higher carcass weights than CD whereas no such difference (p>0.05) was found between male and female ducks. The breed and sex had no significant effects on the breast weight, pH value, and shear force. However, KND had significantly lower cooking loss values than did CD. Creatine, anserine, and L-carnitine contents were significantly higher in KND than in CD and were predominant in female ducks compared to males. The results of this study provide rare information regarding the amounts and the determinants of several bioactive compounds in duck meat, which can be useful for selection and breeding programs, and for popularizing indigenous duck meat. Introduction Meat and meat products are excellent sources for proteins, vitamins such as B 12 , and minerals (Jimenze-Colmenero et al., 2001), however, their risk related to cardiovascular diseases and colon and other cancers has also been reported McAfee et al., 2010;Oostindjer et al., 2014), making consumers more concerned about meat consumption. As consumers understand more that food consumption is one of the important factors and can influence on human health (Goetzke, 2014; Lähteenmäki, 2013), consuming meat and meat products on our meal has been a controversial issue with their benefit and risk. Meanwhile, duck meat consumption in Korea has been increased approximately by 5-folds from 1997 to 2012 (Korea Duck Association, 2013), which might be attributable to consumers' increasing concern about the consumption of healthier meat. Duck meat is considered to be healthier compared to other animal products as it contains higher unsaturated fatty acid, essential fatty acid, and protein contents, in addition to its ability to decrease LDL cholesterol and blood pressure Kim et al., 2010a). With the increase in duck meat consumption, considerable efforts have been made since 1990's to popularize duck meat as a healthier meat source to secure the competitiveness in world market. The present Korean native duck (KND) produced by National Institute of Animal Science (NIAS) is a crossbreed between mallard duck (Anas platyrhynchos) and meat-type duck (Kim et al., 2010a;Kim et al., 2010b). However, still 90% of the breeding ducks in Korea are the breeds from oversea such as Cherry Valley (England) and Grimaud (France) and only 10% of them are KND (Kim et al., 2012a). Hence, it is an urgent goal to preserve pure bloodlines and develop a commercial meat-type KND breed by utilizing indige-nous breeds such as Woorimatori TM [developing Korean indigenous breed by National Institute of Animal Science (NIAS), RDA, Korea] to meet the increasing demand for duck meat. In developing a meat-type KND breed, it is important to carry out more investigations on quality traits of duck meat. When the present KND is further improved, it may have a possibility to secure the competitiveness against other breeds or animal products. Designing meat and meat products as functional foods has received more attention in the field of meat technology. Functional foods with beneficial impact on health (Goetzke et al., 2014) have become a worldwide trend and a craving for healthier food, and its market is continuously expanding based on increasing consumption and interest. Jimenez-Colmenero et al. (2001) and Olmedilla-Alonso et al. (2013) agreed on an optimistic prediction for improving nutritional value and positive image of meat and meat products when they are developed as functional foods. In this regards, improving the bioactive compound content in meat and meat products can be an effective mean. Important bioactive compounds in meat include coenzyme Q 10 , taurine, conjugated linoleic acid, glutathione, lipolic acid, betaine, L-carnitine, creatine, carnosine, and anserine. They are abundant in mammalian skeletal muscles and have distinct biological functions in animal body, including working as a pH buffer, effective antioxidative and anti-aging agent, and an osmolyte. (de Zwart et al., 2003;Hipkiss, 1998;Schmid, 2009;Stenesh and Winnick, 1960). During the development of KND, most of studies on duck meat were focused on productive aspect and basic quality traits Kim et al., 2010a;Kim et al., 2010b;Kim et al., 2012a) rather than its functional properties. Research findings on the availability and amounts of bioactive compounds in duck meat, in particular KND meat, are scarce. Therefore, the aim of this research was to compare the bioactive compound content and other quality traits of the breast meat from KND and commercial ducks (CD). In addition, the effect of sex on the same parameters was tested. Sample preparation Frozen carcasses from three birds of each sex from different breeds, KND and CD (Cherry Valley), were purchased at 6 wk of age from a local farm (Korea), and transported in frozen condition to a laboratory. Then, the carcasses were thawed at 4 o C for 48 h, deskinned, and deboned manually. After collecting breast meat from each carcass, they were minced separately using a mini chopper (CH180, Kenwood, China) for 30 sec and used for the analysis. The carcass and breast weights (g) from each bird were also recorded. pH Each meat sample (1 g) was homogenized with 9 mL of distilled water using a homogenizer (T10 basic, Ika Works, Germany). The homogenates were centrifuged (Union 32R, Hanil Co., Ltd., Korea) at 3,000 rpm for 10 min and filtered (Whatman No. 4, Whatman PLC., UK). The pH value of each filtrate was measured using a pH meter (SevenGo, Mettler-Toledo International Inc. Switzerland) which was pre-calibrated using standard buffers (pH 4.01, 7.00, and 9.21). Cooking loss Cooking loss was determined as the percentage weight loss of each meat sample after cooking. Meat samples (30 g) were vacuum packed (HFV-600L, Hankook Fujee Co., Ltd., Korea), heated in a water bath at 90 o C for 15 min until a core temperature of 72 o C was reached, and cooled in iced water. After recording the final weight, cooking loss was calculated as expressed below: Cooking loss (%) = Shear force Cooked sample was cut into a 10 × 10 × 30 mm to measure shear force. The value was measured using a Warner-Bratzler shear attachment on a texture analyzer (CT3 10K, Brookfield Engineering Laboratories., USA) with a maximum cell load, 10 kg; target load, 10 g; target value, 25 mm; target speed 2.0 mm/sec. The samples were sheared perpendicularly to the direction of muscle fiber. Meat color The surface color measurements (CIE L*, a*, and b* values representing lightness, redness, and yellowness, respectively) of meat samples were measured using a colorimeter (CR-310, Minolta Co., Ltd., Japan) which was calibrated against a white reference tile (Minolta calibration plate, No. 14633072). Three observation readings were taken for each color measurement in order to minimize the possible errors and the average was used as one Weight before cooking Weight after cooking -Weight before cooking Creatine, anserine, and carnosine contents The creatine, anserine, and carnosine contents in the samples were determined as described by Mora et al. (2007). Minced meat sample (2.5 g) were homogenized (T10 basic, Ika Works) with 7.5 mL of 0.01 N HCl at 13,500 rpm for 1 min. The homogenate was centrifuged at 3,000 rpm for 30 min (Union 32R, Hanil Co., Ltd., Korea), and 1 mL of the supernatant was transferred into a microtube and centrifuged at 10,000 rpm for 10 min (HM-150IV, Hanil). After centrifugation, 0.5 mL of the supernatant was mixed with 1.5 mL of acetonitrile, and the mixture was centrifuged at 10,000 rpm for 10 min (HM-150IV, Hanil), and the supernatant was filtered through a membrane filter (0.2 μm) into a glass vial. The samples were injected into a high performance liquid chromatography (HPLC; Ultimate 3000, Thermo Fisher Scientific Inc., USA) system under the gradient condition of two mobile phases; mobile phase A was 0.65 mM ammonium acetate in distilled water and acetonitrile (25:75 v/v, pH 5.5), and mobile phase B was 4.55 mM ammonium acetate in distilled water and acetonitrile (70:30 v/v, pH 5.5). The analytical conditions for HPLC was set up as descri-bed: injection volume, 10 μL; column, Atlantis HILIC silica column, 4.6 × 150 mm, 3 μm (Waters Corp., USA); flow rate, 1.2 mL/min. A detector was used at 214 nm to determine the creatine, anserine, and carnosine contents. The contents of the compounds were calculated using a standard curve obtained from the standard (Sigma, USA) of each compound. Betaine and L-carnitine contents The betaine and L-carnitine contents were quantified following the modified method of Jayasena et al. (2014a). Five grams of each meat sample was added with 10 mL of acetonitrile-methanol (9:1) solution and homogenized (T10 basic, Ika Works) at 13,500 rpm for 30 s. The homogenate was then centrifuged at 3,100 rpm for 5 min at 4 o C (Union 32R, Hanil), and the supernatant was filtered into a 20-mL volumetric flask through a funnel plugged with glass wool. The remaining filtrate was again mixed with 10 mL of acetonitrile-methanol solution and centrifuged (Union 32R, Hanil) under the same conditions. The resulting supernatant was collected in the same volumetric flask which was then filled with acetonitrile-methanol solution. Subsequently, 2 mL of this sample was transferred to a 15-mL tube and then 810 mg of Na 2 HPO 4 and 90 mg of Ag 2 O (9:1 w/w) were added. After vortex-mix-ing the solution, the sample tubes were dried by shaking without their caps in a shaking machine for 20 min and then centrifuged at 3,100 rpm for 5 min (Union 32R, Hanil, Korea). A 0.5-mL aliquot of each supernatant sample was then mixed with 0.5 mL of derivative reagent (0.066 g of 18-crown-6 and 1.39 g of bromoacetophenone in 100 mL of acetonitrile) in a 15-mL tube, voltexed, and heated in a water bath at 80 o C for 60 min. After cooling under running water, this mixture was filtered through a membrane filter (0.2 μm) and analyzed in a HPLC system (Ultimate 3000, Thermo Fisher Scientific Inc., USA) to determine betaine and L-carnitine contents. Two mobile phases (A, 25 mM ammonium acetate in which pH was adjusted to 3.0 using formic acid; B, acetonitrile) were used and the analytical condition for HPLC was set up as described: injection volume, 10 μL; column, Atlantis HILIC silica column, 4.6 × 150 mm, 3 μm (Waters Corp.); flow rate, 1.4 mL/min; detector was used at 254 nm. Standard curves were obtained using the standard (Sigma) for each compound and then used for calculation of betaine and Lcarnitine contents. Statistical analysis Statistical analysis was performed using multifactorial analysis of variance (ANOVA) to estimate the effect of the breed and sex on quality traits and bioactive compound content of duck meat, and the significant differences between the mean values were identified with Tukey's multiple range test using SAS software at a confidence level of p<0.05 (SAS 9.3, SAS Institute Inc., USA). Results and Discussion Carcass and breast weights Table 1 shows the effects of breed and sex on carcass and breast weights of ducks; KND had a significantly higher carcass weight than CD (p<0.05) whereas no such difference was found between male and female ducks ( Table 1). As KND was known for their inferior growth rate compared with commercial breeds, NIAS made considerable efforts to improve the growth rate and developed KND in two main types; small-type and large-type Meat quality traits The breed and sex of duck did not affect the pH value of breast meat in the present study (Table 2), and it is in agreement with the findings of Muhlisin et al. (2013). Furthermore, the pH value of breast meat observed in the present study was closer to that of Pekin ducks (Kim et al., 2012b). In contrast, several other researchers have reported much higher pH values for duck meat from A44 and A55 strains (6.0 and 6.4, respectively). However, the breed × sex interaction (p=0.04) was found to be significant regarding the pH value of duck meat. The pH value is one of the most important factors affecting meat quality traits because it has a direct effect on denaturation of meat proteins, influencing tenderness and water holding capacity and meat color (Hamoen et al., 2013). In our findings, pH value did not seem to have a positive effect on water holding capacity as a significant difference was found in cooking loss values between KND (32.65%) and CD (37.04%). Additionally, the sex and breed × sex interaction had significant effects on cooking loss values, in order of significance. Male ducks showed higher cooking loss values compared with female birds (p<0.05; Table 2). The shear force values of the duck breast meat were comparable between the breeds and sexes ( Table 2). The shear force values of duck meat tested in this study was similar to that observed by Muhlisin et al. (Table 2). In this regards, L*-value was higher (p<0.05) in CD and male duck meats compared to their counterparts. In addition, b*-value was higher in the meat from male ducks compared to that from female ducks, but comparable between the breeds. Joo et al. (2013) explained the relationship between L*-value and water holding capacity of meat. With lowered water holding capacity, a higher loss of myoglobin and a greater reflection of light at the meat surface result in a higher L*-value. The b*-value can be related to L*-value since intracellular fat content affects the increase in b*-value and has a positive relationship with L*-value (Sarries and Beriain, 2006). This explanation agrees with our findings regarding the significant difference observed in the L*-value between breeds and sexes. However, since there was no significant difference in the a*-value, which is known to be affected by the myoglobin content (Quevedo et al., 2013), the reflection of light at the meat surface and b*-value of meat must have contributed a greater proportion towards the higher L*-values observed in the current study. Bioactive compounds No scientific publications that compare the bioactive compounds of different breeds of ducks are available. Only a few studies have reported the presence of these endogenous compounds in meat from other species. Hence, these studies were considered in the following section to put our current findings into context. The contents of creatine, anserine, and L-carnitine in duck breast meat were affected by breed and sex (Table 3). Creatine performs an important role in the energy metabolism of skeletal muscle (Wyss and Kaddurah-Daouk, 2000). Creatine is absorbed into body from meat and meat products after consumption, and it increases muscle power and performance (Schmid, 2009). A strong effect of the breed and sex on the creatine content of duck breast meat was observed in the present study; KND and female birds showed significantly higher creatine contents than their counterparts (Table 3). A previous study showed similar findings regarding the creatine content of Korean native chickens (KNC); female KNC had significantly higher creatine content than male KNC (Jayasena et al., 2015). In contrast, Jung et al. (2013) revealed that sex did not influence the creatine content of KNC meat, but influenced the line of KNC (p<0.0001). Carnosine and anserine are dipeptides composed of βalanine and L-histidine and anserine is derived from carnosine (Hipkiss, 1998;Stenesh and Winnick, 1960). These compounds have pH buffering, antioxidative, and antiaging roles. In addition, meat and meat products are the main dietary source of carnosine and anserine for humans (Schmid, 2009). Comparable carnosine contents were found (p>0.05) between the two breeds and between male and female birds in the current study (Table 3). In contrast, Jayasena et al. (2014b) reported that the carnosine content of KNC meat was significantly higher than that of commercial broiler (CB) meat (p<0.05). Marlin et al. (1989) and Mateescu et al. (2012) demonstrated that carnosine content was not related to sex of the animal. However, Jung et al. (2013) demonstrated a sex effect on the carnosine content of KNC meat; female birds had significantly higher carnosine contents than did male birds. The present study showed that the breed, sex, and breed × sex interaction significantly affected the anserine content of duck breast meat, in order of significance (data not shown). In this regards, KND and female ducks showed higher anserine contents in the breast meat (49.18 and 46.64 mg/100 g, respectively) compared with CD and male ducks (36.72 and 39.27 mg/100 g, respectively). This is in well agreement with the results of Jayasena et al. (2014b) who observed that the anserine content of KNC meat was significantly higher than that of CB meat (p< 0.05). However, Jung et al. (2013) found that the anserine content of KNC meat was also not affected by the bird sex. Peñafiel et al. (2004) explained that carnosine content has a positive correlation with testosterone. In addition, duck meat contained more anserine than carnosine, irrespective of the breed and sex, which is in agreement with previous findings that anserine was the principal histidine dipeptide in poultry meat (Abe and Okuma, 1995; Jayasena et al., 2014a). The anserine contents of meat are governed by muscle type, species, breed, gender, age, and breeding (Abe and Okuma, 1995; Chan and Decker, 1994; Jayasena et al., 2014a). Hence, the higher anserine content of KND meat compared to that of CD meat may be attributed to breed effect. Betaine acts as an osmolyte to preserve osmotic equilibrium and also interacts with fat metabolism resulting in fat reduction (de Zwart et al., 2003;Fernández et al., 1998). According to the findings of the current study, the betaine content of duck breast meat was similar (p>0.05) between KND (4.06 mg/100 g) and CD (4.04 mg/100 g) and between male ducks (3.90 mg/100 g) and females (4.21 mg/100 g; Table 3). KNC had a betaine content of 3.55-5.05 mg/100 g (Jayasena et al., 2014a). In contrast, Jayasena et al. (2014b) revealed that the betaine content of KNC meat was significantly lower than that of CB meat (p<0.05). L-carnitine combines with long chained fatty acids forming L-carnitine esters and fat combustion takes place though β-oxidation in mitochondria (Schmid, 2009). According to our results, L-carnitine content of duck breast meat was influenced both by the breed and sex, in order of significance (data not shown). The L-carnitine contents of KND and CD were 8.33 mg/100 g and 6.76 mg/100 g whereas those of male and female ducks were 6.83 mg/100 g and 8.26 mg/100 g, respectively (Table 3). Similarly, L-carnitine content of KNC meat was significantly higher than that of CB meat (Jayasena et al., 2014b). In general, KNC had higher bioactive compound contents (Jayasena et al., 2014a; Jung et al., 2013), except betaine and L-carnitine, compared to KND. This confirms that bioactive compound content of meat is dependent on the animal species. Table 4 shows that the carnosine and anserine contents were positively related (0.04 and 0.81, respectively) to the breast weight of KND and negatively related (-0.40 and -0.18, respectively) to that of CD. In addition, the betaine, L-carnitine, and creatine contents were related to the breast weight of KND compared with that of CD (Table 4). Conclusion KND had a higher carcass weight and a lower cooking loss value compared to CD. Furthermore, the KND contained higher levels of creatine, anserine, and L-carnitine than CD. The content of same compounds was higher in female ducks than in males. The findings of the present study are useful to disseminate the information regarding the availability and amounts of bioactive compounds in duck meat, particularly in KND meat. However, further studies on endogenous compounds in duck meat from genetically-proven KND such as Woorimatori TM and CD should be investigated.

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2016-05-04T20:20:58.661Z

2015-12-26T00:00:00.000Z

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The effects of water and non‐nutritive sweetened beverages on weight loss and weight maintenance: A randomized clinical trial Objective To evaluate the effects of water versus beverages sweetened with non‐nutritive sweeteners (NNS) on body weight in subjects enrolled in a year‐long behavioral weight loss treatment program. Methods The study used a randomized equivalence design with NNS or water beverages as the main factor in a trial among 303 weight‐stable people with overweight and obesity. All participants participated in a weight loss program plus assignment to consume 24 ounces (710 ml) of water or NNS beverages daily for 1 year. Results NNS and water treatments were non‐equivalent, with NNS treatment showing greater weight loss at the end of 1 year. At 1 year subjects receiving water had maintained a 2.45 ± 5.59 kg weight loss while those receiving NNS beverages maintained a loss of 6.21 ± 7.65 kg (P < 0.001 for difference). Conclusions Water and NNS beverages were not equivalent for weight loss and maintenance during a 1‐year behavioral treatment program. NNS beverages were superior for weight loss and weight maintenance in a population consisting of regular users of NNS beverages who either maintained or discontinued consumption of these beverages and consumed water during a structured weight loss program. These results suggest that NNS beverages can be an effective tool for weight loss and maintenance within the context of a weight management program. Introduction There is continued controversy regarding the potential benefit of non-nutritive sweeteners (NNS) for body weight management (1)(2)(3)(4)(5)(6)(7). Some observational studies have reported a positive association between NNS consumption and BMI and weight gain over time (1,2,8), questioning the benefit of NNS for weight management. Observational studies, however, are unable to establish cause and effect. Randomized trials comparing foods and beverages sweetened with NNS versus caloric sweeteners have generally found that risk of weight gain is reduced among subjects consuming NNS (9)(10)(11). There have been relatively few long-term (1 year) randomized tri-als of NNS for weight loss and maintenance (12)(13)(14). In particular, there have been few studies comparing beverages sweetened with NNS to water (13,15,16), which is the recommended beverage for maintaining good health (17,18). Because of the relative paucity of data examining the long-term effects of NNS consumption on body weight management, the 2015 Dietary Guidelines Advisory Committee recently concluded that there is insufficient evidence to recommend the use of low-calorie sweeteners as a strategy for long-term weight loss and weight maintenance (17). The Committee recommended that further prospective research was needed to establish the effects of low-calorie sweeteners on body weight and other health outcomes. This article reports data from a year-long trial comparing beverages sweetened with NNS to water as part of a behavioral weight management program consisting of 12 weeks of active weight loss and 40 weeks of weight maintenance. Results from the 12-week weight loss phase of this trial were published previously and showed nonequivalence, with the NNS group showing greater weight loss at 12 weeks than the water group (19). Methods Participants Blue Chip Recruiting (www.bluechiprecruiting.ca) recruited participants from the general population through the use of flyers, e-mails, and other advertisements (e.g., radio). Of the 506 applicants screened, 308 subjects were enrolled in the trial between October 2012 and April 2013 at The University of Colorado Denver (n 5 151, in four cohorts) and Temple University (n 5 157, in five cohorts), see Figure 1. Participants were male and female, ages 21 to 65, BMI 27 to 40 kg/m 2 , representing a range of races and ethnicities (Table 1). Of the 308 participants enrolled, five withdrew from the trial prior to the start of the study; 303 participants began treatment. There were no significant differences in attrition by site. Individuals were initially screened over the phone or through an online application and potentially eligible subjects were then screened in person. Eligibility requirements included being weight stable within 10 pounds during the past 6 months, participating in no more than 300 min of physical activity weekly, drinking NNS beverages at least three times per week, and be willing to discontinue NNS beverages if randomized to the water group. Regular NNS users were chosen to ensure exposure to NNS during the trial as many people do not like the taste of NNS and selecting non-users could limit intake reducing the ability to detect effects of NNS beverages on body weight regulation. Women who were lactating or pregnant during the previous 6 months or who were planning on becoming pregnant were excluded. Individuals with diabetes, CVD, and uncontrolled hypertension (as well as other diseases potentially interfering with weight loss, e.g., gastrointestinal or thyroid disease) or who used medications affecting weight and metabolism were excluded. Eligible participants required physician approval stating that the nutrition and exercise requirements were not contraindicated and that they were in good general health. The study was approved by the Temple University IRB and the Western IRB at the University of Colorado site. All participants provided informed consent. Study design This was a 1-year equivalence trial comparing beverages sweetened with NNS to water as part of a behavioral weight management program that included 12 weeks of weight loss followed by 40 weeks of weight maintenance. A computer-generated randomization schedule assigned participants, within each site, to either the NNS beverage or water treatment arms stratified by sex, to ensure an equal distribution of women and men in each treatment group. The study protocol specified preplanned data analyses on the primary outcome of weight loss at 12 weeks (weight loss period) and at the end of 1 year (weight loss maintenance period). Intervention Weight loss. For the 12-week weight loss intervention, all participants received a comprehensive cognitive-behavioral weight loss intervention called The Colorado Weigh (20) involving weekly, 60-min instructional classes. Additional details regarding The Colorado Weigh classes and the weight loss intervention are described elsewhere (19). Weight loss maintenance. All participants attended nine monthly, 60-min group meetings led by registered dieticians or clinical psychologists as part of The Colorado Weigh (20). Participants attended group meetings stratified by treatment (NNS or water) group. Participants were weighed at each monthly meeting. Participants were advised to consume 25% to 35% of calories from fat (using a fat gram counter and total calorie guidebook) and to include 6 days of unsupervised exercise per week in order to meet the weight loss maintenance recommendation of 60 min of moderate activity daily (21). Participants in both treatment groups received the same curriculum, with the only difference being discussion regarding the type of beverages they were instructed to consume. Adherence to the treatment was assessed by study dietitians based on food, beverage, and physical activity logs as well as monitoring weight loss. Daily energy intake targets were individualized and calculated as each participant's estimated resting metabolic rate (RMR) 3 1.6 physical activity level (PAL). Energy intake targets were adjusted to maintain weight based on each individual's PAL. Physical activity was assessed using a Body Media armband activity tracking device (Manufacturer: Body Media, Model AB155) for 1 week at baseline and during weeks 4, 12, 24, 36, and 52. NNS beverage group. Participants randomized to the NNS beverage group were asked to consume at least 24 fluid ounces (710 ml) of NNS beverages per day during the entire year-long trial, with unrestricted water consumption. Premixed beverages containing <5 kcal per 8 ounce serving (237 ml) and containing NNS qualified as NNS beverages. Water group. Participants randomized to the water group were asked to consume at least 24 fluid ounces (710 ml) of water per day during the entire year-long trial and to refrain from NNS beverage consumption. They were instructed to not add NNS (e.g., aspartame-NutraSweet V R or Equal V R ; sucralose-Splenda V R ; or stevia-Truvia V R ; as well as diet creamers) to beverages such as coffee or tea. However, they were permitted to consume foods containing NNS (e.g., artificially sweetened gum, candies, cookies, gelatin, pudding, ice cream, yogurt), although they were not instructed to do so as part of the weight loss program. Participants were given four manufacturer coupons monthly (from The Coca-Cola Company, PepsiCo and Dr. Pepper Snapple Group), redeemable for a monthly supply of NNS beverages or bottled water. Participants were asked to record their daily beverage intake using paper logs. This information was used to assess treatment adherence. While all participants were encouraged to consume only non-caloric beverages as part of the behavioral treatment program, they were allowed to consume any beverages as long as they remained compliant with their required intake of either 24 ounces of NNS beverages or 24 ounces of water daily. Measurements All assessments, except for height, were conducted at baseline, 12 weeks (post-weight loss phase) and 52 weeks (post-weight loss maintenance phase). Height was measured to the nearest 0.1 cm at the screening visit prior to baseline and at 52 weeks with a wallmounted stadiometer. Body weight was measured to the nearest 0.1 kg on a digital scale. Waist circumference, measured at the top of the iliac crest, was determined based on two consecutive measures within 0.5 cm. Blood pressure (while seated) was recorded as the average of two measures. Standard venipuncture method was used to collect fasting blood samples for lipid and glucose measurements. Urine was provided for measurement of urine osmolality. Additional methodological details are described elsewhere (19). Participants completed questionnaires at baseline and at 12, 24, 36, and 52 weeks to assess changes in perceived hunger (using a 100 mm visual analog scale anchored at "not at all hungry" and "extremely hungry"). Beverage treatment adherence was determined from daily beverage logs collected monthly. Participants were compensated at intervals for meeting assessment requirements at weeks 12, 24, 36, and 52 (total compensation 5 $340). Power of the study. The primary outcome addressed in this report is the change in body weight during the 1-year trial. The study was designed as an equivalence trial with the hypothesis that there would be no clinically meaningful difference in weight between those consuming NNS beverages or water. The bounds of equivalence for between-group differences at 1 year of weight loss were pre-specified at 62.2 kg. Assuming the true difference was 0.73 kg (1/3 of the equivalence margin) and common SD of 4.2 kg, a sample size of 63 per arm was required using two, one-sided ttests to ensure at least 80% power with an alpha level of P < 0.05 to establish equivalence. Statistical analysis. Intention-to-treat (baseline observation carried forward (BOCF)) was the primary analysis used for assessing weight loss using monthly body weights as the dependent variable. In a secondary analysis we only examined participants who completed the entire 1-year trial. The primary outcome measure was change in body weight over the 1-year period. The primary hypothesis was that NNS beverage and water treatments would be equivalent with upper and lower bounds of equivalence set at 62.2 kg. The value chosen for body weight difference would not be clinically meaningful. The mean and the upper and lower 90% confidence limits for the difference in weight loss maintenance between the treatment groups would have to be within 62.2 kg in order to be considered equivalent. Other weight-related outcomes analyzed included weight change from the point of maximum weight loss until the end of 1 year and the percentage of participants who lost at least 5% of total body weight after 1 year. A sensitivity analysis for weight loss differences between treatment groups was conducted using several methods: a linear mixed model, multiple imputation, ANCOVA and two independent t-tests (or v 2 when appropriate). All methods showed consistent results. Reported here are the t-test results (two one-sided t-tests; the standard approach for evaluating equivalence (22)) and 90% confidence intervals. This model yielded the most conservative result (i.e., least likely to show a difference) among those tested in the sensitivity analysis. Linear mixed effects models were used to analyze secondary outcomes (waist circumference, systolic blood pressure, blood measures, urine osmolality, hunger, and physical activity) which consisted of classification variables of time (baseline, 52 weeks), group (NNS or water) and their interaction term as fixed effects and compound symmetry covariance. Within-and between-group contrasts were tested under this model. Between-site outcomes were also tested. Results Of the 303 subjects who began treatment, 222 or 73% completed the 1-year trial. Compliance with the beverage consumption prescription at 52 weeks (24 ounces/day of NNS beverages or water) was high based on beverage logs: 98.1% and 97.8% in the NNS and water groups, respectively. Partial compliance (consumption of >0 and <24 ounces/day of water or NNS beverages) was 0.7% and 0.9% for the NNS and water groups, respectively, and non-compliance (consumption of 0 ounces/day) was 1.2% and 1.3% for the NNS and water groups, respectively. These values represent the mean percentage of total study days subjects met the different compliance criteria. Participants in the water and NNS groups attended 84.5 6 23.7% (SD) and 85.1 6 23.1% of the instructional sessions, respectively. The means were not different (P 5 0.8262). Baseline observation carried forward analysis includes all those participants who dropped out of the study in the analysis. This analysis mimics the clinical setting. Completer analysis includes only participants who completed 52 weeks of the trial. Although equivalence cannot be established, participants lost more weight in the non-nutritive sweetener (NNS) group as compared to the water group. All analyses were completed using a Sattherwaite two-sample t-test. All values are mean 6 SD unless otherwise noted. Statistically significant values (P < 0.05) are shown by an asterisk (*) and statistically significant P values are shown in bold. Obesity Diet Beverages and Weight Loss Peters et al. Water and NNS treatments were non-equivalent in this trial. Results using the Sattherwaite two-sample t-test showed the NNS treatment was superior to water for weight loss at both 12 weeks (19) and 1 year. Subjects in both treatment groups achieved and maintained significant weight loss over the 1-year trial ( Table 2 shows BOCF and completer analysis). Maximum mean weight loss occurred at week 20 in the water group (5.5 kg) and at week 28 in the NNS group (8.6 kg) (Figure 2a). Both groups regained weight after reaching the maximum weight loss although the rate of gain was significantly less (P < 0.001) for the NNS group (Figure 2b), which also met the weight maintenance definition of less than 3% weight regain (23). Nearly 19% more subjects in the NNS group lost at least 5% of their body weight from baseline to week 52 compared to the water group (Figure 3). There were no significant between-site differences in weight loss or weight maintenance outcomes (P 5 0.4452). Treatment related effects on blood chemistries, subjective hunger, and physical activity time are shown in Table 3. Waist circumference decreased in both groups with the NNS group losing significantly more girth compared to the water group. Systolic blood pressure was reduced at 52 weeks in the NNS group and was significantly different than in the water group which saw no change from baseline. Both groups experienced significant declines in total cholesterol, LDL cholesterol and triglycerides and increases in HDL cholesterol as a function of weight loss although the difference between groups was only significant for triglycerides (P < 0.001). Urinary osmolality did not change with treatment and was not different between groups. Subjects in the water group reported feeling significantly more hungry at 52 weeks compared to baseline which was different than the NNS group which reported no increase in subjective hunger. Total amount of time spent engaging in moderate and vigorous activity increased similarly for both treatments over the year. Discussion This 1-year randomized clinical trial provides evidence that NNS beverages may be an effective tool to aid in weight loss and maintenance, among regular users of NNS beverages, when used as part of a behavioral weight loss treatment program. In this equivalence trial design, when compared to the most commonly recommended beverage for good health, water (17,18), NNS beverages were shown to be non-equivalent and were superior for both weight loss (19) and maintenance. These findings are important as there continues to be uncertainty about the benefit of NNS for weight management based largely on observational studies showing associations between NNS consumption, obesity and weight gain (1,2,8). In addition, it has been suggested, based on some animal studies, that NNS promote obesity by interfering with normal mechanisms of energy balance through dissociating the link between sweet taste and metabolizable energy (3,24). Results of the present trial are not consistent with the findings from observational studies in humans or studies in animals. The present results are consistent with the few other published longterm human trials that evaluated NNS for weight loss (12,15). In a prospective randomized trial, Blackburn et al. found that people with obesity in a weight loss program using NNS food and beverage products lost more weight and maintained a greater weight loss over 2 years compared to subjects not using NNS (12). Tate et al. (2012) conducted a 6-month randomized trial in people with obesity and found greater weight loss over 6 months and a greater likelihood of achieving a 5% weight loss in participants drinking beverages with NNS compared with participants in an attention control group. There was no difference in the likelihood of achieving a 5% weight loss between participants in the water group versus the control or between the water group versus the NNS group. Observational data from subjects in the National Weight Control Registry indicate that NNS beverages and foods are commonly used as tools to help maintain weight loss among individuals who maintained a weight loss of at least 30 pounds for at least 1 year (25,26). The reason for the difference between results from randomized trials and observational studies in humans cannot be determined from these data. It is possible that results from observational studies are due to reverse causality whereby overweight individuals may choose to consume NNS beverages to reduce their risk of weight gain (9,27). Alternatively, residual confounding may be an issue in cases where insufficient factors about subject characteristics and behaviors were adjusted for in the data analysis (27,28). Furthermore, there may be differences in cognitive behavior between subjects in our study and free living subjects not enrolled in a weight loss program regarding how they use NNS beverages in the context of their diet. In the present study, subjects were in a program designed to teach them healthy behaviors that promote weight loss and maintenance. The observational studies included all users of NNS regardless of participation in any formal weight loss program (10). It is plausible that the effects of NNS on weight loss might be greater when people are actively trying to lose weight in a formal behavior change program compared to when NNS are simply used as a dietary substitute for regular sugar. For example, some people (e.g., those not intentionally focused on losing weight) might cognitively compensate for the absence of energy in the NNS beverages by intentionally consuming more solid food (11) which would mitigate weight loss. It is not possible from the present data to explain why the NNS group lost more weight than the water group despite receiving identical weight loss instruction and beverage interventions that both contained zero calories. Physical activity was measured objectively and was not different between treatments. Both groups increased moderate and vigorous physical activity to the same extent over the course of the study as measured by arm band accelerometers. Caffeine intake was not different between treatments such that potential effects on resting energy expenditure of caffeine withdrawal among participants in the water group cannot explain the smaller weight loss observed in that group. Subjects in the water group did, however, report increased hunger relative to the NNS group which reported no increase in hunger from baseline, consistent with previous data from short-term studies (29). It is possible that, in the water group, limiting access to sweetness in beverages may have promoted a desire to seek sweetness from other aspects of the diet, perhaps to achieve some "reward homeostasis" (30), thereby leading them to consume more sweet foods (31) resulting in greater energy intake and less weight loss. In addition, subjects in the water group were asked to make two changes in their drinking habits, both cessation of NNS consumption and increasing water consumption, which potentially presented a larger behavioral challenge than the NNS group that only had to adhere to twice daily consumption of NNS beverages. Greater weight loss among NNS users in the present study argues against effects of NNS to stimulate consumption of sweet foods or high-energy foods (as has been suggested (1)) compared to the effects of water. Other studies have also found no increase in consumption of sweet foods among consumers of NNS (32,33). Treatment effects on blood lipids were consistent with those expected based on the degree of weight loss for each group. A recent report has suggested that NNS may adversely affect gut microflora in a way that impairs glucose tolerance and promotes diabetes (34). In the present study there was no change in fasting blood glucose after 52 weeks of NNS consumption. There were also no between group differences in fasting blood glucose and values were in the clinically normal range for both treatment groups. These results do not support the notion that NNS hinders weight loss by disrupting normal appetite regulation and glucose homeostasis (3,24,34), at least within the context of a yearlong weight loss program. This study has some limitations. First, only people with overweight and obesity who were regular NNS beverage users were studied and the effects in normal weight and NNS-na€ ıve subjects may be different. Second, subjects were actively trying to lose weight using a formal behavior management program and therefore may not represent effects on weight in people not enrolled in such a program. Conclusion In conclusion, among participants enrolled in a comprehensive weight loss program, regular users of NNS beverages who were asked to consume NNS beverages lost significantly more weight, and maintained significantly greater weight losses, over 1 year than subjects asked to stop NNS beverage consumption and consume water alone. These results provide support for the use of NNS beverages as a tool to help with weight loss and maintenance.O

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2019-04-01T13:12:11.507Z

2015-04-01T00:00:00.000Z

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Responses of Hydroponically Grown Common Bean Fed with Nitrogen-free Nutrient Solution to Root Inoculation with N2-fixing Bacteria To date, few attempts have been made to assess the impact of Rhizobium inoculation on N2 fixation and plant yield in soilless cultivations of common bean. In the present study, common bean (P. vulgaris L.) grown on an inert medium (pumice) was inoculated with either Rhizobium tropici CIAT899 or a commercial product containing a mix of N2-fixing bacteria, specifically rhizobia, and Azotobacter sp. The plants treated with both inoculants were supplied with nitrogen (N)-free (0% N) nutrient solution (NS) throughout the cropping period. A third treatment with non-inoculated plants, which were supplied with a standard (100% N) NS was applied as a control. Inoculation with R. tropici significantly increased the total number of root nodules (80 nodules per plant on average) in comparison with the other two treatments (nine nodules per plant on average). The supply of N-free NS restricted markedly both total plant biomass and pod yield, whereas the inoculation with R. tropici mitigated this effect. The aboveground tissues of plants fed with N-free NS contained appreciably less N than those fed with standard solution when they were inoculated with the commercial inoculant (1.7 vs. 29 mg·g dry weight, respectively). The shoot total N concentration 45, 65, and 90 days after transplanting (32, 31, and 29 mg·g dry weight, respectively) was not reduced by the supply of N-free NS when the plants were inoculated with R. tropici. This finding indicates that, at least from the first sampling date onward, the tissue N level was not a limiting factor for growth and yield in plants inoculated with R. tropici. The supply of N-free NS restricted appreciably the potassium (K), magnesium (Mg), and zinc (Zn) levels in the aboveground plant biomass, regardless of inoculation treatment. The impaired growth and yield in plants fed with N-free NS and inoculated with R. tropici is ascribed to both a N shortage at early growth stages and a reduced K uptake aimed at electrochemically balancing the anion-to-cation uptake ratio under conditions of no external NO3 – supply. Root inoculation of legumes with efficient nodulating bacteria of the genus Rhizobium aims to enhance biological N fixation and increase crop yield and quality. Legume inoculation with N2-fixing bacteria is an old practice in agriculture, because Rhizobium inoculants have been used since the 19th century (Catroux et al., 2001; Stephens and Rask, 2000). Biological N fixation by common bean (P. vulgaris L.) in the field is often low compared with that of other legumes (Remans et al., 2008). Nevertheless, several investigators showed that rhizobial inoculation of common bean can enhance root nodulation and yield (Graham, 1981; Hardarson, 1993; Thies et al., 1991). Older studies (Singleton and Tavares, 1986) have indicated many factors that may limit the efficacy of rhizobial inoculation and one of the main limiting factors is the presence of highly competitive indigenous Rhizobium populations in the soil (Segovia et al., 1991; Thies et al., 1991). Nevertheless, very little research work has adequately addressed the impact of Rhizobium inoculation on N2 fixation and yield performance in soilless cultivation of common bean. Jebara et al. (2001) inoculated five common bean lines grown in two hydroponic systems (gravel in pots and aerated nutrient solution in bottles)with eitherRhizobium tropiciCIAT899 or native rhizobia from Tunesia using both saline and non-saline nutrient solutions. This research revealed that strain · line interactions should be considered for selecting the legume most adapted to salinity and that the aerated solution system is efficient for selecting highly efficient rhizobial symbioses. In another study, Zaman-Allah et al. (2007) investigated the effect of different rhizobial strains and phosphorus (P) supply in bean crops grown in a hydroponic system and found that inoculation with suitable rhizobia with a supply of additional P can improve symbiotic N2 fixation and yield in common bean. Nevertheless, to our best knowledge, the effects of rhizobia inoculation on common bean grown in N-free soilless media without N supply have not been reported in the international peer-reviewed literature to date. Soilless cropping systems often start with a ‘‘microbiological vacuum,’’ lacking a diverse and competitive microflora (Postma et al., 2008), and thus competitiveness from indigenous microorganisms is not expected when inoculating soilless bean crops with rhizobia. Consequently, rhizobial inoculation in soilless bean crops may be more effective than in soil-grown crops in terms of both nodulation and yield. In the present article, common bean grown hydroponically on pumice was inoculated with Rhizobium tropici strain CIAT899 or a commercial inoculum containing a mixture of rhizobia sp. and Azotobacter sp. The aim of this research was to compare the nodulation efficiency of these inoculants and to test whether biological N2 fixation is capable of covering the plant N requirements of common bean grown hydroponically when no inorganic N is provided through nutrient solution to the plants. The Rhizobium tropici strain CIAT899 was selected because previous research proved its efficiency in nodulating roots of common bean in soil-based production systems (Martinez-Romero et al., 1991). The commercial mix was selected because previous reports highlighted the potential of inoculating legumes with a combination of rhizobia and Azotobacter (Rodelas et al., 1999). Polymerase chain reaction (PCR)-sequencing analysis was used to test whether the applied inoculants successfully colonized the roots of common bean. Furthermore, root nodulation, plant biomass production, pod yield, N status in roots and shoot, and tissue nutrient concentrations were determined to test the impact of inoculation on growth, yield, and plant nutrient status in the hydroponically grown common bean plants. Materials and Methods Plant material and growth conditions. The experiment was conducted in a glasshouse at the Agricultural University of Athens (Greece) from 18 Oct. 2011 (date of sowing) to 27 Jan. 2012. Common beans (Phaseolus vulgaris L., cv. Contender) were Received for publication 24 Nov. 2014. Accepted for publication 25 Jan. 2015. This work was supported by the European Commission within the project ‘‘Legume-supported cropping systems for Europe—LEGUME FUTURES’’ (grant agreement no. 245216 CP-FP), whichwas carried out under the EFP7Cooperation Theme ‘‘Food, Fisheries and Biotechnologies.’’ P.P.M. Iannetta is also supported by the Scottish Government. We thank Dr. Jean-Jacques Drevon (INRA) for providing the R. tropici CIAT899 and Prof. E.K. James, Laura Lopez (The James Hutton Institute, U.K.), and Prof. Peter Young (Uni. York, U.K.) for their support and guidance with the isolation and identification of nodule bacteria. To whom reprint requests should be addressed; e-mail [email protected]. HORTSCIENCE VOL. 50(4) APRIL 2015 597 sown in seedling trays filled with pumice granules (0 to 8 mm diameter) and placed in a temperature-controlled (23 to 26 C) incubation chamber. The seedlings were transplanted to 12 closed-loop hydroponic circuits (experimental plots) 15 d after sowing, when they were at the two-cotyledon stage. Each loopwas isolated and suppliedwith a separate NS. Each circuit comprised one channel (3.0 · 0.2 · 0.3 m, length · width · height), which accommodated three bags (1 · 0.2 · 0.2 m, length · width · height) filled with pumice. Four seedlings were planted in each bag and this resulted in 12 plants per circuit. Three treatments (described in more detail below) were applied in a randomized complete block experimental design with four plots (replicates) per treatment. Immediately after transplanting, seedlings in two of the three treatments were inoculated either with a liquid culture (10 mL/plant) containing Rhizobium tropici, strain CIAT899 (10 colony-forming units/mL) orwith a commercial product. The R. tropici inoculum was provided by Dr. Jean-Jacques Drevon (INRA, France). The commercial product contained a mix of N2-fixing bacteria, specifically rhizobia and Azotobacter sp. according to the information provided by the manufacturer on the product label (AzoRiz; Humofert, Athens, Greece). These applications were repeated after 6 d. In both cases, the total N concentration in the fresh NS supplied to the plants to compensate for plant uptake was adjusted to zero level [denoted as ‘‘N-free NS (0%N) + R. tropici’’ and ‘‘N-free NS (0%N) + com. mix’’]. The concentrations of macronutrients (mM) in the N-free NS were: K 5.00, Ca 2.50, Mg 1.20, NO3 – 0.00, NH4 + 0.00, SO4 2– 7.20, H2PO4 – 1.80. In the third treatment, the seedlings were not inoculated, and the total N concentration in the NS was adjusted to a standard level for green bean grown hydroponically (standard nutrient solution henceforth denoted as SNS, 100% N, or control treatment). The composition of the SNS is based on an already published recipe for the hydroponic cultivation of common bean in Mediterranean greenhouses (Savvas et al., 2013) with the following macronutrient concentrations (mM): K 5.00, Ca 2.50, Mg 1.20, NO3 – 10.00, NH4 + 1.50, SO4 2– 2.60, H2PO4 – 1.20. The concentrations of micronutrients, i.e., iron, manganese, Zn, copper, boron, and molybdenum, in both the SNS and the N-free NS were 12, 6, 4, 0.5, 20, and 0.5 mm, respectively. The seedlingswithin each circuit were automatically supplied with NS from a dedicated ‘‘supplycistern’’ (i.e., a constant NS level was maintained using a floater) through a drip irrigation system. The level of NS in the supply-cistern was fed by a ‘‘replenishment tank’’ positioned above it, which contained either SNS or Nfree NS, depending on the treatment. Also, and for all treatments, the pH of the NS was adjusted daily to 5.6 to 5.7 by adding appropriate amounts of phosphoric acid. Yield, growth measurements, and mineral composition. Fresh and dry aboveground biomass was recorded 45, 65, and 90 d after transplanting (DAT) by sampling two randomly selected plants per experimental plot on each sampling date. The roots of the plants collected on the mentioned dates were rinsed with water, blotted dry on filter paper, and the number of nodules per plant was recorded. Subsamples of the crushed dried aboveground plant biomass were powdered using a ball mill and passed through a 40-mesh sieve. Total N was determined using an automated Dumas procedure on a Carlo Erba NA 1500 elemental analyzer (Erba Science, U.K.). Mineral nutrients [K, P, magnesium (Mg), Zn] were assayed after ashing dried plant biomass samples in a muffle furnace at 550 C for 5 h and extraction of the ash using 1NHCl. The obtained liquid extract was used to measure K through flame photometry, P colorimetrically as phosphomolybdate blue complex at 880 nm using a spectrophotometer (Eaton et al., 1995) and Mg and Zn by atomic absorption spectrophotometry. Green pods were harvested when they reached marketable size (over 23 cm) to estimate total number of pods/plant and total yield (kg/plant). Pod dry weights per plant were determined by drying representative samples of fresh green pods at 65 C until their mass was stabilized to a constant level. Root nodule bacteria isolation and identification of rhizobial types. Frozen (–80 C) clean healthy root nodules were surface-sterilized [2.5% (v/v) NaClO, 30 s, and rinsed three times in sterile distilled water] and placed on sterile yeast mannitol agar medium (YM; Vincent, 1970) plus congo-red dye [0.0025% (w/v)] contained within a petri dish. The nodule was crushed using sterile tweezers and the exudate spread over the agar surface before incubation (28 C, 24 to 48 h). Single colonies were reisolated by streaking onto a fresh YM-agar plate and prepared for long-term storage by culturing overnight in 3 mL tryptone-yeast media (TY; Beringer, 1974). Equal volumes (0.75 mL) of the culture and 50% (v/v) sterile glycerol were mixed well before freezing in liquid N and storage at –80 C. Isolateswere cultured on liquid TYmedia for 24 h and at log-phase 4 mL was pelleted by centrifugation and the pellet re-suspended in TE buffer (400 mL) containing greater than 800 mm·mL proteinase-K (Sigma #P4850; Signma, Poole, U.K.). After incubation (37 C, 1 h), an equal volume of phenol:chloroform:isoamyl alcohol [25:24:1 (v/v/v); Sigma #P2069] was added and the mixture vortexed (1 min) and centrifuged. The aqueous phase (200 mL) was recovered and combined with 10% (v/v) 3M sodium acetate (NaOAc; pH 5.2) and a 3· volume of chilled isopropanol (Sigma #I9030). The DNA was precipitated by incubation (–80 C, 15min, or –20 C, overnight), pelleted by centrifugation, washed [200 mL/70% (v/v) ethanol], re-centrifuging (2 min), and dried (37 C, 15 min) after removal of the supernatant. The DNA pellets were re-suspended in TE buffer (25 mL). All centrifugation steps were performed at 14,000 rpm and at 4 C. A portion of the 16S ribosomal RNA gene was amplified using the primers Bac27F (Lane, 1991) and Univ1492R (Jiang et al., 2006), according to the manufacturer’s recommendations (Promega #M3175; Promega, Southampton, U.K.) using the following thermal profile: denaturation at 95 C, 2 min; 35 cycles of 94 C 30 s, 52 C 1min, 72 C 1min, and 72 C for 5 min (using a G-Storm GS1 thermal cycler; GRI Ltd., Braintree, U.K.). PCR products ( 1400 bp) were purified using QIAquick Spin columns (Qiagen, Inc., Chatsworth, CA) and sequenced (ABI3730 DNA analyzer with a 36 cm · 48-cm capillary array; Applied Biosystems, by Life Technologies, Paisley, U.K.) using the internal-primer 519R (Lane et al., 1985). The resultant sequences were edited (BioEdit Sequence Alignment Editor Version 7.2; Tom Hall, Ibis Biosciences, Carlsbad, CA) and screened against nucleotide databases using Basic Local Alignment Search Tool (BLASTN, National Center for Biotechnology Information, National Library of Medicine, Bethesda, MD; Altschul et al., 1997) on the National Center for Biotechnology Information web site. Statistical analysis. All data were statistically evaluated by applying analysis of variance using the STATISTICA Version 9.0 software package (StatSoft Inc. 2010, Tulsa, OK). Duncan’s multiple-range test was performed on each of the significant (P < 0.05) variables measured. Standard errors are displayed in graphs to depict statistical significance of the differences between treatment means. Results and Discussion PCR sequencing. PCR-sequencing analysis revealed the presence of R. tropici in all tested plants inoculated with this N2-fixing bacterium, whereas no R. tropici could be isolated in plant roots originating from the other two treatments (Table 1). This result confirms the successful inoculation of P. vulgaris plants with R. tropici in the relevant treatment and is in agreement with previous reports pointing to a high efficiency of R. tropici in nodulating common bean (Hungria et al., 2000, 2003; Mostasso et al., 2002). However, from the nodules of plants inoculated with the commercial mix, no R. tropici or Azotobacter species could be isolated. This finding indicates that either the inoculation technique used to apply the commercial mix failed or the N2-fixing species included in this commercial mix are incapable of colonizing the roots of common bean and form symbiotic associations. Furthermore, the Agrobacterium radiobacter K84 strain was isolated in plant roots from all treatments, including the control in which plants were not inoculated with any N2-fixing bacteria, which indicates that this microorganism does not originate from the inoculation treatments. Because the growing medium (pumice) used to grow bean plants in the present experiment is considered chemically and biologically inert (Postma et al., 2008), A. radiobacter either was present in the seeds of the bean or had been introduced from atmospheric deposition. Nodulation. Inoculation with the R. tropici strain CIAT899 enhanced nodulation compared with both non-inoculation and inoculation 598 HORTSCIENCE VOL. 50(4) APRIL 2015 with the commercial mix (Fig. 1). The total number of nodules per plant in the treatment inoculated with R. tropici was significantly higher (80 nodules per plant, average calculated across all samplings dates) in comparison not only with that recorded in non-inoculated plants (four nodules per plant, average calculated across all samplings dates), but also with the plants inoculated with the commercial mix (six nodules per plant, average calculated across all samplings dates). Previous studies (e.g., Thies et al., 1991; Zaman-Allah et al., 2007) also reported an increase in total number of nodules in legume plants inoculated with R. tropici. According to Singleton and Tavares (1986), a significant increase in nodule number did not always result in a N2-fixation increase, but only when the number of nodules in the inoculated plants was at least 2.5 times higher than in the non-inoculated controls. However, the number of nodules in plants inoculated with R. tropici increased to a much higher order of magnitude than 2.5-fold in the present study. Thus, it is reasonable to assume that inoculation with R. tropici CIAT899 had a positive impact on biological N2 fixation as well. Nodulation in the plants supplied with N-free NS and inoculated with the commercial mix also increased compared with the noninoculated treatment, but a significant difference was found only in one of the three sampling dates (12 vs. five nodules per plant, 65 DAT). As reported byMhamdi et al. (2005), inoculation of P. vulgaris plants grown on sterile gravel with Agrobacterium strains had no effect on the total number of root nodules. In the present study, only A. radiobacter but no N2-fixing bacteria were isolated from the roots of plants inoculated with the commercial mix. Thus, it seems that, in plants inoculated with the commercial mix, the weak increase in the number of nodules per plant was the result of N starvation, which is known to stimulate nodulation (Gan et al., 2004; Li et al., 2009) rather than the result of microorganisms included in the commercial mix. Tissue nitrogen status. The establishment of an efficient symbiosis in bean plants inoculated with the R. tropici strain CIAT899 is further confirmed by the total N concentrations measured in the aboveground tissues of plants inoculated with R. tropici. As shown in Figure 2, the total N concentrations in the shoot of plants fed with an N-free NS and inoculated with R. tropici were not lower than in the non-inoculated plants fed with standard NS (100% N), at least from Day 45 after transplanting and onward. Especially 65 DAT, the total N in the shoot of plants treated with N-free NS and inoculated with the strain CIAT899 was even higher (3.10% vs. 2.73% in dry weight) than in non-inoculated plants treated with standard N levels (100% of the standard recommendation). These results are in agreement with those reported by Hungria et al. (2000), who found similar total N levels in P. vulgaris plants inoculated with R. tropici strain CIAT899 and supplied with N-free NS with those measured in plants supplied with mineral N in a greenhouse trial. On the other hand, the shoots of plants fed with N-free NS in the present study contained less N (17.4 mg·g dry weight, average calculated across all samplings dates) than those fed with SNS when the former were inoculated with the commercial inoculants. However, this is reasonable given the poor root nodulation and the results of PCR analysis, which revealed the absence of N2fixing microorganisms in the roots of plants inoculated with the commercial mix. Yield and biomass production. Despite the sufficiently high N levels in the shoot of plants inoculated with the strain CIAT899 and fed with an N-free NS, at least from Day 45 after transplanting and onward, both the total biomass production (Fig. 3) and the green pod yield (Table 2) were appreciably lower than in the non-inoculated plants supplied with 100% N. However, the similar shoot N concentrations indicate that the appreciable suppression of growth (221.43 g/plant vs. 32.31 g/plant 90 DAS) and pod yield (107.1 g/plant vs. 18.51 g/plant) in P. vulgaris plants inoculated with R. tropici in comparison with the control was not associated with deficient tissue N levels throughout the cropping period, despite their cultivation on an inert medium lacking any N and the continuous supply of N-free NS. This was surprising and a likely explanation is that the plant biomass was restricted as a result of a shortage of N in the early growth stage of common bean, before R. tropici bacteroids were developed enough to be capable of providing sufficient N to the plants. The occurrence of a severe N shortage in the plants treated with N-free NS despite inoculation with R. tropici CIAT899 during the first 3 to 5 weeks after inoculation is reasonable. Indeed, it is well known that a period of 3 to 5 weeks is needed between infection with N2-fixing microorganisms and the onset of N2 fixation, and during this period, the plant has to provide N both to itself and to the bacteroids (Kucey, 1989; Marschner, 1995). Tissue mineral composition. The supply of N-free NS restricted appreciably the K, Mg, and Zn levels in the aboveground plant biomass, regardless of inoculation treatment. Only on the third sampling date did the shoot Mg concentration in the plants treated with N-free NS and inoculated with the commercial mix (3.87 mg·g) tend to approach that of the control plants (4.41 mg·g). K and Mg, together with Ca (which was not measured), constitute the three major nutrient cations for plants. Both K and Mg are actively Fig. 1. Root nodulation in a hydroponic crop of common bean inoculated either with R. tropici CIAT 899 or with a commercial mix containing various rhizobia and Azotobacter sp. strains or non-inoculated. The inoculated plants were supplied with nitrogen (N)-free nutrient solution, whereas the non-inoculated plants were fed with a nutrient solution containing 100% of the standard recommended N level. SNS = standard nutrient solution.Vertical bars depict ± SEs (n = 4). Table 1. Polymerase chain reaction-sequencing analysis of root nodule bacteria isolated from a hydroponic crop of common bean inoculated with either R. tropici CIAT 899 or a commercial mix containing various rhizobia and Azotobacter sp. strains or non-inoculated. Treatment BlastN results Percent similarity Rhizobia/Agro-bacterium + 100% N A. radiobacter strain K84 16S ribosomal RNA, complete sequence 99 + 0% N + R. tropici Rhizobium tropici CIAT 899 strain USDA 9030 16S ribosomal RNA, partial sequence/A. radiobacter strain K84 16S ribosomal RNA, complete sequence 99 + 0% N + commercial mix A. radiobacter strain K84 16S ribosomal RNA, complete sequence 99 + The inoculated plants were supplied with N-free nutrient solution, whereas the non-inoculated plants were fed with a nutrient solution containing 100% of the standard recommended nitrogen (N) level. Fig. 2. Aboveground fresh plant biomass in a hydroponic crop of common bean inoculated either with R. tropici CIAT 899 or with a commercial mix containing various rhizobia and Azotobacter sp. strains or non-inoculated. The inoculated plants were supplied with nitrogen (N)-free nutrient solution, whereas the non-inoculated plants were fed with a nutrient solution containing 100% of the standard recommended N level. SNS = standard nutrient solution. Vertical bars depict ± SEs (n = 4). HORTSCIENCE VOL. 50(4) APRIL 2015 599 absorbed through dual transport mechanisms so as to electrochemically balance the cytosol (Marschner, 1995). On the other hand, NO3 – is the major nutrient anion, which accounts for 70% to 80% of the total ion uptake in higher plants (Van Beusichem et al., 1988). Plants supplied with N-free NS cannot take up any NO3 – and thus the total anion uptake is accordingly suppressed. The low NO3 – availability in the roots may signal a marked suppression of K and Mg uptake through the active transport mechanisms localized in the root cells (Mengel and Kirkby, 2001) to maintain electrochemical balance in the cytosol. As reported by Raab and Terry (1994), low NO3 – concentrations in the roots restrict cation uptake because NO3 – anions are needed to counterbalance cations in the xylem. In legumes, N2 fixation results in a net release of H ions (Bolan et al., 1991) and this may entail a further restriction of K andMg uptake by roots in the absence of NO3 – in the root zone aiming at maintaining the electrochemical balance in the root cells. The K levels in the shoot of bean plants treated with N-free NS were markedly lower (15.2 mg·g, average calculated across all sampling dates) than the lower critical level suggested byMills and Jones (1996), in contrast to those of Mg (4.06 mg·g, average calculated across all samplings dates), which were still within the sufficiency range. Also the shoot Zn levels (28.09 mg·g, average calculated across all samplings dates) were clearly below the critical level of sufficiency in the bean plants treated with N-free NS. The supply of N-free NS reduced significantly the shoot P concentration 65 DAT, irrespective of inoculation treatment (Fig. 4). This is attributed to the increased number of nodules at this growth stage in both inoculated treatments, because many studies have shown that nodules are strong sinks for P (Drevon and Hartwig, 1997; Kleinert et al., 2014). Indeed, as reported by Vadez et al. (1996), the P concentration in nodules formed by N2-fixing bacteria is 3-fold higher than in plant tissues. As stated by O’Hara et al. (1988), nutrient deficiencies constitute a major constraint limiting biological N2 fixation and yield in legume crops. Thus, a partial involvement of K and/or Zn deficiency in the appreciable suppression of growth and pod yield in P. vulgaris plants treated with N-free NS is likely. Nevertheless, based on the results of the present study, it is not possible to estimate the relative contribution of N deficiency during the early developmental stage vs. K and Zn deficiencies to growth and yield suppression in bean plants treated with N-free NS and inoculated with R. tropici CIAT899. Legume crops may need to be inoculated only when they are grown in regions outside their centers of diversity or where they have not traditionally been grown or have not been grown for a number of years (Brockwell et al., Table 2. Fresh pod yield and yield components, dry pod yield, and dry matter content (DMC) in a hydroponic crop of common bean inoculated either with R. tropici CIAT 899 or with a commercial mix containing various rhizobia and Azotobacter sp. strains or non-inoculated. Treatment Pod number per plant Fresh pod wt (g/plant) Dry pod wt (g/plant) Mean pod wt (g) DMC (%) 100% N 11.5 ± 0.5 a 107.1 ± 7.5 a 8.6 ± 0.81 a 9.3 ± 0.55 a 8.01 ± 0.2 b 0% N + R. tropici 3.1 ± 0.1 b 18.5 ± 1.1 b 1.7 ± 0.13 b 6.5 ± 0.54 b 9.04 ± 0.3 a 0% N + commercial mixture 1.4 ± 0.4 c 3.8 ± 0.2 c 0.3 ± 0.05 c 2.3 ± 0.37 c 7.53 ± 0.2 b Statistical significance Root inoculation of legumes with efficient nodulating bacteria of the genus Rhizobium aims to enhance biological N fixation and increase crop yield and quality. Legume inoculation with N 2 -fixing bacteria is an old practice in agriculture, because Rhizobium inoculants have been used since the 19th century (Catroux et al., 2001;Stephens and Rask, 2000). Biological N fixation by common bean (P. vulgaris L.) in the field is often low compared with that of other legumes (Remans et al., 2008). Nevertheless, several investigators showed that rhizobial inoculation of common bean can enhance root nodulation and yield (Graham, 1981;Hardarson, 1993;Thies et al., 1991). Older studies (Singleton and Tavares, 1986) have indicated many factors that may limit the efficacy of rhizobial inoculation and one of the main limiting factors is the presence of highly competitive indigenous Rhizobium populations in the soil Thies et al., 1991). Nevertheless, very little research work has adequately addressed the impact of Rhizobium inoculation on N 2 fixation and yield performance in soilless cultivation of common bean. Jebara et al. (2001) inoculated five common bean lines grown in two hydroponic systems (gravel in pots and aerated nutrient solution in bottles) with either Rhizobium tropici CIAT899 or native rhizobia from Tunesia using both saline and non-saline nutrient solutions. This research revealed that strain · line interactions should be considered for selecting the legume most adapted to salinity and that the aerated solution system is efficient for selecting highly efficient rhizobial symbioses. In another study, Zaman-Allah et al. (2007) investigated the effect of different rhizobial strains and phosphorus (P) supply in bean crops grown in a hydroponic system and found that inoculation with suitable rhizobia with a supply of additional P can improve symbiotic N 2 fixation and yield in common bean. Nevertheless, to our best knowledge, the effects of rhizobia inoculation on common bean grown in N-free soilless media without N supply have not been reported in the international peer-reviewed literature to date. Soilless cropping systems often start with a ''microbiological vacuum,'' lacking a diverse and competitive microflora (Postma et al., 2008), and thus competitiveness from indigenous microorganisms is not expected when inoculating soilless bean crops with rhizobia. Consequently, rhizobial inoculation in soilless bean crops may be more effective than in soil-grown crops in terms of both nodulation and yield. In the present article, common bean grown hydroponically on pumice was inoculated with Rhizobium tropici strain CIAT899 or a commercial inoculum containing a mixture of rhizobia sp. and Azotobacter sp. The aim of this research was to compare the nodulation efficiency of these inoculants and to test whether biological N 2 fixation is capable of covering the plant N requirements of common bean grown hydroponically when no inorganic N is provided through nutrient solution to the plants. The Rhizobium tropici strain CIAT899 was selected because previous research proved its efficiency in nodulating roots of common bean in soil-based production systems . The commercial mix was selected because previous reports highlighted the potential of inoculating legumes with a combination of rhizobia and Azotobacter (Rodelas et al., 1999). Polymerase chain reaction (PCR)-sequencing analysis was used to test whether the applied inoculants successfully colonized the roots of common bean. Furthermore, root nodulation, plant biomass production, pod yield, N status in roots and shoot, and tissue nutrient concentrations were determined to test the impact of inoculation on growth, yield, and plant nutrient status in the hydroponically grown common bean plants. Materials and Methods Plant material and growth conditions. The experiment was conducted in a glasshouse at the Agricultural University of Athens (Greece) from 18 Oct. 2011 (date of sowing) to 27 Jan. 2012. Common beans (Phaseolus vulgaris L., cv. Contender) were sown in seedling trays filled with pumice granules (0 to 8 mm diameter) and placed in a temperature-controlled (23 to 26°C) incubation chamber. The seedlings were transplanted to 12 closed-loop hydroponic circuits (experimental plots) 15 d after sowing, when they were at the two-cotyledon stage. Each loop was isolated and supplied with a separate NS. Each circuit comprised one channel (3.0 · 0.2 · 0.3 m, length · width · height), which accommodated three bags (1 · 0.2 · 0.2 m, length · width · height) filled with pumice. Four seedlings were planted in each bag and this resulted in 12 plants per circuit. Three treatments (described in more detail below) were applied in a randomized complete block experimental design with four plots (replicates) per treatment. Immediately after transplanting, seedlings in two of the three treatments were inoculated either with a liquid culture (10 mL/plant) containing Rhizobium tropici, strain CIAT899 (10 9 colony-forming units/mL) or with a commercial product. The R. tropici inoculum was provided by Dr. Jean-Jacques Drevon (INRA, France). The commercial product contained a mix of N 2 -fixing bacteria, specifically rhizobia and Azotobacter sp. according to the information provided by the manufacturer on the product label (AzoRiz; Humofert, Athens, Greece). These applications were repeated after 6 d. In both cases, the total N concentration in the fresh NS supplied to the plants to compensate for plant uptake was adjusted to zero level [denoted as ''N-free NS (0%N) + R. tropici'' and ''N-free NS (0% N) + com. mix'']. The concentrations of macronutrients (mM) in the N-free NS were: K + 5.00, Ca 2+ 2.50, Mg 2+ 1.20, NO 3 -0.00, NH 4 + 0.00, SO 4 2-7.20, H 2 PO 4 -1.80. In the third treatment, the seedlings were not inoculated, and the total N concentration in the NS was adjusted to a standard level for green bean grown hydroponically (standard nutrient solution henceforth denoted as SNS, 100% N, or control treatment). The composition of the SNS is based on an already published recipe for the hydroponic cultivation of common bean in Mediterranean greenhouses (Savvas et al., 2013) with the following macronutrient concentrations (mM): K + 5.00, Ca 2+ 2.50, Mg 2+ 1.20, NO 3 -10.00, NH 4 + 1.50, SO 4 2-2.60, H 2 PO 4 -1.20. The concentrations of micronutrients, i.e., iron, manganese, Zn, copper, boron, and molybdenum, in both the SNS and the N-free NS were 12, 6, 4, 0.5, 20, and 0.5 mm, respectively. The seedlings within each circuit were automatically supplied with NS from a dedicated ''supplycistern'' (i.e., a constant NS level was maintained using a floater) through a drip irrigation system. The level of NS in the supply-cistern was fed by a ''replenishment tank'' positioned above it, which contained either SNS or Nfree NS, depending on the treatment. Also, and for all treatments, the pH of the NS was adjusted daily to 5.6 to 5.7 by adding appropriate amounts of phosphoric acid. Yield, growth measurements, and mineral composition. Fresh and dry aboveground biomass was recorded 45, 65, and 90 d after transplanting (DAT) by sampling two randomly selected plants per experimental plot on each sampling date. The roots of the plants collected on the mentioned dates were rinsed with water, blotted dry on filter paper, and the number of nodules per plant was recorded. Subsamples of the crushed dried aboveground plant biomass were powdered using a ball mill and passed through a 40-mesh sieve. Total N was determined using an automated Dumas procedure on a Carlo Erba NA 1500 elemental analyzer (Erba Science, U.K.). Mineral nutrients [K, P, magnesium (Mg), Zn] were assayed after ashing dried plant biomass samples in a muffle furnace at 550°C for 5 h and extraction of the ash using 1 N HCl. The obtained liquid extract was used to measure K through flame photometry, P colorimetrically as phosphomolybdate blue complex at 880 nm using a spectrophotometer (Eaton et al., 1995) and Mg and Zn by atomic absorption spectrophotometry. Green pods were harvested when they reached marketable size (over 23 cm) to estimate total number of pods/plant and total yield (kg/plant). Pod dry weights per plant were determined by drying representative samples of fresh green pods at 65°C until their mass was stabilized to a constant level. Root nodule bacteria isolation and identification of rhizobial types. Frozen (-80°C) clean healthy root nodules were surface-sterilized [2.5% (v/v) NaClO, 30 s, and rinsed three times in sterile distilled water] and placed on sterile yeast mannitol agar medium (YM; Vincent, 1970) plus congo-red dye [0.0025% (w/v)] contained within a petri dish. The nodule was crushed using sterile tweezers and the exudate spread over the agar surface before incubation (28°C, 24 to 48 h). Single colonies were reisolated by streaking onto a fresh YM-agar plate and prepared for long-term storage by culturing overnight in 3 mL tryptone-yeast media (TY; Beringer, 1974). Equal volumes (0.75 mL) of the culture and 50% (v/v) sterile glycerol were mixed well before freezing in liquid N and storage at -80°C. Statistical analysis. All data were statistically evaluated by applying analysis of variance using the STATISTICA Version 9.0 software package (StatSoft Inc. 2010, Tulsa, OK). Duncan's multiple-range test was performed on each of the significant (P < 0.05) variables measured. Standard errors are displayed in graphs to depict statistical significance of the differences between treatment means. Results and Discussion PCR sequencing. PCR-sequencing analysis revealed the presence of R. tropici in all tested plants inoculated with this N 2 -fixing bacterium, whereas no R. tropici could be isolated in plant roots originating from the other two treatments (Table 1). This result confirms the successful inoculation of P. vulgaris plants with R. tropici in the relevant treatment and is in agreement with previous reports pointing to a high efficiency of R. tropici in nodulating common bean (Hungria et al., 2000(Hungria et al., , 2003Mostasso et al., 2002). However, from the nodules of plants inoculated with the commercial mix, no R. tropici or Azotobacter species could be isolated. This finding indicates that either the inoculation technique used to apply the commercial mix failed or the N 2 -fixing species included in this commercial mix are incapable of colonizing the roots of common bean and form symbiotic associations. Furthermore, the Agrobacterium radiobacter K84 strain was isolated in plant roots from all treatments, including the control in which plants were not inoculated with any N 2 -fixing bacteria, which indicates that this microorganism does not originate from the inoculation treatments. Because the growing medium (pumice) used to grow bean plants in the present experiment is considered chemically and biologically inert (Postma et al., 2008), A. radiobacter either was present in the seeds of the bean or had been introduced from atmospheric deposition. Nodulation. Inoculation with the R. tropici strain CIAT899 enhanced nodulation compared with both non-inoculation and inoculation with the commercial mix (Fig. 1). The total number of nodules per plant in the treatment inoculated with R. tropici was significantly higher (80 nodules per plant, average calculated across all samplings dates) in comparison not only with that recorded in non-inoculated plants (four nodules per plant, average calculated across all samplings dates), but also with the plants inoculated with the commercial mix (six nodules per plant, average calculated across all samplings dates). Previous studies (e.g., Thies et al., 1991;Zaman-Allah et al., 2007) also reported an increase in total number of nodules in legume plants inoculated with R. tropici. According to Singleton and Tavares (1986), a significant increase in nodule number did not always result in a N 2 -fixation increase, but only when the number of nodules in the inoculated plants was at least 2.5 times higher than in the non-inoculated controls. However, the number of nodules in plants inoculated with R. tropici increased to a much higher order of magnitude than 2.5-fold in the present study. Thus, it is reasonable to assume that inoculation with R. tropici CIAT899 had a positive impact on biological N 2 fixation as well. Nodulation in the plants supplied with N-free NS and inoculated with the commercial mix also increased compared with the noninoculated treatment, but a significant difference was found only in one of the three sampling dates (12 vs. five nodules per plant, 65 DAT). As reported by Mhamdi et al. (2005), inoculation of P. vulgaris plants grown on sterile gravel with Agrobacterium strains had no effect on the total number of root nodules. In the present study, only A. radiobacter but no N 2 -fixing bacteria were isolated from the roots of plants inoculated with the commercial mix. Thus, it seems that, in plants inoculated with the commercial mix, the weak increase in the number of nodules per plant was the result of N starvation, which is known to stimulate nodulation (Gan et al., 2004;Li et al., 2009) rather than the result of microorganisms included in the commercial mix. Tissue nitrogen status. The establishment of an efficient symbiosis in bean plants inoculated with the R. tropici strain CIAT899 is further confirmed by the total N concentrations measured in the aboveground tissues of plants inoculated with R. tropici. As shown in Figure 2, the total N concentrations in the shoot of plants fed with an N-free NS and inoculated with R. tropici were not lower than in the non-inoculated plants fed with standard NS (100% N), at least from Day 45 after transplanting and onward. Especially 65 DAT, the total N in the shoot of plants treated with N-free NS and inoculated with the strain CIAT899 was even higher (3.10% vs. 2.73% in dry weight) than in non-inoculated plants treated with standard N levels (100% of the standard recommendation). These results are in agreement with those reported by Hungria et al. (2000), who found similar total N levels in P. vulgaris plants inoculated with R. tropici strain CIAT899 and supplied with N-free NS with those measured in plants supplied with mineral N in a greenhouse trial. On the other hand, the shoots of plants fed with N-free NS in the present study contained less N (17.4 mg · g -1 dry weight, average calculated across all samplings dates) than those fed with SNS when the former were inoculated with the commercial inoculants. However, this is reasonable given the poor root nodulation and the results of PCR analysis, which revealed the absence of N 2fixing microorganisms in the roots of plants inoculated with the commercial mix. Yield and biomass production. Despite the sufficiently high N levels in the shoot of plants inoculated with the strain CIAT899 and fed with an N-free NS, at least from Day 45 after transplanting and onward, both the total biomass production (Fig. 3) and the green pod yield (Table 2) were appreciably lower than in the non-inoculated plants supplied with 100% N. However, the similar shoot N concentrations indicate that the appreciable suppression of growth (221.43 g/plant vs. 32.31 g/plant 90 DAS) and pod yield (107.1 g/plant vs. 18.51 g/plant) in P. vulgaris plants inoculated with R. tropici in comparison with the control was not associated with deficient tissue N levels throughout the cropping period, despite their cultivation on an inert medium lacking any N and the continuous supply of N-free NS. This was surprising and a likely explanation is that the plant biomass was restricted as a result of a shortage of N in the early growth stage of common bean, before R. tropici bacteroids were developed enough to be capable of providing sufficient N to the plants. The occurrence of a severe N shortage in the plants treated with N-free NS despite inoculation with R. tropici CIAT899 during the first 3 to 5 weeks after inoculation is reasonable. Indeed, it is well known that a period of 3 to 5 weeks is needed between infection with N 2 -fixing microorganisms and the onset of N 2 fixation, and during this period, the plant has to provide N both to itself and to the bacteroids (Kucey, 1989;Marschner, 1995). Tissue mineral composition. The supply of N-free NS restricted appreciably the K + , Mg 2+ , and Zn 2+ levels in the aboveground plant biomass, regardless of inoculation treatment. Only on the third sampling date did the shoot Mg 2+ concentration in the plants treated with N-free NS and inoculated with the commercial mix (3.87 mg · g -1 ) tend to approach that of the control plants (4.41 mg · g -1 ). K + and Mg 2+ , together with Ca 2+ (which was not measured), constitute the three major nutrient cations for plants. Both K + and Mg 2+ are actively absorbed through dual transport mechanisms so as to electrochemically balance the cytosol (Marschner, 1995). On the other hand, NO 3 is the major nutrient anion, which accounts for 70% to 80% of the total ion uptake in higher plants (Van Beusichem et al., 1988). Plants supplied with N-free NS cannot take up any NO 3 and thus the total anion uptake is accordingly suppressed. The low NO 3 availability in the roots may signal a marked suppression of K + and Mg 2+ uptake through the active transport mechanisms localized in the root cells (Mengel and Kirkby, 2001) to maintain electrochemical balance in the cytosol. As reported by Raab and Terry (1994), low NO 3 concentrations in the roots restrict cation uptake because NO 3 anions are needed to counterbalance cations in the xylem. In legumes, N 2 fixation results in a net release of H + ions (Bolan et al., 1991) and this may entail a further restriction of K + and Mg 2+ uptake by roots in the absence of NO 3 in the root zone aiming at maintaining the electrochemical balance in the root cells. The K levels in the shoot of bean plants treated with N-free NS were markedly lower (15.2 mg · g -1 , average calculated across all sampling dates) than the lower critical level suggested by Mills and Jones (1996), in contrast to those of Mg (4.06 mg · g -1 , average calculated across all samplings dates), which were still within the sufficiency range. Also the shoot Zn levels (28.09 mg · g -1 , average calculated across all samplings dates) were clearly below the critical level of sufficiency in the bean plants treated with N-free NS. The supply of N-free NS reduced significantly the shoot P concentration 65 DAT, irrespective of inoculation treatment (Fig. 4). This is attributed to the increased number of nodules at this growth stage in both inoculated treatments, because many studies have shown that nodules are strong sinks for P (Drevon and Hartwig, 1997;Kleinert et al., 2014). Indeed, as reported by Vadez et al. (1996), the P concentration in nodules formed by N 2 -fixing bacteria is 3-fold higher than in plant tissues. As stated by O'Hara et al. (1988), nutrient deficiencies constitute a major constraint limiting biological N 2 fixation and yield in legume crops. Thus, a partial involvement of K and/or Zn deficiency in the appreciable suppression of growth and pod yield in P. vulgaris plants treated with N-free NS is likely. Nevertheless, based on the results of the present study, it is not possible to estimate the relative contribution of N deficiency during the early developmental stage vs. K and Zn deficiencies to growth and yield suppression in bean plants treated with N-free NS and inoculated with R. tropici CIAT899. Legume crops may need to be inoculated only when they are grown in regions outside their centers of diversity or where they have not traditionally been grown or have not been grown for a number of years (Brockwell et al., 11.5 ± 0.5 a 107.1 ± 7.5 a 8.6 ± 0.81 a 9.3 ± 0.55 a 8.01 ± 0.2 b 0% N + R. tropici 3.1 ± 0.1 b 18.5 ± 1.1 b 1.7 ± 0.13 b 6.5 ± 0.54 b 9.04 ± 0.3 a 0% N + commercial mixture 1.4 ± 0.4 c 3.8 ± 0.2 c 0.3 ± 0.05 c 2.3 ± 0.37 c 7.53 ± 0.2 b Statistical significance *** *** *** *** ** z The inoculated plants were supplied with nitrogen (N)-free nutrient solution, whereas the non-inoculated plants were fed with a nutrient solution containing 100% of the standard recommended N level. Significant differences at P # 1% and 0.1%, are denoted by ** and ***, respectively. 1995). Substrates on which common bean may be grown for the production of green pods do not host naturally efficient rhizobia strains for N 2 fixation. Thus, inoculation with efficient rhizobia strains for bean such as R. tropici CIAT899 is essential for the reduction of external N supply in inorganic form through biological N 2 fixation. The results of the present study showed that inoculation with R. tropici CIAT899 results in extensive nodulation and adequate biological N 2 fixation for maintenance of sufficient N levels in the plant tissues. As a general rule, common bean plants fix 15 kg shoot N for every ton of shoot dry matter accumulated (Unkovich et al., 2008). As reported by the same authors, the true value of N 2 fixation can be known only under defined conditions when the organism in question either fixes no N 2 or is totally dependent on N 2 fixation for growth. Thus, plants growing with their competent N 2 -fixing bacteria in the absence of any other plant-available sources of N will derive all N (100%) from N 2 fixation, except for a very small amount originating from the sown seed (Unkovich et al., 2008). In the present study, the plants inoculated with R. tropici CIAT899 did not receive any external N except for that of the seed. Thus, the total N concentration measured in the shoots of these plants (30 mg · kg -1 , i.e., 30 g per ton shoot dry weight) can be considered net N 2 fixation. This amount is three times as high as that reported by Unkovich et al. (2008) indicating that N 2 fixation in bean crops grown on substrates is highly efficient after inoculation with R. tropici CIAT899. Conclusion Inoculation of bean plants with R. tropici CIAT899 results in efficient nodulation and biological N 2 fixation in bean crops grown on N-free substrates when N-starved nutrient solution is supplied. However, this is the case only after the first 3 to 5 weeks of growth. During the early developmental period, the availability of sufficient mineral N in the root environment is critical, because the bean/ Rhizobium symbiosis is not able to supply adequate amounts of N for plant growth. In the present study, inoculation with R. tropici CIAT899 was combined with the supply of N-free NS even during the early growth stages and thus both vegetative growth and yield were severely restricted. In addition, the absence of any NO 3 supply restricted the uptake of K and other nutrient cations. Based on these results it is concluded that an adequate supply of mineral N during the first 3 to 5 weeks of cropping and a continuous supply of some NO 3 -(but less than in noninoculated crops) throughout the cropping period are essential practices to benefit from rhizobia inoculation in common bean crops grown hydroponically.

v3-fos

2018-04-03T01:17:28.903Z

2015-03-12T00:00:00.000Z

28951998

s2

Prevalence of carcass bruises as an indicator of welfare in beef cattle and the relation to the economic impact Abstract The objective of this work was to characterize bruises in bovine carcasses in Uruguay and to evaluate the economic impact. Thirteen abattoirs were visited during 2 years and bruises were identified, classified, and quantified by zone and degree (depth and size). One hundred carcasses were separated and bruises were cut out and weighed separately. From a total of 15 157 carcasses observed, 60.0% had at least one bruise; 42.0% of these had bruises on both sides. The expensive butt zone was the most damaged, followed by rib, shoulder and loin, respectively. The mean weight and standard error of the condemned trimmed meat was 1602 ± 212 g. It suppose a loss of 899 g per animal slaugtered in Uruguay. In a country sending 2.5 million heads of cattle to be slaughter yearly, this indicates an important financial loss. Improving transport conditions and personnel skills will probably result in a better welfare for the animals as well as better financial profit. Introduction Economic losses due to carcass bruises are a substantial problem in the meat chain and have been estimated at several million dollars annually by several authors (McCausland & Millar 1982;Lorenzen et al. 1993;Van Donkersgoed et al. 2001;McKenna et al. 2002). Bruises are defined as tissue damage with rupture of the vascular supply and accumulation of blood and serum (Hoffman et al. 1998). These can occur during most of the pre-slaughter stages, including loading at the farm level, transportation and unloading with or without a lairage period at the abattoirs (Kenny & Tarrant 1987;Warriss et al. 1990;Jarvis et al. 1995). In Uruguay, it is unlikely that they occur earlier because the animals are raised in a pasture (Strappini et al. 2009;Huertas et al. 2010). Bruises in cattle have a significant association with improperly maintained trucks for transporting animals, long distances shipments, bad state of roads, inappropriate handling of the cattle at the preslaughter stages and presence of horned animals (Warriss et al. 1995;Huertas et al. 2010;Strappini et al. 2010). Therefore, carcass damages result, not only in an economic loss to the meat chain but also it is a strong indicator of poor animal welfare. (Grandin 1997(Grandin , 2000. Cattle coming from auction markets are loaded and unloaded several times, increasing the probability of injury (McNally & Warris 1996;Grandin 2000;Costa 2006). Bruises in bovine carcasses affect the quality of the meat and bruised carcasses are downgraded, reducing the economic value of the whole carcass (Gallo et al. 1999). Immediate consequences are 'dark cuts', condemned zones and low quality meat (Kelly et al. 1998;Knowles & Warriss 1999). Extent, depth and localization of bruises are important factors, thus the amount of meat trimmed from carcasses varies according to different authors between 0.5 and 6.0 kg per carcass (Marshall 1976;McNally & Warris 1996). Moreover, from the economic point of view, bruises located at the hindquarter affect the most expensive meat cuts and extra costs due to trimming also add to the losses caused by bruises (Costa 2006). The objective of this work was to evaluate the most frequent bruises in bovine carcasses observed during slaughter in Uruguay and to estimate the economic losses due to partial condemnation of the carcass and extrapolate the severity of the impact on the welfare of cattle. Materials and methods An assessment of carcass bruises in 13 Uruguayan abattoirs licensed by the Livestock, Agriculture and Fisheries Ministry and authorized to export to the European Union (EU) and the North American countries, was performed during the 2-year period (2002)(2003). The visited abattoirs were representative for the country of Uruguay because 85% of the total beef cattle were slaughtered here. The slaughtered cattle were mostly Hereford steers weighing a mean of 450 kg. The abattoirs were located as follows: 46% in the south zone of the country; 15% in the Middle West zone and 39% of the plants located in the North zone. Each abattoir slaughtered an average of six hundred animals per day and they were visited at least twice during the study period, including different weather seasons. Scoring of bruises Bruises and carcass identification were recorded by three of the authors and four veterinary students who were trained as observers. Concordance levels between observers were found acceptable for a subjective method (80%, Kappa = 0.5) (Huertas et al. 2010). Each pair of observers assessed all carcasses slaughtered the day of the previous visit at the abattoir using an adapted subjective scoring methodology (Strappini et al. 2009). Although all bruises are important from the animal welfare point of view; we only recorded the fresh ones and those with a bright red colour. Older bruises, most probably, originated at the farm and were very few and not the subject of the present study. Localization of the bruises The visual appraisal of bruising was confined to four areas (butt, loin, rib and shoulder) performing the identification, classification and quantification of bruises by zone and degree of muscle participation. Classification of bruises according to depth or severity Severity was classified into two grades according to the amount of tissue affected: superficial tissue (subcutaneous) = grade 1, and involving muscular tissue and sometimes bone = grade 2. Separation of carcasses to weigh the bruises One hundred carcasses with all type of bruises were separated for convenience off the line and the meat trimmed for each bruise was saved in a plastic bag. Later that same day, the bag was weighed and each bruise was recorded separately using a digital scale (Rimont model MT 8461 RIMONT Sistemas -Grupo Access, http://www.rimont.com, Argentina) with a capacity to weigh up to 15 kg with a precision of 0.005 kg. In addition, zone and depth of each individual bruise was also recorded. Data analysis Descriptive analysis was performed with a statistical package Intercooled Stata 11.2 (StataCorp LP, USA, 2009) in order to obtain the frequencies of each variable considered. The association between the categorical variables (zone in the carcass and degree of deepness) were tested by the chi square test. To evaluate the economic losses, the following formula was developed: where x is the estimated average loss by carcass; z is the number of zones considered, in this case 4; i is the index of areas ranging from 1 to z (z = 4); Gr is the degree of bruise depth in two categories: superficial and deep; j is the index of grades varying from 1 to 2; p ij is the frequency of bruises in zone i, grade j; weight is the weight of bruises in zone i, grade j; Σ is the sum; and N is the number of carcasses observed. Potential changes in the destination of the meat cuts or downgrading of the carcasses as well as the higher costs at the industry to trim the damaged carcasses were not considered in this study. Amount of bruises found in carcasses The total number of carcasses observed during the study was 15 157, of which 60.0% (9105) presented at least one bruise. In damaged carcasses, the number of bruises varied from 1 to more than 4 as shown in Fig. 1. As shown in Table 1, the prevalence of bruises grade 1 was 71.0% and bruises grade 2 was 29.0%. According to the localization of bruises, the butt zone had the greatest number of bruises compared with other zones of the carcass, with 58.25% of grade 1 and 21.78% grade 2. An association between zone and depth was found in carcass bruises [v 2 ð3Þ = 51.63 P < 0.001] meaning that the butt zone had the most bruises and also the deepest ones. Quantification of losses The mean weight and standard error of the trimmed parts (n = 100) was 1602 AE 212 g with a minimum of 50 and a maximum of 4900 g. Table 2 presents the mean weight of trimmed bruises in grams by localization and depth. The estimation of direct losses was calculated as the product of the number of bruises by the estimated weight of condemnation, divided by the total number of slaughter cattle observed, reaching a loss of 899 g per animal slaughtered in Uruguay (Table 3). Discussion and conclusions The percentage of bruised carcasses observed in Uruguayan slaughter plants was high (60.0%). This result agrees with those found in other countries by Van Donkersgoed et al. 2010) observed a relatively low prevalence of bruises (varying from 8.0% to 20.0%) in slaughterhouses in Chile. These variations in the results between authors and regions may be due to different production systems and/or different methodologies for the diagnosis and recording of bruises. So far, there are no indications reported for a better welfare situation during transport in Chile. Differences do exist between feedlot and grazing systems, since cattle in feedlots are more in contact with humans. Furthermore, an association between distance to slaughterhouses and the number of bruises is reported (Huertas et al. 2010). The method to register the bruises can vary with the observer, the training, experience and particular conditions in each slaughter plant such as the speed of the slaughterline, the place where the observer is located and lighting. It is worth mentioning that in our study, trained observers recorded the bruises so that most of the above-mentioned factors were minimized. This was confirmed by the concordance levels between observers (80%, Kappa = 0.5) (Huertas et al. 2010). The high percentage of bruises, observed in the present study, could be explained in part by the conditions for animals during transport, including bad maintenance of vehicles, the presence of 'guillotinetype' door at the rear end in most of the trucks, the state of the roads and the frequent use of devices to force animals to move, such as: electric prod (75%), sticks (3%), loud shouts (40%) and a combination of all of them as we found in previous studies (Huertas et al. 2010). It is well demonstrated that animals sold through the auction markets have the highest risk of showing bruises compared to those delivered by dealers (Weeks et al. 2002), however, in the present study, all the animals came directly from the farms to the slaughterhouse by trucks. Clearly, transportation, loading and unloading are very important criteria pertaining to animal welfare in the pre-slaughter stages (Wythes et al. 1985). Considering the number of bruises recorded per animal, it is remarkable that more than 4000 carcasses had one bruise, indicating that these animals had at least one injury. Moreover, several animals had two, three or more than four bruises. With respect to localization and depth of bruises, the butt zone was the most damaged (80.03%) with the majority of bruises also in depth (21.78%), followed by the rib zone (8.5%), the shoulder (6.02%) and the loin (5.45%), respectively. The high percentage of bruises in the butt zone could be due to the truck doors that can fall on the back of the cattle when they are passing, perhaps because of the lack of trained personnel. Furthermore, it is the region with the most valuable meat cuts from the economic point of view. Carcass bruises are probably caused by the method of handling the animals, poorly maintained trucks and failures at the trailer gate openings rather than butting or mounting. This behaviour is more because the animals are not familiar when they are let in the lairage pens (Huertas et al. 2010;Strappini et al. 2012). Rib zone involves a very popular cut called asado by Uruguayan people and almost 22.0% of deep bruises in this region imply a great disservice to the local consumers and stakeholders. It is common practice in some countries to pay producers only after the trimmed meat is removed; this implies a decrease in the money that the farmers receive when selling their animals. If the estimated prevalence of carcass bruises was 60.0%, the Uruguayan meat chain would lose at least 899 grams of high quality meat per animal slaughtered. In Uruguay, the mean number of cattle slaughtered per year is about 2.5 million heads, thus almost 2 million tons of high quality meat is wasted each year. In 2012, the average meat price 'on hook' in the country, was approximately US$4 per kg implying a loss of approximately 8 billion American Dollars (US$) per year in the country due to bruises, not taking into consideration that the most valuable parts of the carcasses was damaged. Even though in some countries quality audits have been performed, very few studies considered this economic point of view, taking into account losses in terms of beef export and income to producers. However, apart from the economic losses, the welfare of the animals involved is also seriously impaired. Bruises are painful and may be caused by conditions during transport and handling of the animals. These are already stressful events because they are living the entire year outside on pasture and are not used to the proximity of humans or being transported in a truck. Adding pain to this stress will have a detrimental effect on the welfare of these animals during the last hours of their life. As a consequence, bruising is associated with stress and subsequent appearance of dark, firm and dry (DFD) meat which is also downgraded and an economic loss (McNally & Warris 1996;Strappini et al. 2010). Particularly in developing countries, sometimes, the economic perspective of welfare has a greater impact than ethical or moral aspects of animal welfare (Appleby & Huertas 2011) and could be a strong drive (incentive) for improvement. Results from the present study show that the amount of bruises that appear on the carcass clearly indicates a failure in the management of livestock. Animal welfare is seriously compromised but the bruises also cause important economic losses to the entire meat sector, including farmers who are most affected. Further studies at all levels (farms, transport and industry) are needed. As previously shown (Huertas et al. 2010), most bruises are related to handling and transport to the slaughterhouse. Maybe that the economic factors may stimulate the people involved to alter their behaviour and routine in order to minimize the unnecessary losses and to improve the conditions for the animals.

v3-fos

2019-04-05T03:37:07.064Z

2014-04-22T00:00:00.000Z

95710364

s2

Ash reduction of corn stover by mild hydrothermal preprocessing Lignocellulosic biomass such as corn stover can contain high ash content, which may act as an inhibitor in downstream conversion processes. Most of the structural ash in biomass is located in the cross-linked structure of lignin, which is mildly reactive in basic solutions. Four organic acids (formic, oxalic, tartaric, and citric) were evaluated for effectiveness in ash reduction, with limited success. Because of sodium citrate’s chelating and basic characteristics, it is effective in ash removal. More than 75 % of structural and 85 % of whole ash was removed from the biomass by treatment with 0.1 g of sodium citrate per gram of biomass at 130 °C and 2.7 bar. FTIR, fiber analysis, and chemical analyses show that cellulose and hemicellulose were unaffected by the treatment. ICP–AES showed that all inorganics measured were reduced within the biomass feedstock, except sodium due to the addition of Na through the treatment. Sodium citrate addition to the preconversion process of corn stover is an effective way to reduced physiological ash content of the feedstock without negatively impacting carbohydrate and lignin content. Introduction Lignocellulosic biomass (wood, grasses, and agricultural residues) are an alternative, renewable, and sustainable energy source with a large potential to address the increasing demands for alternative liquid fuels and green chemicals. These feedstocks do not directly compete with the food supply, but have a constrained usage due to their inherit characteristics and storage limitations [1]. About 450 million dry tons (Mt) of wood, energy crops, and agricultural residues, both primary and secondary, are available currently in the USA, and this amount is expected to increase to more than 1,000 Mt by 2030 [2]. Feedstock supply and logistics of lignocellulosic biomass, such as wood, rice hulls, straw, and switchgrass, are challenging due to low bulk density, low energy density, and high ash content [3,4]. Chemically and physically consistent feedstocks can reduce or remove barriers that limit access to much of the potential US biomass resources, while helping to reduce biofuel production costs and enabling a national-scale biorefining industry [5]. In many thermochemical conversion processes (e.g., pyrolysis, fast pyrolysis, liquefaction) high mineral content has an adverse effect on the product output. The presence of alkali and earth alkali metals drastically decreases the production of levoglucosan in pyrolysis [6][7][8]. High content of alkali and silicon contributes to slagging and fouling in boilers and heat transfer surfaces of biomass gasifiers thus decreasing the overall thermal efficiency [9,10]. For biochemical conversion, increased mineral directly correlates to a reduction in carbohydrate content of the feedstock reducing sugars' yield. Weiss et al. [11] determined that pretreatment performance was negatively correlated to neutralization capacity and ash content, reducing both monomeric xylose and total xylose yield from pretreatment. Results also suggest that acid-neutralizing compounds were in the soil and not in the ash. Importantly, both soil (non-physiological ash) and physiological-bound ash, which is bound in the cell walls and incorporated into the vascular structure increased mineral content, will most likely increase water treatment cost and will likely be solubilized in the pretreatment and biological conversion processes resulting in mineral contaminants being observed in the sugars' intermediate products. In all biomass conversion processes, the direct impact of high ash content, whether from physiological ash or from soil contamination, is the increased cost and logistical implications of solid waste disposal. An increase of 5 % ash in a biomass feedstock for a 2,000-T/day conversion refinery would correlate to~32,000 T of solid waste annually (assuming~320 days of operation). Humbird et al. [12] estimated waste disposal costs of US$28.86 per ton, accounting for 2.5 cents of the $2.15 per gallon minimum ethanol selling price, and results in waste disposal costs exceeding $1 M annually. Entrained ash, i.e., soil, is largely a property of feedstock handling methods and can be mitigated through harvesting operations, best management practices, and mechanical separation [13]. Physiological-bound minerals, termed "structural ash," results from intrinsic biomass properties such as plant type, maturity, and anatomical fractions and will require advanced preprocessing methods to effectively remove the bound minerals. Structural ash can vary widely, both in quantity and in composition, in different types of biomass. Pine pulp generally has a very low ash content (0.5 %), while miscanthus and corn stover have ash content of about 8-10 %, and rice hulls have ash content as high as 21 % [9,10]. An effective process of the demineralization of biomass ash without degrading hemicellulose, cellulose, and lignin is necessary for improving the conversion processes and reducing waste disposal costs. Previous research has reported various methods for the demineralization of woody and grassy biomass. Mourant et al. [14] reported that an extraction using hot, deionized water was effective for removing the monovalent cations Na and K and divalent cation Mg. Another effective procedure was reported by Scott et al. [15] using a strong acid (0.1 wt% HNO 3 ) hydrolysis for 60 min at 30°C. The reaction removed most of the alkaline ions, but hemicellulose was also removed, and the degree of polymerization (DP) of cellulose was significantly reduced. Dobele et al. [16] reported that a 2 wt% H 3 PO 4 hydrolysis procedure was effective for demineralization, but most of the levoglucosan produced in subsequent pyrolysis was converted to levoglucosone, a less desirable intermediate product. Chelating agents are commonly used for removing metal (inorganic) ions in soil research and other industries [17]. A chelating agent, or chelant, contains two or more electron donor atoms that can form coordinate bonds to a single metal atom. After such initial coordinate bond, each successive donor atom that binds creates a ring containing the metal atom. This cyclic structure is called a chelation complex or chelate, the name deriving from the Greek word chele meaning "the great claw of the lobster" [17]. Chelation is a system based on chemical equilibrium and may be used to control metal ion concentrations. A chelation complex usually has solubility properties that are markedly different from both the free metal ion and chelating agent. Chelation has many common industrial applications, including metal buffering, corrosion inhibition, solubilization, and cancer therapy [18]. The citrate ion is a common organic chelating agent which is biodegradable, environmentally friendly, and cost effective compared to other chelates or even other organic acids. The chelation characteristics of citric acid and its derivatives are such that it is widely used in various applications, from soil amendment to consumer products [18]. The chelation of essential metal nutrients with citric acid is very popular in the food and fertilizer industries. The citrate ion can form bi-, tri-, and multi-dentate complexes, depending upon the type of metal ion [19]. For example, metals like iron and nickel form bi-dentate, mononuclear complexes with two of the carboxylic acid groups of the citric acid molecule. Copper, cadmium, and lead form tri-dentate, mononuclear complexes with citric acid utilizing two carboxylic acid groups and a hydroxyl group [20]. Tetra-dentate compounds with silicate anion can be formed with two citric acid molecules utilizing two carboxylic acid groups of each citric acid ion. The main goal of this work was to examine the demineralization capabilities of several organic chelating agents on corn stover. Because hemicelluloses and cellulose are reactive in acidic environments, acids have the potential to hydrolyze biomass and degrade hemicellulose and cellulose. Using the conjugate base alleviates this problem of hydrolysis and still utilizes the chelating properties of the organic anion. Demineralization of corn stover using four organic acids (formic, oxalic, citric, and tartaric) and sodium citrate was examined. The demineralized products were analyzed for the degradation of hemicellulose, cellulose, and lignin. Materials The INL procured three bales of corn stover from Emmetsburg, Iowa, and initially ground them using a Bliss Hammermill (Ponca City, OK) to 6.4 mm. The material was then sequentially ground to 2 mm using the Thomas Wiley Model 4 Mill (Ramsey, MN), and the samples were stored in 5-gal buckets until analysis and/or treatment. Four buckets of ground corn stover were provided to the University of Nevada, Reno, where the samples were mixed manually prior to hydrothermal preprocessing. Particles have the arithmetic mean diameter of 0.72±0.20 mm. Organic chelation treatments Chelation reactions of raw corn stover were performed in a 2-l Parr reactor, and data reported here are the average and standard deviation for at least three repetitions. Chelating agents bind metal ions in covalent bonds and thus can remove the metal ions from the cross-linked structure of the biomass. Several organic chelating agents, including citric acid, sodium citrate, tartaric acid, oxalic acid, and formic acid, were evaluated for the efficiency of structural inorganic removal from corn stover. Figure 1 shows the experimental procedure of chelation using chelating agents. For the effective removal of structurally bound inorganics (i.e., structural ash) by chelating agents, the loose dirt and other non-structural, polar inorganics were removed first by washing the sample with boiling water. Acetone was then used for the removal of non-polar extractives. This treatment prepares a dirt-free extractive-free biomass, which allows for more consistency when studying the effects of chelation on structural ash. The solid residue was subjected to chelation and mild hydrothermal treatment in a 2l Parr reactor with various concentrations of chelating agent for 2 h at 130°C. The degradation temperature of citric acid is 170°C, which can be reduced in a pressurized system [21], so the temperature of 130°C was chosen to avoid the possible degradation of the chelant. Reactor pressure was monitored and was consistently at, or near, the vapor pressure of water (2.7 bar). After chelation, the Parr reactor was cooled by submersion in an ice-water bath. Wet filtration through a 100 Tyler mesh was used to filter the solid residue from a liquid filtrate. Boiling water was applied for 5 min to remove the chelated metals on the surface of the solid residue. Acetone was used afterward to precipitate unreacted citrate ions into aldoles for removal. Aliquots from each of the washing steps was taken and refrigerated for further analyses. Finally, the solid fraction was collected and placed into a 105°C oven to dry and then stored in a ziplock bag until further use. Compositional analysis Chemical analysis of the untreated corn stover and the chelation-treated samples was conducted using the following standard methods from the NREL biomass program [ Proximate analysis The Na citrate-treated and untreated corn stovers were analyzed with a LECO TGA701 Thermogravimetric Analyzer (St. Joseph, MI) for moisture, volatiles, ash, and fixed carbon content. The instrument was heated to 107°C and held there until a constant mass was reached under 10 liters per minute (lpm) UHP nitrogen flow to measure the moisture content. The crucibles were capped with ceramic covers, and the temperature was then ramped to 950°C and held there for 7 min to determine volatiles. The instrument was cooled to 600°C, the covers were removed, and the gas was switched to a flow of 3.5 lpm of oxygen. The temperature was then increased to 750°C and was held until a constant mass was reached for an ash measurement. Fixed carbon was determined by the weight loss between the volatile measurement and the ash measurement. Ultimate analysis The determination of CHN and S for both the treated and untreated corn stover was performed using a LECO TruSpec CHN and S add-on module (St. Joseph, MI), while oxygen was determined by difference. The moisture from the proximate analysis (performed simultaneously) was used to correct for a "dry-basis" analysis. Step 1 Step 2 Step 3 Step 4 Step 5 Washed with 1 L boiling water Step 6 Fig. 1 Block diagram illustrating the experimental procedure for the hydrothermal treatment of corn stover with chelation 2.6 Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) A Perkin-Elmer Spectrum 2,000 ATR-FTIR with mid-and far-IR capabilities was used on the raw and pretreated biomass. IR spectra of raw corn stover as well as sodium citratetreated corn stover were recorded at 30°C using ATR-FTIR. All samples were milled into fine powder for homogeneity and dried at 105°C for 24 h in an oven prior to FTIR. Only 5-10 mg of dry sample was placed in the FTIR for this analysis and pressed against the instrument's diamond surface with its metal rod. All spectra were obtained using 200 scans for the background (air) and 32 scans for the samples, which were scanned from 500-4,000 cm −1 . To study the organic bands of sodium citrate-treated samples from IR spectra, only fingerprint region (800-1,800 cm −1 ) is presented here. Inductively coupled plasma (ICP) analyses A Varian Vista Pro ICP-AES (Cary, NC) was used for inorganic analysis. Acid digestion was used to dissolve solid samples for inductively coupled plasma atomic emission spectroscopy (ICP-AES). A volume of 5 ml of 99.5 % HNO 3 was added to 0.4 g of dry solid sample. A volume of 0.5 ml of 50 % (v/v) hydrofluoric acid (HF) was added to the solution to dissolve SiO 2 . Liquid argon, at the rate of 88 ml/min, was used as carrier. The liquid solution was heated to 80°C and maintained at that temperature for 4 h. After 4 h, the sample was removed from the oven and cooled for 5 h. To prevent HF from reacting with the torch, a 0.5-g solid 98 % boric acid was put into the solution so that the unreacted HF would react with B(OH) 3 to form fluoroboric acid (HBF 4 ), which is invisible and not harmful for the ICP-AES torch at room temperature [23]. The solution was diluted 20-200 times before injection into the ICP-AES instrument with a 5 % ethanol/water solution used as the solvent. A Thermo Scientific iCAP 6,000 Series ICP optical emission spectrometer (ICP-OES) (Waltham, MA) was used to analyze the elements K, Ca, P, S, Mg, Mn, Na, and Si in the liquid fractions from the chelation treatment for a select number of samples. The samples that were analyzed were the liquid fractions and their solid precipitates from the five steps in Fig. 2 from the 0.05-g Na citrate treatment. These samples were treated with the same digestion procedure as the ICP-AES analysis listed above. The reproducibility of these ICP measurements is quite good, with a standard deviation generally less than 0.1 %, and the error bars of ICP measurements is left off the figures for clarity. Ion chromatography (IC) analysis The liquid fractions and washings were also analyzed for their anions using IC analysis. A Dionex ICS-3,000 instrument was used to measure the anions F, Cl, and Br with a Dionex ASRS 300 (anion self-regenerator suppressor) and Dionex IonPac AS18 anion-exchange column (Sunnyvale, CA). The sample preparation was the same for the ICP-OES analysis. All standards used in the procedure met the ISO 9001 qualifications. Higher heating value The higher heating values (HHV) for the untreated biomass and the Na citrate-treated samples were measured following the ASTM D1826 method using a Parr 1,241 adiabatic oxygen bomb calorimeter (Moline, IL) fitted with continuous temperature recording. All samples (0.5 g each) were dried at 105°C for 24 h prior to analysis, and HHVare reported on a dry basis (ash included), distinct from the more common reporting of dry ash-free basis. Results and discussion 3.1 Treatment of corn stover with various organic chelates As a preliminary screening tool, total ash analysis of solid samples was used to identify promising chelants. The content of raw corn stover was 10.5 % whole ash (including the loose soil from harvesting), 7.0 % structural ash, and 9.2 % extractives (including starches, proteins, simple sugars, organic acids, etc.) The following chelation experiments were conducted at conditions at which those extractives would be removed from the biomass. A baseline was established for structural ash on an "extractive-free" basis, which is 7.7 %. Any treatment that resulted in a solid with structural ash less than 7.7 % on this basis was identified as one that reduced ash content. In all cases, fractions of biomass are reported as a mass fraction. The ash content of various treated solid residues is presented in Fig. 2. With the exception of that treated in formic acid, each dry solid residue had less than 7.7 % ash content, meaning each of them was effective at removing structural ash. Formic acid proved to be the least effective for removing structural ash, as it has only one carboxyl "hand," and so its capacity for binding to metals is limited. Oxalic acid has two carboxylic groups and reduced the structural ash content more than formic acid but was still not the most effective treatment. Citric acid and sodium citrate each have three carboxylic groups along with one hydroxyl group and are excellent chelating agents. Corn stover treated with 10 % citric acid (mass of acid per mass of dry biomass) had a 6.0 % ash content, meaning that 23 % of the structural ash was removed. Sodium citrate showed the highest reduction of structural inorganics among the chelating agents considered. Treatment with as little as 5 % sodium citrate, with respect to dry corn stover, reduced the structural ash by 66 %, and treatment with 10 and 25 % sodium citrate resulted in a further removal of structural ash. One possible explanation of sodium citrate superiority relative to citric acid might be the higher pH of the solution. A 10 % citric acid solution has a pH of 3.8 while a 4 % sodium citrate solution has the pH 7.5. At lower temperatures, hemicellulose and cellulose are reactive in acidic media while lignin remains inert [24]. But in a slightly basic solution, lignin is reactive, and cellulose and hemicelluloses are inert [25]. Because structural inorganics form covalent bonds within the cross-linked structure of lignin, the solution should be slightly basic to dissolve lignin-bound metals [8]. Cellulose has a unique structure, and there is little or no possibility of forming bonds between the inorganic molecules and cellulose. Hemicellulose has the potential to bind some inorganics, but research has shown that a hydrothermal carbonization treatment at 200°C degrades all hemicelluloses while the ash content is not reduced significantly [9,24]. The sodium citrate makes it possible for the lignin to depolymerize and for the citrate ion to form bonds with the metal ions. As a result, the inorganic metal is removed as a chelate. Note that the cation Na is left behind on the biomass and cannot be completely removed by water and acetone washings alone. Therefore, the potential exists to use a different chelating agent, such as ammonium citrate, to further reduce ash content. Chemical analysis of sodium citrate treated solid residue The previous section describes the promising results of inorganic removal from corn stover using sodium citrate as a chelating agent. Adding 10 and 25 g of sodium citrate to 100 g of dry raw corn stover yields similar ash contents, about 80 % reduction from the raw corn stover. This may indicate that an optimal concentration of Na citrate is between 5 and10 g for the 100 g of corn stover under the experimental conditions of 130°C, 2 h, and a 10:1 water to biomass ratio. In this section the chemical characteristics of sodium-citrate treated corn stover will be discussed compared with raw corn stover. Corn stover treated by two different concentrations of sodium citrate, 0.05 and 0.10 g/g, were selected for analysis. Table 1 shows the chemical analyses of sodium citratetreated solid residues. Because of the washing steps before and after the chelation process ( Fig. 1), extractable inorganics, protein content, and total extractives are significantly reduced. Concentrations of extractive-free glucan (cellulose) and xylan, galactan, and arabinan (hemicellulose) remain similar after treatment compared to raw biomass. This indicates that cellulose and hemicellulose were not degraded during the sodium citrate treatment. Lignin content was slightly reduced by treatment with sodium citrate. Proximate and ultimate analysis The results of proximate and ultimate analysis of raw corn stover and sodium citrate-treated corn stover solid residues are shown in Table 2. Raw corn stover has 78.1 % volatiles, 11.2 % fixed carbon, and 10.5 % ash. The proximate analysis of solid residues treated by 5 and by 10 % sodium citrate is similar. The content of volatiles in samples treated with sodium citrate increases, and ash decreases when compared to raw corn stover. As mineral content is removed, the remaining organic fractions makeup a larger fraction of the treated solid. Ash content is lower in treated samples than raw, but fixed carbon did not increase accordingly. This may indicate some loss of fixed carbon during the treatment. It is possible that lignin partially depolymerizes during the treatment, and the product is volatilized more readily than untreated lignin. The ultimate analysis of sodium citrate treatment solid residue shows similar hydrogen and nitrogen content compared to the raw corn stover. But an increase of oxygen and carbon percentage can be observed in the treated solid residue compared to raw corn stover. However, calculation of C-and O-content on an ash-free basis, as a fraction only of the organic content, shows that oxygen is essentially constant (44 %) while carbon may decrease slightly, from 50 to about 48 % (treatment with 5 % sodium citrate) and 47 % (10 % sodium citrate). The decrease in carbon content is small and may reflect experimental error only. The HHV increases in the treated corn stover compared to that in the untreated samples but are similar between the two treatments. Even on an ashfree basis, the HHV content of the solid residue is increased. Near-complete removal of extractive compounds with relatively low HHV might explain this increase in fuel value. 3.4 FTIR analysis of sodium citrate-treated corn stover FTIR spectroscopy has been extensively used in biomass research, as it shows the bond energies of characteristic groups in biomass, and can indicate changes in molecular formulation resulting from various treatments [26]. By identifying the peaks of FTIR spectra, which are caused by the vibrations of functional groups, FTIR allows us to identify and analyze the chemical structure of a sample. Table 3 identifies functional groups for particular wavelengths and the corresponding chemical compounds. Aromatic compounds have weak bonds, and therefore they stretch (vibrate) at lower wave numbers usually from 500-2,000 cm −1 . Aliphatic compounds stretch in the higher wave numbers (usually 2,500-4,000 cm −1 ). Biomass is a complex mixture of aromatic compounds, so the fingerprint region (800-1,800 cm −1 ) is the range of interest here. Figure 3 shows the FTIR spectra of raw and treated corn stover. Raw corn stover shows strong bonds at 896, 1,036, 1,186, Data are reported as a mass fraction on an extractive-free basis of the biomass sample. Note: ash reported here is on a total mass basis (not the extractive-free basis) [32]. The bond at 985 cm −1 is shifted to 975 cm −1 for the treated samples, which might result from the degradation of the extractives. Every bond present in raw corn stover is either stronger or unaffected by the treatment, except those bonds corresponding to extractive compounds. The FTIR spectra demonstrate that mild hydrothermal treatment with sodium citrate does not change the chemical structure of hemicelluloses, cellulose, or lignin. Inorganic analysis of sodium citrate-treated solid residue The inorganic content of raw corn stover is presented in Table 4. Silicate anion, a tetra-valent element, is the dominant inorganic element of the raw corn stover. Potassium, calcium, aluminum, and sodium are the other main inorganics. ICP and IC analyses were performed on the solid residues treated by three different concentrations of sodium citrate and compared with inorganic analysis of raw corn stover (Fig. 4). Because sodium citrate is a poly-dentate ligand and can be used to remove tetra-valent ions, it can potentially remove silicate anion from biomass. The experimental results seem to confirm this hypothesis. With the increasing Na citrate strength, the inorganic elements' concentrations decrease; and as a result, the overall ash decreases. Each Mg, S, P, Fe, Cl, and K all seem to decrease rapidly with a little addition of Na citrate. Potassium and Cl were removed almost completely with a 0.05-g/g Na citrate addition. A possible explanation might be the forms of K and Cl present in corn stover. Potassium, a monovalent cation, is typically found in the form of a halogen salt [33]. Similarly, Cl, a monovalent anion measured with IC, is also frequently found as an ionic-bound salt such as NaCl or KCl. These salts are readily dissolved in hot water and can be removed almost completely (90 %) with no further treatment [10,33]. Because the first step of the treatment process is a hot-water wash, which likely removes the majority of K and Cl, the sodium citrate's strength is likely not a factor for the removal of these monovalent ions. Conversely, the extent of removal of divalent ions Ca, Mg, and Fe is correlated with sodium citrate concentration. A 0.05-g/g sodium citrate reaction removes 80 % of these ions while a 0.25-g/g sodium citrate removes more than 93 % of Ca, Mg, and Fe. Sulfur and Al content are also effectively reduced with the sodium citrate treatments. With only 0.05 g/g of sodium citrate, more than 85 and 89 % of S and Al, respectively, were removed. More than 95 % of both S and Al were removed when the sodium citrate concentration was increased to 0.25 g/g. Silicates, the most abundant Fig. 3 FTIR of raw corn stover and Na citrate-treated corn stover (fingerprint region); blue raw corn stover, red 0.05 g/g Na citrate, green 0.1 g/g Na citrate, cyan blue 0.25 g/g Na citrate) mineral in raw corn stover, is also removed by sodium citrate. Silica can possess multiple valances in biomass, e.g., tetra-, tri-, and/or bi-dentate. With the addition of the citrate ion, poly-dentate Si and other poly-dentate cations form a soluble chelate and are thus released from the structure of the biomass. With the addition of 0.05 g/g of sodium citrate, more than 65 % of Si was removed; and with 0.25 g/g of sodium citrate, 75 % of the total Si was removed. The increase of sodium citrate concentration is effective at removing most metals, but the Na in sodium citrate itself is apparently deposited on the surface of the treated solid. Sodium deposition by the treatment process masks the reduction in total ash, since the additional Na deposited accounts for 0.05 % ash increase. Sodium concentration in biomass shown in Fig. 4 also reflects this effect. The sodium concentration apparently decreases when the amount of chelant used in treatment is increased from 0.05 to 0.1 g/g, but seems to increase when the chelant dosage is further increased to 0.25 g/g. As discussed above, it is likely that Na is removed from biomass by the hot-water wash (step 1 of the treatment process, Fig. 1). Thus, it is likely that the sodium shown in Fig. 4 is a by-product of the treatment with sodium citrate. It is possible that a more rigorous rinsing step after chelation would be effective to further reduce the sodium content. Inorganics' mass balance in sodium citrate treatment There are five main steps in each demineralization experiment (Fig. 1). Liquid aliquots from every step were collected, digested, and analyzed by ICP. Inorganics found from steps 1 and 2 were assumed to be the non-structural inorganics prior to sodium citrate treatment. As the main goal of this research is to evaluate the removal of structural ash by sodium citrate chelation, the results from steps 3 to 5 are of most interest. It is possible to calculate the amount of each element removed from the biomass for each of the five steps separately. For this inorganic mass balance, the mass of inorganics found in the liquid aliquots by ICP-OES were subtracted from the mass present in the raw solid and are presented in Fig. 5. The elemental metal composition in the biomass remaining after each step of the treatment is seen to decrease with each subsequent step of treatment. Inorganics remaining after step 2 are assumed to be structural inorganics. The inorganic composition remaining in the corn stover after treatment is shown in the last two bars; one calculated by subtracting the sum of all five steps (five liquid samples) from that elemental content in the raw biomass, and the last column shows the elemental composition of treated biomass measured by ICP. The accuracy of this mass balance is determined by comparing the last two columns of Fig. 5 for each element. The amount of most of the inorganics present in the treated biomass (last column), a direct measurement, is similar to the amount calculated by subtracting the amount found in all the liquid aliquots from the amount present in the untreated biomass (penultimate column). Solids in the aliquots tend to settle, and some dissolved species precipitate slowly over time, which may cause the detected concentrations to be too low, so that the amount predicted in the treated biomass would be higher than that actually found by direct digestion and ICP analysis. This was in fact observed for 10 of the 11 measured elements, Al being the sole exception. A major portion of monovalent ions sodium, potassium, and chlorine were leached by steps 1 and 2 (hot-water and acetone washings), as shown in Fig. 5 Fig. 4 Inorganic analysis of sodium citrate-treated corn stover. Data are presented as a percentage of the element remaining in the solid after treatment, relative to the amount in raw corn stover silicate anion was removed by hot-water and acetone wash, which indicates the presence of loose dirt in the raw biomass. After step 2, the calculated remaining inorganic in the treated solid is 6.55 %, which is similar to the measured structural ash (7.0 %). Additional metals are removed during steps 3, 4, and 5, and the chelation reaction is followed by hot-water and acetone washings. Due to the chelation reaction, the inorganics are extracted from the biomass and bonded with a ligand to form a metal chelate complex. Metal chelates are very soluble and thus require washing to remove them from the external and internal surfaces of the pretreated solid. Steps 4 and 5 help to remove the metal chelates from the solid surfaces; those steps by themselves are not capable of dissolving inorganics, without the prior chelation of step 3. Silicon is significantly leached due to the chelation reaction. Citrate ions have a higher affinity for higher valence (+4, +3) cations than for lower ones (+2, +1) [18]. Silicon ion is a poly-valent cation (+4, +2), and thus citrate is effective for bonding with and dissolving Si. Based on the stoichiometry of the citrate ion, which has three active binding sites, two citrate ions are required to make a chelation complex with one Si +4 ion. But the other two empty available sites of those two citrate ions are available and can bind another divalent ion. If only Si is considered, four citrate ions may be required to bind three Si ions and make a citrate silica organometallic complex. Based on this assumption, then, 5 g of sodium citrate can bind as much as 0.36 g of Si. Because structural Si was decreased by 0.74 from 100 g of raw biomass sample treated with 5 g sodium citrate, citrate might not act strictly as a trivalent binder. Sometimes, citrate can release a hydroxy ion and become a tetra-valent chelating agent [19], in which case it can potentially bind as much as one Si atom per citrate. The exact electronic state of Si in corn stover is not known, and further research is required. For example, X-ray diffraction or dynamic NMR might shed light on the chelation binding mechanisms. Similar to Si removal, sodium citrate treatment also decreases the structural (i.e., ash remaining after step 2) Al, Fe, Ca, Mg, S, and K. About 32-64 % of those structural elements were removed by sodium citrate chelation in steps 3, 4, and 5. Sodium decreased significantly with the hot-water and acetone washing (step 2). But with the addition of sodium citrate, sodium concentration in the solid increased. In fact, adding 5 g of tri-sodium citrate means an introduction of 1.17 g of Na. Due to the high concentration, only a portion of this additional sodium was able to be removed by rinsing in steps 4 and 5. This is likely the reason for the sodium concentration increasing from 0.06 to 0.7 % after step 3. As shown in Fig. 5, after step 4, about 45 % of sodium is removed compared to step 3, and it further leached with step 5 to 54 % compared to step 3. The use of acetone in step 5 does not reduce significantly inorganic content. It is possible that step 5 could be omitted to achieve the same amount of ash reduction. Corn stover treated with 0.05 g of sodium citrate per gram of raw biomass shows structural ash reduced from 7.0 to 2.5 %. Based on the stoichiometric balance, 5 % sodium citrate is not adequate to remove all structural ash. This is why further reduction of ash is found for treatment with a higher strength of sodium citrate (Section 3.1). With the addition of sodium citrate, structural ash is reduced, but sodium is deposited simultaneously. With the intended goal of reducing overall ash, it is obviously counterproductive to add one metal (sodium) as part of the process. Use of alternative conjugate citrate bases like ammonium citrate might resolve this issue. Citrate, in this work, was not recovered. Further Structural inorganics Inorganics remaining after step 3 Inorganics remaining after step 4 Inorganics remaining after step 5 Inorganics measured in treated CS Fig. 5 Analysis of inorganic content in corn stover after different steps of the treatment process. Step numbers refer to Fig. 1; structural inorganics is that remaining after step 2. Treatment is with 0.05 g of sodium citrate per gram corn stover. Values are reported on a mass basis as a percentage of the treated corn stover development of this technology will require the identification of a process for citrate recovery, perhaps by ion exchange or with strong acid resin. Conclusions Sodium citrate removes structural ash from corn stover without affecting cellulose, hemicellulose, and lignin. Adding 25 g of sodium citrate to 100 g of dry corn stover can reduce 77 % of the structural ash. However, sodium concentration is increased in the solid product by this treatment process. It is likely that the use of other citrates, such as ammonium citrate, can further reduce ash. Further investigation to the reaction chemistry is indicated. If an economic process for chelant recovery and regeneration is identified, then this chelation preprocess might play a significant role in providing a commodity-grade biomass for a commercial biorefinery industry.

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2019-04-25T13:11:41.989Z

2015-01-10T00:00:00.000Z

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The Pollen Spectrum of Apis mellifera Honey from Reconcavo of Bahia, Brazil Background: The use of pollen grains to establish the geographical and botanical origin of honey samples, and for supplying information concerning to the floral class of a honey produced in a region, has been employed since 1895. Pollen analysis has relevant significance for the quality control of a honey that may include numerous pollen grains and elements of honeydew. The objective of this work was to determine the botanical origin of pollen from plants contributing in the composition of honey from the region of Reconcavo of Bahia, Brazil. Methodology: Form each honey sample, a 10 g subsample was diluted in 20 mL of distilled warm water (40oC) and centrifuged before the supernatant was drawn out. After centrifugation the pollen sediment was acetolysed following for better observation of the pollen grains. Subsequently, this sediment was mounted in microscopy slides with glycerin jelly for latter pollen grains counting and identification. The identification of the pollen grains found in the samples was determined by comparison with a reference collection from the Palynotheca of the Nucleus of Insect Studies from the Federal University of Reconcavo da Bahia and with descriptions obtained from specialized literature. A total of 70 honey samples of Apis mellifera L. were obtained from beekeepers of 17 Original Research Article Nascimento et al.; JSRR, 6(6): 426-438, 2015; Article no.JSRR.2015.167 427 counties in the Reconcavo of Bahia region, from March 2009 to February 2010, and palynologically analysed. Acetolyzed pollen were count and identified. Results: One hundred and twelve pollen types were identified, distributed within 35 families. Among these pollen types, 67.00% occurred with low frequency (rare) within the samples analyzed. Inside abundance class, 63.00% occurred as minor pollen. Mimosaceae was the richest accounting for 13.00% of the pollen types. There was similarity among the sources of trophic resources used by A. mellifera in the counties studied, with the highest similarity index of trophic resources found between the counties of Cabaceiras do Paraguaçu and São Felipe (Cs = 0.68). Conclusion: Pollen analysis demonstrated that honey produced by A. mellifera in the region of Reconcavo of Bahia, Brazil, is predominantly multifloral, with emphasis the Mimosaceae. INTRODUCTION Pollen grains are spontaneously collected by bees while they collect nectar from flowers. As a consequence, pollen is present in honey composition and for this reason it becomes an important marker of the botanical and geographical origin of honey. Those grains also help to determine honey variety [1,2]. A quantitative and qualitative palynological survey of a honey sample constitutes its pollen spectrum. This spectrum identifies the nectar producing plant species, the non producer species, the contaminants, and when compared to known base line data can be used to detect mixtures of honey and falsification in labeling [3]. Trough the quantitative analysis of pollen grains is possible to establish the proportion in which each nectariferous plant contributes in honey composition, thus determining the botanical species that gave origin to such honey [4]. This information is important as it identifies those species that should be conserved or restored into the landscape in order to assist in the establishment of a sustainable beekeeping industry [5,6]. While pollen grains from nectar supplying plants, known as nectariferous plants, are honey components, a percentage of honey pollen may also originate from anemophilous plants. There is also a third category of plants, the polliniferous plants, that in addition to supplying small nectar quantities, supply significant quantities of pollen [7]. Nectariferous plants are usually under represented in pollen spectra as they supply a lot of nectar but little pollen. Thus, the presence of a small number of grains may indicate a great quantity of nectar. In pure honey samples from this category, few pollen grains and sediment are present. On the other hand, polliniferous plants are over represented in pollen spectra, supplying few nectar quantities but great quantities of pollen. The presence of vast amounts of pollen may indicate less nectar percentages. When a honey sample contains more than 98% of pollen from a polliniferous plant, it must be considered a monofloral honey [3,7]. The pollen analysis is a method used to describe floral origin of honey and is useful to estimate the diversity of local flora. The knowledge of honey origin allows to map the regions suitable for apiculture. The decline in floral resources used by bees is considered one of the main causes involved in loss of pollinators, therefore, the pollen analysis has become an important tool to identify apicultural flora that can be propagated to increase food resources for honeybees. Thus, considering the "global collapse of honeybees" and "pollination crisis", studies on the identification of apicultural flora are important. [8]. In the Reconcavo of Bahia region, beekeeping is based on a familiar agricultural system, lacking enough information about floral diversity to sustain efficiently such activity. In this context, the objective of the present study was to determine the botanical origin of pollen contained in honey samples of Apis mellifera L. (Hymenoptera: Apidae), contributing to the knowledge of the regional honey flora. , 2004). Form each honey sample, a 10 g subsample was diluted in 20 mL of distilled water and centrifuged before the supernatant was drawn out. After centrifugation the pollen sediment was acetolysed following Erdtman [11] for better observation of the pollen grains. Subsequently, this sediment was mounted in microscopy slides with glycerin jelly for latter pollen grains counting and identification. The identification of the pollen grains found in the samples was determined by comparison with a reference collection from the Palynotheca of the Nucleus of Insect Studies from the Federal University of Reconcavo da Bahia and with descriptions obtained from specialized literature as Barth [3,12], Barth et al. [13], Moreti et al. [14]. MATERIALS AND METHODS After the determination of botanical origin of pollen samples, at least 1000 pollen grains/sample were counted consecutively [15]. The relative frequency of each pollen type was established trough the formula: f = (n i /N) x100, where f = relative frequency of the pollen type i within the sample; n i = number of pollen grains of the i type within the sample; N = total number of pollen grains within sample [16]. Subsequently, values of the mean and the confidence interval were calculated for each pollen type (i), with its respective Superior Limit (LS) and Inferior Limit (LI) at 1% and 5% significance level, and the frequency (rare = n i  LI 5% ; Frequent = LI 5% < n i < LS 5% ; and Very Frequent = n i  LS 5% ) and abundance classes (predominant pollen = n i  LS 1% ; secondary pollen = LI 5%  n i < LS 1% ; Important minor pollen = LI 1%  n i < LI 5% ; minor pollen = n i < LI 1% ) acquired [17]. The pollen similarity among honey samples of A. mellifera by county from the Reconcavo of Bahia region was obtained by the use of the similarity coefficient of Sörensen, expressed as the formula: Cs = 2c / (s1 + s2), where: s1 is the number of pollen types within the county 1 samples, s2 is the number of pollen types within the county 2 samples and c is the number of common pollen types for both counties. RESULTS By means of pollen analysis of the samples, the botanical origin of 112 pollen types was determined, distributed in 35 families (Table 1). Among families found in the present study, Mimosaceae had the largest number of pollen types, with 13.00% of the total, followed by Asteraceae and Fabaceae, both with 9.00% and Myrtaceae, Caesalpiniaceae and Rubiaceae with 8.00%, 7.00% and 5.00%, respectively. DISCUSSION According to various authors, the Mimosaceae has significant potential for beekeeping due to its abundant distribution in the distinct regions in Brazil and as a source of trophic resources (pollen and nectar) for bees [16,[18][19][20]. Moreti et al. [21] showed the importance of the participation of various species of Mimosaceae in the constitution of honey within the State of Bahia, Brazil. The Asteraceae is considered one of the richest in number of species visited by social bees in different regions of Brazil [22]. In the present study this family was one of the most diverse in pollen types and some of these types were classified as predominant and secondary (Table 1). Locatelli and Machado [23] suggest that this is probably due to the fact this family is one of the largest and greater geographical distribution among angiosperms. Novais et al. [24] identified 46 pollen types from 22 botanical families in honey samples of A. mellifera from the semi-arid region in Bahia, Brazil, with the Fabaceae having an exceptional number of pollen types, followed by Malvaceae, Asteraceae, Euphorbiaceae, Rubiaceae and Lamiaceae. This is similar to the results found in this study. According to Melo [25], pollen type of M. pudica was the most frequent in honey samples of Africanized bees in Mundo Novo, Bahia, being predominant pollen in the majority of the samples where it was observed. Costa [17] working with bee pollen in Cruz das Almas, Bahia, observed that 60.00% of the pollen types were rare and 61.00% minor pollen. In the county of Castro Alves, Carvalho and Marchini [16] observed that 58.33% of the plant species visited by bees were rare versus 41.67% considered frequent. The greatest quantity of pollen types identified by these authors were rare, as occurred also in the present work. Some pollen types that occurred as predominant and very frequent pollen belongs to the Mimosaceae and the Mimosa, having their species classified as polliniferous plants. Members of the Anacardiaceae, Asteraceae, Euphorbiaceae and Sapindaceae were observed as secondary and frequent pollen, with species classified as nectariferous plants (Table 1). Oliveira [26] Nascimento et al. [9] conducted a study in the Recôncavo Baiano and identified 240 plant species visited by A. mellifera. The authors observed that most plants are herbaceous (44%), followed by arboreal (26%) and shrubby (18%). Lianas (4%), vines (4%) and palm trees (1%) had less representation. The greatest preference for bee visitation to the flowers of herbaceous plants may be associated with the abundance of these plants in the region, the flowering period with a higher concentration of species between March and June, and the rainy season in the region. Additional plant species have also important participation in A. mellifera honey, as for example those considered as weeds, possessing honey production potential. Among these species, Almeida et al. [31] highlights Commelina benghalensis, Croton campestri and Portulaca sp. Similar to the results found by these authors, pollen types with affinity with those species were identified in the Reconcavo da Bahia samples, as for example Borreria verticillata, Commelina benghalensis, Crotalaria incana, Croton and Portulaca oleracea (Table 1), stressing the provable potential of these plants for beekeeping in the region. Samples from the counties of Amargosa, Cachoeira, Cruz das Almas, Governador Mangabeira and Sapeaçu, had M. arenosa, M. pudica and Syagrus coronata as common types. This region had a mean of 27 pollen types among samples. Oliveira [26], working with honey form this region observed Borreria verticillata, Eupatorium, M. sensitiva, M. tenuiflora, S. coronata and Vernonia types in all the samples, with predominant pollen types found in samples from Amargosa (Schinus), Cruz das Almas (M. tenuiflora) and Sapeaçu (S. coronata). Santos Jr. and Santos [32] demonstrated that S. coronata pollen type was the most frequent, being observed in the monosulcate and tricotomosulcate pollen forms in the counties of the micro-region of the river Paraguassu, in Bahia, Brazil. In the present study, species of the Arecaceae, Asteraceae and Fabaceae families had relevant pollen contribution in honey samples of the same micro-region. The S. coronata type was also preponderant in samples collected in the present work, being observed in both pollen forms (Fig. 2). Due to its diversified flora, honey classification as monofloral is not common in Brazil. According to Zander and Maurizio [33] honey of Citrus and Lavandula (10-20% of the total pollen grains), are considered pure honey subrepresented in pollen. Considering the percentages referred by these authors, two samples originated from the county of Cruz das Almas, with accessory pollen of Citrus may be classified as being monofloral. In this county, the installation of apiaries next to orange tree orchards is common, contributing for the occurrence of such pollen type. Pollen types of anemophilous species found in the honey pollen spectrum are considered not important for its composition, but significant for the geographical identification of the honey and for protein supplying for the colonies [7]. Among the three pollen types of anemophilous species identified in the samples of the Reconcavo da Bahia region, the type Cecropia was prominent. CONCLUSION Honey produced by Apis mellifera in the Reconcavo of Bahia, Brazil, has a multi-floral pattern with great contribution of the Mimosaceae species. In that sense, programs of implementation or expansion for beekeeping foraging in the region must consider members of this family in the floral composition.

v3-fos

2019-03-20T13:04:42.459Z

2015-05-08T00:00:00.000Z

83969464

s2

Optimization of carbon and nitrogen sources and substrate feeding strategy to increase the cell density of Streptococcus suis The swine pathogen Streptococcus suis is a worldwide emerging threat to public health. Efforts to develop an effective vaccine require the economic production of sufficient quantities of bacteria. To address this issue, the goal of the present study was to optimize culture conditions by reducing the accumulation of lactate through optimization of the medium components and feeding strategy. Sucrose (5 g/L) and a mixture of yeast extract and peptone (4 g/L each) were the most effective carbon and nitrogen sources, respectively, for S. suis ST171 fermentation. Further, from the results of S. suis ST171 fermentation with different ratios of carbon/nitrogen, 2 g/L sucrose was used as a carbon source and a mixture of 4 g/L yeast extract and 4 g/L peptone was used as a nitrogen source. Using these optimized conditions, S. suis ST171 fermentation with glucose-stat feeding strategy cultures accumulated a low concentration of lactate (3.47 g/L), which was accompanied by a high cell yield (2.712) and viability (1.192 × 1010 colony forming units/mL). Introduction Streptococcus suis is a Gram-positive, facultative anaerobic bacterium [1] that is economically important, because it causes a wide range of diseases in pigs, [2] including meningitis, septicaemia, pneumonia, endocarditis and arthritis. [3,4] Moreover, zoonotic infection of humans with S. suis, particularly serotype 2, causes serious diseases in many countries engaged in intensive swine production. [5] Two large outbreaks of S. suis human infections occurred in China in 1998 and 2005, causing 229 infections and 52 deaths because of toxic shock syndrome and meningitis. [6,7] The emergence of S. suis as a zoonotic agent is causing great public concern worldwide, particularly because a vaccine is not available. [4,8] The vaccine strain S. suis ST171 is a special strain for producing live vaccine against septic pig streptococcus disease. [9] Although S. suis ST171 is an attenuated strain that prevents infection, it is unsuitable for practical use, because it cannot be obtained in sufficient yields. Increased cell yields will likely reduce production costs, expand the market for the vaccine [9] and help protect the swine production industry and public health. Lactate is the primary metabolite that inhibits the growth of S. suis ST171 cultures. [9] The Emb-denÀMeyerhofÀParnas (EMP) pathway, the pentose phosphate pathway and the tricarboxylic acid (TCA) cycle operate in a number of Streptomyces species. [10,11] The excretion of lactate is not caused from the increased expression of lactate dehydrogenase (ldhA), but from the accumulation of pyruvate. [12] For example, the cell density of S. suis ST171 cultures is significantly improved by decreasing the accumulation of lactate and the excretion of lactate is decreased by optimization of the culture conditions, including the concentration of dissolved oxygen (DO), initial and residual glucose, and regulation of the strain growth rate. [9] The formation of by-products by Escherichia coli is strongly affected by the composition of the culture medium. [13] Replacing of glucose with mannose or fructose, as well as supplementing the medium with yeast extract, methionine or glycine reduces the accumulation of acetate in E. coli cultures. [14,15] Protein synthesis is sensitive to decreasing of C/N ratios and the carbon flux between protein and oil synthesis is regulated by the composition of the medium. [16] The levels of the transcripts of certain genes, required for the uptake and metabolism of carbon sources and the formation of by-products, are affected by the C/N ratio as well. [17] Moreover, an appropriate feeding method is important for achieving high cell densities of S. suis cultures. [9,18] The rate of formation of by-products by E. coli, such as acetate, is directly related to the growth rate or to the rate *Corresponding author. Email: [email protected] 1 Contributed equally to this work. of glucose consumption. [13] The excretion of by-products decreases when the glucose concentration is maintained at a low level. [19] In cultures of S. suis vaccine strain SD11, lactate accumulation decreases and cell density increases with the application of a pH-feedback feeding strategy. [18] Further, a strategy that combines exponential feeding and pH-stat feeding of E. coli cultures that produce tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), prevents the accumulation of acetate. Also, the dry cell weight and active soluble TRAIL were increased by 58.54% and 47.37%, respectively, when compared with a constant feeding rate strategy. [20] A useful approach involves applying glucose pulses to the feed and observing the changes in the DO concentration. The DOstat feeding avoids substrate overfeeding and O 2 limitation. [19] In the present study, the excretion of lactate during a fermentation process with S. suis ST171 was reduced by optimizing the composition of the medium, including the carbon and nitrogen sources, and the C/N ratio. In addition, the effect of intermittent feeding, DO-feedback feeding and glucose-stat feeding on S. suis ST171 fermentation were investigated to select an appropriate feeding strategy for a high cell density cultivation of S. suis. Materials and methods Bacteria and culture medium The strain S. suis ST171, used in this study, was obtained from China Institute of Veterinary Drugs Control (CVCC 563) and stored at the culture collection of the Shandong Binzhou Animal Science & Veterinary Medicine Institute. The seed medium contained the following components: 5 g/L sucrose, 5 g/L yeast extract, 3 g/L MgSO 4 ¢7H 2 O, 3 g/L KH 2 PO 4 and 0.1 g/L vitamin B 1 . The fermentation medium for S. suis ST171 contained the following components: 10 g/L sucrose, 5 g/L yeast extract, 2 g/L MgSO 4 ¢7H 2 O, 2 g/L KH 2 PO 4 and 0.1 g/L vitamin B 1 . The pH of the seed and fermentation media was adjusted to 7.0 with 4 mol/L NaOH. Culture methods Automated microbiology growth analysis systems A 250-mL baffled flask, containing 50 mL of seed medium was inoculated with a single colony of S. suis ST171 and cultivated at 37 C for 6 h. Thirty microlitres of this culture was inoculated into 400 mL of the inoculum medium in an Automated Microbiology Growth Analysis System (Bioscreen C, Oy Growth Curves Ab Ltd, Finland) containing 300 mL of fermentation medium and was cultivated at 37 C for 8 h. Ten litre fermenter A single colony of S. suis ST171 was inoculated into a 250-mL baffled flask containing 50 mL of seed medium and cultivated at 37 C with shaking at 200 rpm for 8 h. The 10-L fermenter containing 6 L of fermentation medium was inoculated aseptically with the culture grown in the baffled flask (2%, v/v). The temperature was maintained at 37 C and the pH was adjusted to 7.0 with 4 mol/L NaOH. The DO level was maintained at approximately 20% saturation by adjusting the agitation and aeration rates. When the initial sucrose was depleted, the feeding medium was added to the fermenter to meet the specific experimental requirements described below. Carbon and nitrogen sources. Feeding media and feeding strategies Different carbon and nitrogen sources were used during the study. Glucose, sucrose, lactose and galactose (4 g/L) were added into the fermentation medium in order to be used as carbon sources for S. suis ST171 fermentation process. Further, we obtained results from the fermentation processes by using yeast extract (5 g/L), peptone (5 g/L), beef extract (5 g/L) and a mixture of yeast extract (2.5 g/L) and peptone (2.5 g/L) as nitrogen sources. To determine the most appropriate fermentation conditions, we added different concentrations of the carbon and nitrogen sources, which gave the best results during the fermentation process. The used concentrations were 2, 5, 8 and 10 g/L for the chosen carbon source and 1, 2, 4 and 5 g/L for the chosen nitrogen sources. The used C/N ratios for the next trial were 1:1, 1:2, 1:4 and 1:10. Cell density, viable count [colony forming units (CFU)/mL] and accumulation of lactate (g/L) were measured during the study. In addition, the sucrose solution and glucose solution were selected as feeding media to investigate their effect on S. suis ST171 fermentation. The effect of intermittent feeding, DO-feedback feeding and glucose-stat feeding on S. suis ST171 fermentation were investigated to select the best feeding strategy for a high cell density cultivation. Analysis of fermentation products The DO, pH and temperature were measured automatically with electrodes attached to the fermenters. The optical density was monitored at 600 nm using an Automated Microbiology Growth Analysis System and a spectrophotometer (722N, INESA, China). The viable bacterial count in 1 mL of fermentation broth was determined as described previously. [9] The concentrations of glucose and lactate were monitored using an SBA-40E Biosensor Analyser (Biology Institute of Shandong Academy of Sciences, China). The concentrations of sucrose, lactose and galactose were determined using an Agilent 1206 (Agilent Technologies, Santa Clara, CA, USA) high-pressure liquid chromatography system. Statistical analysis All experiments were conducted in triplicate and the data were averaged and are presented as the mean § standard deviation. One-way analysis of variance followed by Dunnett's multiple comparison tests were used to determine significant differences. Statistical significance was defined as p < 0.05. Results and discussion Effect of carbon source on S. suis ST171 fermentation Carbon sources Certain carbohydrates serve as carbon sources to increase the growth rate and synthesis of cellular components of S. suis. Glucose is the most frequently used raw material for industrial fermentations, because it is relatively inexpensive and the most effective carbon and energy source. [21] The lac and gal gene clusters, which are arranged tandemly in the genome of Streptococcus gordonii, encode the components of the tagatose pathway (lacABCD) and the sugar phosphotransferase system. The permease-designated EII Lac transports lactose and galactose, whereas EII Gal transports galactose. [22] Glucose, sucrose, lactose and galactose were added into the fermentation medium in order to be used as carbon sources for S. suis ST171 fermentation process. The results displayed in Figure 1 indicated that when glucose was used as the carbon source, the cell density (0.884) and viable count (1.42 £ 10 9 CFU/mL) were the lowest and the accumulation of lactate (5.04 g/L) was the highest. The highest cell density (1.185) and viable count (1.98 £ 10 9 CFU/mL) and the lowest accumulation of lactate (3.14 g/L) occurred when using sucrose as the carbon source. Compared with galactose, growth in the presence of lactose yielded an increase in cell density (1.023) and viable count (1.72 £ 10 9 CFU/mL) by 6.89% and 2.38%, respectively. Also, the accumulation of lactate (3.74 g/L) decreased by 5.56%. The highest cell density and viable count were obtained with sucrose because of the low concentration of lactate. It has been proven that the accumulation of lactate is reduced in the presence of low concentrations of nicotinamide adenine dinucleotide hydrogen (NADH) that is generated primarily by the EMP pathway and TCA cycle [23] and NADH levels are low when carbon flow into these metabolic pathways is reduced. [24] The accumulation of lactate is lower in the presence of sucrose because of its complicated metabolic pathways. [25] We selected sucrose as the carbon source for S. suis ST171 fermentations. In S. suis SD11 fermentation, cell density and viable count, obtained with sucrose, were higher than that with glucose. [18] Sucrose concentration The results of fermentations using different concentrations of sucrose are presented in Figure 2. The accumulation of lactate was increased by increasing the concentration of sucrose. The highest cell density (1.213) and viable count (2.13 £ 10 9 CFU/mL) were obtained by using 5 g/L sucrose. Growth of bacteria in the presence of excess amount of a carbon source under aerobic conditions generates acidic by-products in the so-called 'overflow' metabolism. [26] Cell density and viable count decreased when the concentration of sucrose was above 5 g/L. Although low concentrations of lactate were accumulated in low sucrose concentrations, the latter did not satisfy the requirement for the growth amount of the S. suis vaccine strain SD11. [18] Compared with the 5 g/L surcose, the cell density and viable count obtained at 2 g/L sucrose were decreased by 8.17% and 11.26%, respectively. The cell density and viable count obtained with 2 g/L sucrose were higher than those obtained with 8 and 10 g/L sucrose. Effect of nitrogen sources on S. suis ST171 fermentation Nitrogen sources Nitrogen is an absolute requirement for cell growth and is assimilated by the synthesis of glutamine and glutamate. [27] The results of the fermentation processes by using yeast extract, peptone and beef extract as nitrogen sources, are displayed in Figure 3. The accumulation of lactate was increased with higher cell densities, which was in accordance with the study, described previously, which indicated that the concentration of lactate was directly proportional to the cell density. [28] The cell density and viable count obtained when using beef extract were lower compared with the values obtained when using yeast extract and peptone. Cell density (1.023) and viable count (1.92 £ 10 9 CFU/mL) obtained when using yeast extract were increased by 3.86% and 1.59% compared with the values obtained when using peptone, respectively. When a mixture of yeast extract and peptone was used, the highest cell density (1.147) and viable count (1.98 £ 10 9 CFU/mL) were obtained. Yeast extract and peptone are rich in free amino acids, which support cell growth with a low consumption rate of the carbon source and minimize the accumulation of by-products. [29] We selected a 1:1 mixture of yeast extract and peptone as a nitrogen source for S. suis ST171 fermentations. Figure 4 shows the effects of varying concentrations of 1:1 ratio of yeast extract combined with peptone. The cell density, viable count and accumulation of lactate increased when increasing the concentrations of these nitrogen sources. In the presence of mixture containing 1 g/L of each component, the cell density, viable count and lactate accumulation were 0.745, 1.42 £ 10 9 CFU/ mL and 2.74 g/L, respectively. With 4 g/L of each component, these values were 1.127, 2.01 £ 10 9 CFU/mL and 3.18 g/L, respectively. Compared with the obtained values when using 4 g/L of each component, the obtained values when using 5 g/L of each component were increased by 0.98%, 1.49% and 1.89%. Because there was not a significant difference between the values when using 4 or 5 g/L mixtures, we used the 4 g/L mixture to reduce the costs for further analysis. Mixture of yeast extract and peptone concentration Effect of the C/N ratio on S. suis ST171 fermentation The C/N ratio is more important than the nitrogen concentration for increasing the cell density and desired product concentration in fermentations. [30] Next, we maintained a constant amount of the nitrogen source mixture (4 g/L yeast extract and 4 g/L peptone) and added varying amounts of sucrose, so that the C/N ratios by weight [ D sucrose/(yeast extract C peptone)] were 1:1, 1:2, 1:4 and 1:10 ( Figure 5). As the C/N ratio increased, so did the production of lactate by S. suis ST171. On the other hand, the cell density and viable count decreased. These findings are consistent with the effect of the C/N ratio on acetate accumulation in cultures of E. coli. [17] With 1:1 and 1:10 ratios of C/N, the lactate concentrations were 4.32 g/L and 2.23 g/L, respectively. The highest cell density (1.147) and viable count (2.08 £ 10 9 CFU/mL) were obtained when the C/N ratio was 1:4; that is, 2 g/L sucrose and a mixture of 4 g/L yeast extract and 4 g/L peptone. The ratio of C/N affects the levels of transcription of genes (pfkA and pykF) related to the transport of carbohydrates. For example, as the C/N ratio increases, the expression of TCA cycle genes, such as gltA, icdA, fumC, sdhC and mdh increase, which also increases the accumulation of by-products. [17] Due to the higher nitrogen source in the fermentation medium with C/N ratio maintained at 1:4, higher cell density and viable count were obtained at 2 g/L sucrose, which indicated that sucrose of 2 g/L was used as carbon source for S. suis ST171 fermentation. Effect of feeding substrate on S. suis ST171 fermentation Feeding media Fed-batch processes are employed to achieve high cell densities, whereas the composition of the feeding medium is important for cell growth. [30] Figure 6 shows the results of S. suis ST171 fermentation with different feeding media, which indicated that the lactate concentrations were 5.32 and 4.51 g/L at the end of fermentation in the presence of glucose or sucrose, respectively. Cell density (2.478) and viable count (0.835 £ 10 10 CFU/mL) were 1.16-fold and 1.05-fold higher during the use of glucose, respectively, when compared with sucrose. Due to the increased accumulation of lactate, the increase in viable count and the consumption rate of carbon source with glucose were lower than these with sucrose during the late fermentation period, which was consistent with the reduction of production rate of desired product with higher excretion of acetate. [31] Because of the more complex metabolism of the sucrose, this carbon source did not satisfy the requirement of cell growth during the late fermentation period, which led to lower cell density and viable count, although lactate concentrations were lower. Therefore, glucose was selected as the feeding medium. Feeding strategy By adjusting the feeding rate of glucose in fed-batch fermentation, the concentration of glucose is maintained below a certain critical value required for the formation of by-products. [13] This is why the accumulation of byproducts is decreased by maintaining a low glucose concentration. [31] The results from using intermittent feeding, DO-feedback feeding and glucose-stat feeding are presented in Figure 7. The concentration of glucose was maintained at approximately 0.5 g/L using intermittent feeding and at a low level (0.1 g/L) with DO-feedback and glucose-stat feeding, but glucose starvation occurred with DO-feedback feeding. Using intermittent feeding, the lowest cell density (2.343) and viable count (0.875 £ 10 10 CFU/mL) and the highest accumulation of lactate (5.12 g/L) were obtained. Compared with cell density (2.652), viable count (1.078 £ 10 10 CFU/mL) and accumulation of lactate (3.31 g/L) obtained using DO-feedback feeding, these respective values were increased by using glucose-stat feeding, which were 2.712, 1.192 £ 10 10 CFU/mL and 3.47 g/L, respectively. The lactate that was accumulated using DO-feedback feeding was reused during the late fermentation period and the production rate of lactate using glucose-stat feeding was lower compared with that of intermittent feeding. Lactate that accumulated during growth is recaptured because of the change in expression levels of ldhA and Dld (encoding NAD C -dependent and NAD C -independent lactate dehydrogenases) in the stationary phase. [32] The consumption rate of glucose with intermittent feeding was higher, compared with those of DO-feedback and glucose-stat feeding. The glucose consumption rate was the lowest with DO-feedback feeding. Glucose-stat feeding maintains a low lactate concentration and serves as a sufficient source of carbon to support cell growth. [31] Here, the highest cell density and viable count were obtained using glucose-stat feeding. Conclusions In summary, we showed that the accumulation of lactate in fermentation cultures of S. suis ST171 was decreased by optimizing the concentrations of medium constituents and feeding strategy. Higher cell density and viable count were also obtained. The study, therefore, illustrates a useful approach for large-scale production of a vaccine strain of S. suis ST171. This enhances the application of a vaccine, leading to prevention of the occurrence of septic pig streptococcus disease and promotion of the pig industry development. Disclosure statement No potential conflict of interest was reported by the authors.

v3-fos

2018-04-03T02:21:46.369Z

2015-03-30T00:00:00.000Z

207359464

s2

Correction: Transcriptome Analysis of Resistant and Susceptible Alfalfa Cultivars Infected With Root-Knot Nematode Meloidogyne incognita Nematodes are one of the major limiting factors in alfalfa production. Root-knot nematodes (RKN, Meloidogyne spp.) are widely distributed and economically important sedentary endoparasites of agricultural crops and they may inflict significant damage to alfalfa fields. As of today, no studies have been published on global gene expression profiling in alfalfa infected with RKN or any other plant parasitic nematode. Very little information is available about molecular mechanisms that contribute to pathogenesis and defense responses in alfalfa against these pests and specifically against RKN. In this work, we performed root transcriptome analysis of resistant (cv. Moapa 69) and susceptible (cv. Lahontan) alfalfa cultivars infected with RKN Meloidogyne incognita, widespread root-knot nematode species and a major pest worldwide. A total of 1,701,622,580 pair-end reads were generated on an Illumina Hi-Seq 2000 platform from the roots of both cultivars and assembled into 45,595 and 47,590 transcripts in cvs Moapa 69 and Lahontan, respectively. Bioinformatic analysis revealed a number of common and unique genes that were differentially expressed in susceptible and resistant lines as a result of nematode infection. Although the susceptible cultivar showed a more pronounced defense response to the infection, feeding sites were successfully established in its roots. Characteristically, basal gene expression levels under normal conditions differed between the two cultivars as well, which may confer advantage to one of the genotypes toward resistance to nematodes. Differentially expressed genes were subsequently assigned to known Gene Ontology categories to predict their functional roles and associated biological processes. Real-time PCR validated expression changes in genes arbitrarily selected for experimental confirmation. Candidate genes that contribute to protection against M. incognita in alfalfa were proposed and alfalfa-nematode interactions with respect to resistance are discussed. Introduction After the arthropods, nematodes are the most populated phylum comprising~80,000 of currently described species and originating about one billion years ago [1]. A large number of nematodes (40% of the described species) are free-living organisms that consume bacteria, fungi, other nematodes and protozoa [2]. Other species are parasites of animals (44% of the described species) and obligate parasites of plants (15% of the described species) [2]. Average worldwide crop losses due to plant-parasitic nematodes constitute 12-14% or more than $100 billion per year [3][4][5]. Ubiquitous root-knot nematodes (RKN) of the genus Meloidogyne are among the most damaging and economically important species [6][7][8]. As a result of RKN feeding, large galls or "knots" are formed on the roots of infected plants. They consist of giant multinucleate cells that never divide and serve as nutrition reservoirs for nematodes, providing diminished resources for the rest of the plant [4]. Among nearly 100 described species in the genus Meloidogyne, four represent a major threat to agricultural crops: Meloidogyne incognita, Meloidogyne arenaria, Meloidogyne javanica and Meloidogyne hapla [8]. RKN M. incognita is the most common and destructive nematode species found in all agricultural regions worldwide [4] and is considered as "the single most damaging crop pathogen in the world" [9]. According to Commonwealth Agricultural Bureaux International (CABI), it is widespread in 25 states of the USA (CABI Invasive species compendium, http://www.cabi.org/isc/). Alfalfa (Medicago sativa), the most extensively cultivated forage legume and the fourth most widely grown crop in the US [10], is a host for several important nematode species, namely stem nematode (Ditylenchus dipsaci), root lesion nematode (Pratylenchus spp.), cyst nematode (Heterodera spp.) and root-knot nematode (Meloidogyne spp.). Five species of RKN are of importance on alfalfa, including the southern RKN M. incognita, the northern RKN Meloidogyne hapla, the Columbia RKN, M. chitwoodi, the Javanese RKN M. javanica, and the peanut RKN M. arenaria. These nematodes are a significant economic concern to alfalfa production because of their wide distribution, broad host range and the serious damage they can cause to the crops grown in rotation with alfalfa [11,12]. Although many sources of genetic resistance to RKN have been identified and characterized in other crops [5], little information exists on the mechanisms of resistance responses to RKN in alfalfa. Similarly to many commercially available alfalfa cultivars resistant to other environmental factors, alfalfa plants resistant to RKN are selected from germplasm with field resistance or resistance obtained under experimentally controlled conditions. In most cases, alfalfa-nematode interactions and mechanisms underlying the observed resistance to nematode infection remain uncharacterized and poorly understood at the molecular level. Whereas early attempts to address this question provided new insights into the basis of alfalfa susceptibility and resistance to RKN [13,14], recently developed genomic resources and technologies that quantify changing expression levels during development and under different conditions [15] have not yet been applied towards improving our understanding of these complex interactions. In the meantime, it was noted that the alfalfa resistance response to RKN lacks the typical hypersensitive reaction (HR) seen in tomato and tobacco [14]. This observation was later confirmed in a model legume, Medicago truncatula, a close relative of alfalfa [16]. Thus, it appears that the biological processes that underline resistance to nematodes in alfalfa are quite unique, which may also be reflected in the structure of alfalfa transcriptome. Here, we approached this problem by means of transcriptome profiling of two alfalfa cultivars contrasting in response to RKN M. incognita. Prior to this work, RNA-sequencing had never been used to study alfalfa-nematode interactions even though this radical technology can transform research in the field and open up new possibilities for the investigation of survival strategies employed by both a host plant and a parasitic nematode. Plant material, inoculum preparation and inoculation Two alfalfa cultivars contrasting in response to M. incognita were used in the experiments: Moapa 69 (resistant) and Lahontan (susceptible) [13,17]. For germination on the medium, seeds were scarified with H 2 SO 4 , surface sterilized with 70% ethanol for 3 min and with 1.2% sodium hypochlorite solution for 10 min, rinsed with distilled water and placed on 1% water agar, pH 5.7. Three-day old seedlings were cut at hypocotyls and the roots were transferred to individual Petri dishes (3 roots per dish) containing Gamborg's B5 medium with vitamins and sucrose supplemented with 0.1% PPM [18,19]. Excised roots were left to grow until they filled up a significant amount of the media plate and developed lateral roots. Inoculum of M. incognita was prepared by extracting nematode eggs from infected roots of pepper plants, (cv. PA136), using agitation in 10% household bleach (adaptation of [20]). The eggs were rinsed with distilled water on 500 μm-pore sieves. A population of 800-900 eggs in 1 ml of water was transferred onto excised root cultures, sealed with Parafilm and kept in an incubator at 28°C. Before samples were collected, separate sets of root cultures were observed for a period of one month post inoculation to confirm establishment of nematode infection. Infected roots, stained with acid fuchsine were scanned for the presence of nematodes under the Zeiss light microscope. Conventional PCR with M. incognita-specific primers (S1 Table) was also used to confirm infection. For RNA-seq, roots were collected 10 dpi, thoroughly rinsed with distilled water and used for total RNA extraction. Four replicates (one Petri dish with excised roots represented one replicate) were used for the inoculated roots and non-inoculated control. According to Potenza et al. [13], changes in gene expression in both cultivars were noticeable 24 hrs after inoculation, more pronounced 72 hrs after inoculation, and were not observed 14 dpi. Since we collected roots 10 dpi and it took on average 3 days for J2 nematodes to hatch from eggs, our time point represents 7 dpi. RNA extraction, first-strand synthesis and quantitative real-time PCR Total RNA extraction, first-strand synthesis and quantitative real-time PCR (qPCR) were performed essentially as described in Postnikova et al [21]. Amplification was conducted with an iQ SYBR Green Supermix kit (Bio-Rad Laboratories, Inc.) on the MiniOpticon Real-Time PCR system (Bio-Rad) in three to four biological and two technical replicas using the following parameters: 94°C/1 min (one cycle); 94°C/30 s, 60°C/30 s, 72°C /30 s (30 cycles). cDNA for qPCRs was made from the same RNA samples that were used for RNA-sequencing. Previously detected NP_001237047, an unknown gene with little variation in expression levels, was used as a reference in all qPCR experiments [21]. The specificity of all amplifications was confirmed by single-peak melting curves. Delta Delta C (T) method (2 −ΔΔC T ) was used for analysis of relative expression. A ratio between each of the nematode-infected samples and a corresponding average of the mock-inoculated samples was calculated. To obtain a final ratio for any given gene, an average and a standard deviation (SD) for all biological replicates were calculated. RNA-seq, transcriptome assembly and analysis RNA-sequencing was performed by GENEWIZ, Inc (South Plainfield, NJ, USA) for a fee using the Illumina HiSeq 2000 system. There were four replicates for each experimental condition. For each of the two cultivars, eight samples representing roots from individual Petri dishes were sequenced (four control and four infected). Two types of cDNA libraries were generated (polyA selection and rRNA depletion) and combined for the transcriptome assembly. Only the polyA selection libraries were used for the expression profiling since the cDNA libraries derived from rRNA depletion method did not form correct clusters (S1 Fig.). To achieve a high quality alfalfa root transcriptome, de-novo transcripts were acquired using a range of k-mer sizes (k-mers 49, 53, 55 and 57) by Oases 0.2.08 and Velvet 1.2.07 genome assemblers. Since the libraries were obtained by paired-end sequencing, the 'shortPaired' parameter was used in Velvet. The file sizes for each library on average ranged from 4.GB to 6GB, which corresponds to 15 million to 25 million reads for each file, respectively, or 30 million to 50 million reads for each pair. On average, each assembly with varius k-mers yielded between 120,000 to 200,000 contigs. In order to generate and improve a complete alfalfa transcriptome and utilize many de novo assemblies, these results were processed with the Evi-dentialGene pipeline, as described in Nakasugi et al. [22]. The process started with CD-HIT program that removes identical fragments, thus reducing the redundancy from combining the assemblies (http://weizhongli-lab.org/cd-hit/). Next, the EvidentialGene pipline extracted biologically meaningful transcripts from the resulting CD-HIT pool of sequences utilizing the coding potential. The EvidentialGene pipeline produces the best coding sequences (CDS and amino acid sequences) among highly similar transcripts though blast alignments. The resultant transcriptome served as a template for gene expression profiling. The DESeq 2 package from Bioconductor [23] was used to estimate sample quality (PCA) and the expression level of transcripts. The DESeq package is a program in the R statistical software suite that accepts raw counts of sequencing reads from high throughput experiments as input into the program. The DEseq program performs normalization, variance estimation and differential expression of the raw read counts and works best with experiments with replicates. Differential expression [>2 fold, false discovery rate (FDR) < 0.025] of alfalfa genes was assessed by mapping reads to the de novo-assembled TCs using Bowtie2-2.1.0. Raw counts of the mapped reads were extracted from the Bowtie alignment for each library. After transcriptome assembly, genes were annotated and assigned to known functional groups as previously described [21]. For detection of differentially expressed transcripts in nematodes a publicly available M. incognita database was used (www.nematode.net). M. incognita on alfalfa root culture Preliminary experiments demonstrated that M. incognita grew and reproduced well on alfalfa excised root culture. Primary signs of infection represented by root galls were first visually detected on susceptible cv. Lahontan at 10 days post-inoculation (dpi). In most cases, with few exceptions, galls were not formed on roots of resistant cv. Moapa 69, which has an approximate expected resistance to M. incognita of 50% [17]. Four weeks after inoculation, infective juveniles and sedentary female nematodes were readily observed on excised roots of cv. Lahontan ( Fig. 1A and B); females were often surrounded by laid eggs (Fig. 1C). Overview of the generated assemblies A total of 1,701,622,580 short reads were obtained. Seven different transcriptomes were assembled using four k-mers values (49, 53, 55 and 57) and then combined into a high quality single transcriptome with the EvidentialGene pipeline [22], (Table 1). We were able to map back to the largest assembly~84% of paired end reads in each individual sample with Bowtie2 Aligner [24]. Between 80% and 81.5% of all assembled tentative consensus sequences (TCs) had significant (e-value < 0.00001) blast hits with the protein database and Medicago truncatula database M.t.4.1, respectively (S2A and S2B Tables). Based on the nr database the total assembly contained 25,151 unique genes. This number is close enough to 31,661 high confidence gene models found in M. truncatula up to date. Thus, root transcriptome assessment showed that data obtained by RNAseq are sufficient for gene expression profiling ( Table 1). Identification of the nematode-responsive transcripts and their functional categorization Differential expression [>2 fold, false discovery rate (FDR) <0.025] of alfalfa genes of both cultivars was evaluated using DESeq 2 tool [23]. Count data were obtained by mapping reads to the de novo-assembled TCs with Bowtie 2 [24]. Cv. Lahontan. We found 1143 differentially expressed TCs in this susceptible to M. incognita cultivar ( Table 2). These TCs included unknown sequences with no blast hits. All TCs with sequence similarity to the nematode were removed from the list. Among 1143 TCs, 923 had high similarity scores with M. truncatula (e-value <0.00001) and 712 of them were unique (non-redundant). Relative to all non-redundant TCs with sequence similarity to M. truncatula, DEG represented 3%. The number of induced and repressed unigenes in cv Lahontan during nematode infection was similar (373 vs 339, respectively; S3 Table). Using AgriGO software [25], 712 unigenes were further assigned to Gene Ontology (GO) terms for annotation and description of their prospective biological functions. GO overrepresentation analysis showed that GO terms "response to stress", "response to stimulus" and "defense response" in the domain "biological process" were significantly overrepresented among both up-and down-regulated genes (S3 Table). Other categories, such as "regulation of cellular process" "biological regulation", "translation", etc were significantly overrepresented only among the up-regulated gene set, suggesting involvement of the corresponding biological processes in the general response of this cultivar to nematode infection. The number of genes in the overrepresented GO categories is shown in Fig. 2. Comparison of the DEGs between susceptible and resistant cultivars. Sets of the DEGs revealed in Lahontan and Moapa 69 were compared with each other. Contrasting the responses of DEGs that are common to both cultivars and distinguishing the set of DEGs that are unique for each line can help to identify genes that may be critical for genetic susceptibility or resistance to M. incognita. We found that 51 common genes were differentially expressed in the two cultivars while 827 DEGs were unique to one or the other (Fig. 3 and S5 Table). Among the common genes, 36 and 21 DEGs were up-regulated while 15 and 30 DEGs were downregulated in Lahontan and Moapa 69, respectively (S5 Table). Expression of some common genes was discordant between the lines; two clusters of these discordantly expressed genes had elevated basal expression levels in the resistant ecotype (S5 Table), suggesting their specific roles in response to the infection. Interestingly, when the composition of the common and distinct DEGs was evaluated, we found that both sets contain a significant number of genes that are associated with resistance against pathogens (R genes), (S5 Table). Putative R genes were detected by blastp search against manually curated R genes of the PRGdb (112 genes), an open-source database of plant resistant genes [26]. The PRGdb recognizes at least five R protein types: TIR-NBS-LRR, CC-NBS-LRR, RLK (receptor-like kinases), RLP (receptor-like protein) and RLK (kinase-like protein) [26,27]. However, because many R genes are missing from the manually curated PRGdb or not classified as such, we added to the blastp output known, annotated resistant genes from M. truncatula (12 genes) and screened the final list against our DEGs. This analysis revealed that almost one-third (16) of the common DEGs are R genes. Two of them, orthologous to M. truncatula's Medtr3g056585 and Medtr0277s0020.3, were induced in Moapa 69 and repressed in Lahontan. The distinct DEGs of the susceptible line contained 121 (17%) putative R-genes, 95 of them were induced and 26 repressed (S4 Table). The resistant cultivar contained 36 (16%) distinct DE R-genes: 14 of them were up-regulated and 22 repressed (S5 Table). Apparently, since cv. Lahontan is a susceptible cultivar, the presence of non-specific DE R-genes has no influence on a nematode's ability to cause disease and may be part of a general or passive defense reaction. On the contrary, distinct DE R-genes found in Moapa 69, especially 14 induced genes, may contain a candidate gene(s) responsible for the unique resistance interactions between this cultivar and RKN [14,16]. The same is true for the two common R-genes upregulated in Moapa. GO analysis of the distinct DEGs in cv. Lahontan revealed other interesting categories, not only terms related to "defense response", are overrepresented as well. Distinct up-regulated genes of cv. Lahontan were also distributed among categories "biological regulation", "translation", "hydrolase activity", "ribonucleoprotein complex", "cytoplasm" etc. (S5 Table). Downregulated genes, in addition to "defense response" and "signaling" terms, were classified into categories "response to oxidative stress", "response to chemical stimulus", "antioxidant activity", "oxidoreductase activity", "peroxidase activity". These terms were not found among overrepresented GO categories of cv. Moapa. Apparently, induction and repression of genes associated with these biological processes in cv. Lahontan reflect not only host basal defense responses during compatible interaction, but also activities related to the successful establishment of nematode parasitism. Using MapMan annotation software to organize and display our data sets in the context of biological pathways [28], we found 92 genes related to biotic stress responses among distinct DEGs of cv. Lahontan and 17 in cv. Moapa (S2 Fig. and S6 Table). The majority of them are pathogenesis-related proteins (PRs) whose expression varies as much as 36-fold (-4 to > +4, log 2 ). Production of PRs in response to biotic stress is well-known since most of them possess antimicrobial activity [29]. The presence of significant numbers of induced PRs in the susceptible line (69) as compared to the resistant one (5) may be explained by their involvement in basal defense responses that do not play a critical role in resistance pathways to specific pathogens. DE transcripts associated with ubiquitin dependent proteasome degradation pathway were noticeable in both cultivars, especially in Lahontan, where they were significantly induced. Most intracellular proteins are degraded by this pathway and selectivity of proteolytic processes can indicate different rates of protein synthesis and degradation. In general, a larger variety of MapMan-outlined metabolic processes in the susceptible line suggests a steady, continuous defense response that is overcome by the pathogen. When we assessed the final transcriptome for the presence of DE transcription factors using TF domain footprints [30], only 24 TFs were located among all DEGs in both cultivars: four in cv. Moapa and 20 in Lahontan (S7 Table). Almost all of them were down-regulated, with the exception of several TFs (4) in cv. Lahontan. No common DEG TFs were detected in both lines. Theoretically, the limited number of DE TFs during infection may be attributed to the temporal difference between transcriptional regulation and changes in gene expression levels. Basal gene expression levels in normal conditions. Comparing basal gene expression levels in uninfected plants of susceptible and resistant cultivars can provide important clues to the behavior of genes that may influence plant responses to nematode infection. That is, these genes may already be "preconditioned" toward disease, as evident from differences in their basal expression profiles. A total of 1433 DEGs were found when two cultivars were compared to each other under normal conditions (a ratio between Moapa 69/Lahontan, S8 Table). Among them, basal expression of 769 genes was elevated in the resistant line. When these 769 genes were screened against 82 DEGs up-regulated during RKN infection, 13 genes matched, i.e. had concordant expression in healthy and infected plants of cv. Moapa 69 (S8 Table). In this group, there were six R genes orthologous to M. truncatula Medtr6g088245.1, Medtr6g072450.1, Medtr6g074810.1, Medtr6g088250.1, Medtr0277s0020.3 and Medtr6g472230.1 and one RNAbinding protein with CCCH-type zinc finger domain orthologous to Medtr3g056160.1. Comparison between 1433 genes differentially expressed under control conditions and 51 common DEGs found in both cultivars during RKN infection revealed 26 DEGs (Fig. 3 and S8 Table). Nine of them were concordant in healthy and infected plants of cv. Moapa 69 (5 induced and 4 repressed). In this cluster, the R gene of the TIR-NBS-LRR class, orthologous to Medtr0277s0020.3, was notably up-regulated in healthy and infected roots of Moapa, while its expression was reduced in the susceptible cultivar during RKN infection. Discordant DEGs in cv. Moapa 69 included 16 genes: 14 of them changed their expression pattern from being induced under normal conditions to becoming repressed under infection, and two downregulated genes became induced during infection. Promising DEGs obtained by basal expression profiling are summarized in S8 Table. Confirmation of the transcriptomic data by Quantitative Real-time PCR QPCR was performed with 33 arbitrarily selected unique genes classified as differentially expressed based on the transcriptomic analysis. Most of them were chosen from DEGs common between two lines so that expression could be experimentally validated in both cultivars. QPCR results showed a strong correlation with transcriptomic data (Pearson correlation coefficients r = 0.7, Table 3). Particularly interesting are two genes that were up-regulated during infection in Moapa 69 and repressed in Lahontan ( Table 3). One of the DEGs is likely to be an R gene with a characteristic NBS-LRR domain (Medtr3g056585.1) and another DEG (Medtr5g087320) was a receptor-like protein kinase that also has a blast hit with resistant genes. [24]. On average, alignment rates for both lines were 0.4% from the total number of reads obtained. Only 348 M. incognita transcripts were assembled by more than 50 short reads (S9 Table). Unfortunately, current genome annotation of M. incognita (http://www7.inra.fr/ meloidogyne_incognita/genomic_resources/downloads) that would enable a comprehensive description of our TCs, is in poor condition: complete annotation tables and GFF files with start and end coordinates, are not accessible for downloading. For this reason, we attempted to classify nematode-related TCs into GO terms using the annotated genome of C. elegans (http://www.uniprot.org/uniprot/?query = taxonomy:6239). Since C. elegans is not a parasitic nematode, this analysis was meant to place genes into general biological pathways and metabolic processes that may not necessarily be involved in parasitism in M. incognita. Among the 348 TCs, 208 had blast hits with C. elegans, 16 with other species and 124 did not have any blast hits (S9 Table). Out of 208 TCs, only 78 were distributed into GO terms based on the similarity with C. elegans genome (S9 Table). Some potentially interesting or highly expressed M. incognita TCs, which might be important for parasitic relationship did not have any match with C. elegans. Two of these genes (M. incognita TCs BM882635, the C-type lectin-like domain superfamily and TA989_6306, putative NADH-ubiquinone/plastoquinone, complex I protein), along with the gene orthologous to C. elegans (BM774159, protein of the CD36 superfamily), were down-regulated in nematodes infecting roots of resistant cultivar Moapa 69, as compared to the nematodes from susceptible cv. Lahontan (S9 Table). Three other genes without any blast hits with C. elegans genome (M. incognita TCs TA2213_6306, no blast hits, TA189_6306, blast hit with an unknown protein of parasitic roundworm Ancylostoma ceylanicum and CF099765, blast hit with a small integral membrane protein 20 of red flour beetle Tribolium castaneum), were highly expressed in nematodes infecting both cultivars. Discussion As of today, no studies have been published on global gene expression profiling in alfalfa infected with RKN or any other plant parasitic nematode. Very little information is available about molecular mechanisms that contribute to pathogenesis and defense responses in alfalfa against these pests, and specifically against RKN. A few available published reports, however, pointed to the unique resistant pathway implemented in alfalfa against RKN [13,14,16]. As the response of resistant alfalfa does not preclude nematode penetration into the plant, it appears to be cardinally different from the mode of resistance to M. incognita identified in tomato and tobacco due to the lack of localized hypersensitive response (HR). According to Williamson and Kumar [5], nematode resistance, as a general rule, is characterized by a localized programmed cell death at or near the feeding site. This reaction is normally controlled by a single or multiple resistance genes. Pioneering studies of Potenza and co-authors [13,14] revealed that resistance to M. incognita in alfalfa cv. Moapa 69 does not rely on apoptotic cell death but may occur due to the inability of the RKN to enter developing vascular cylinder of the root, instead remaining clumped at the root apex as early as 48-72 hours after inoculation. Using Northern blot and genomic DNA blot analyses, they further identified four genes that are potentially involved in Moapa 69 resistant interactions with M. incognita: glycine-rich RNAbinding protein, phosphoenolpyruvate carboxykinase, isoflavone reductase-like protein, and metallothionein-like protein. While screening the world-wide accession collection of M. truncatula with several RKNs, Dhandaydham and co-authors [16] found that one variety, DZA045, was resistant to M. incognita without exhibiting an HR response. The authors suggested that a single gene may control resistance in DZA045 by a unique pathway. Judging from their observations showing the absence of HR symptoms and poor development of RKN in the roots, this pathway could be similar to the one described by Potenza et al. in alfalfa [14]. Thus, it appears that in those cases plants may not implement an R gene-based response, instead using other defensive strategies of a basal nature which depend on different gene products. Alternatively, if host R-genes are involved in the process, their action may differ from typical gene-for-gene interplay leading to the HR after pathogen's recognition by a specific R gene [5]. One known example of this kind of interaction is "defense, no death" (dnd) phenotypes in Arabidopsis thaliana, which exhibit loss of hypersensitive response (HR) cell death without loss of genefor-gene resistance [31]. In this work, we attempted to shed more light on these questions by performing root transcriptomic analysis of resistant (cv. Moapa 69) and susceptible (cv. Lahontan) alfalfa cultivars infected with RKN Meloidogyne incognita. By generating whole transcriptome profiles, Illumina RNA-seq not only permits a precise estimation of gene expression but is also capable of identifying new genes, transcript variants and genetic polymorphisms. It is especially informative for species without annotated genomes, such as alfalfa. Identification of DEGs in resistant and susceptible cultivars was of primary importance since differential expression of some of the gene products during nematode infection may be a key to the resistance interactions. Differential expression of the genes found in common between the two lines not only points to the genes participating in general response to infection and to the changes initiated by pathogen to establish parasitism, but also demonstrates contrasting behavior of the same gene products under the infection background. The latter could suggest specific biological roles associated with mechanisms of resistance. In this sense, a discordant expression of common genes between the two lines was of particular interest. In total, we have found 23 common discordantly expressed DEGs (S5 Table). The majority of them (19) were down-regulated in the resistant cultivar. Moreover, 11 of those genes were induced in Moapa in healthy plants, indicating that their declining levels in infected plants are not coincidental. Four common genes, up-regulated during infection in Moapa and repressed in cv. Lahontan, (Medtr3g056585, Medtr5g087320.1, Medtr0277s0020.3, and Medtr7g105720.1), in our opinion, may be of special importance to the resistance pathway in cv. Moapa 69. Presence of at least two R genes among them (Medtr3g056585, an LRR and NB-ARC domain disease resistance protein and Medtr0277s0020.3, a disease resistance protein of TIR-NBS-LRR class) support the idea of atypical gene-for-gene interactions. Furthermore, among 936 distinct DEGs found in both cultivars, 154 (16%) were putative R genes, classified as such according to the PRGdb. [26,27]. Considering that genome of M. truncatula (45,888 protein-coding transcripts, Mt3.5 v4) contains approximately 2,084 (4.5%) putative R genes (predicted from Phytozome, http://www.phytozome.net/), this is an impressive number. Although the level of R-genes expression is usually low [5], individual R genes may be induced in response to nematode infection and this induction can play a role in signal transduction pathways, leading to resistance [5]. When we examined all DE R genes detected in both lines using PDGdb, five of them were found to be involved in nematode resistance: Medtr6g015510.1 and Medtr6g088250.1 in Lahontan and Medtr6g015510.1, Medtr6g088250.1, Medtr6g087200.1, Medtr4g080777.1 and Medtr6g084360.1 in Moapa. All these R genes, representing different transcripts, mapped to the same ortholog in M. truncatula: Solanum tuberosum nematode resistance protein (Gro1-4) gene, conferring resistance to pathotype Ro1 of the root cyst nematode Globodera rostochiensis [32]. We were not able to locate any information on the occurring of the HR-like phenotype in potato cultivars resistant to G. rostochiensis pathotype Ro1. Apparently, two R genes induced in cv. Lahontan, (orthologous to Medtr6g088250.1 and Medtr6g015510.1) are irrelevant for resistant pathways. However, both of them are upregulated in cv. Moapa and one R-gene, Medtr6g088250.1 is induced in the resistant line during normal conditions. In other words, the level of the Medtr6g088250.1 transcripts, being already significantly elevated in healthy plants as compared to cv. Lahontan, is even more induced after nematode infection. Four genes previously found to be induced in both cultivars during infection with M. incognita [14] were not among the set of DEGs detected in this work. One possible explanation is the timeline of the experiment: early response to infection [14] vs 7 dpi (this study). To search for similarities between our data and plant-RKN reactions in other species, we compared them with two published reports: a study on comprehensive gene expression profiling in tomato resistant to RKN [33] and transcription profiling of soybean-root-knot nematode interaction [34]. Responses to RKN in other species varied. Gene expression profiling of tomato resistant to RKN (resistant gene Mi-1) revealed 58 differentially expressed genes in resistant roots. Among them only one common gene, orthologous to Medtr3g054080.1 (Solyc11g072630.1.1), was up-regulated both in resistant alfalfa cultivar and in resistant tomato in response to the RKN. In M. truncatula, it encodes cyclin-dependent (CDK)-activating kinase and in tomato mitogen-activated protein kinase (MAPK). CDKs and MAPKs are related and regulate a variety of cellular functions, such as cell cycle and proliferation, transcription, mRNA processing, response to environmental stimuli etc. Based on alfalfa transcriptome analysis presented in this study, the gene (ID evgevgevgvelvLoc27212t3, S2A and S2B Table) was annotated as a cyclindependent kinase. Comparison of our data with the expression profiling of soybean PI 595099 (resistant line)-RKN pathosystem [34] by direct blast search, revealed 16 common differentially expressed genes in resistant cv Moapa 69 and RKN-resistant soybean line (S10 Table). Interestingly, that two orthologous genes not particularly mentioned by the Beneventi and co-authors [34] as playing a role in resistant reaction, are on our list of gene-candidates involved in resistant response in cv. Moapa (Medtr3g054080.1 and Medtr2g096970.1). One of them is again CDK (Medtr3g054080.1). Both genes were up-regulated in cv Moapa and resistant soybean line. Activation of CDKs and/or MAPKs in resistant lines of three different species in response to M. incognita may indicate their involvement in molecular mechanisms controlling resistance to root-knot nematode in legumes. Only 24 TFs were found to be differentially expressed in both lines during nematode infection (S7 Table). Speculatively, the limited number of DE TFs as well as a general decrease in their activity may be attributed to the temporal difference between transcriptional regulation and changes in gene expression levels. On the other hand, down-regulation of this small group of TFs can be a specific requirement for their functional roles in plant defense mechanisms or otherwise in establishment of parasitism. Little is known about molecular mechanisms of RKN parasitism in alfalfa and nematode genes involved in interaction with this plant host. This question was not a focus of this study; but, since we needed to clearly differentiate nematode-originated sequences from alfalfa transcripts, it created an opportunity to look at the main biological processes involved in parasitism. We were able to find common GO terms corresponding to biological pathways responsive to interactions of nematodes with resistant and susceptible hosts (S9 Table). M. incognita genes orthologous to the C-type lectin-like domain superfamily, NADH-ubiquinone/ plastoquinone (complex I) protein and CD36 protein family were down-regulated in nematodes infecting resistant cv Moapa. The Metazoan proteins with C-type lectin-like domains (CTLDs) have diverse functions including cell adhesion, intracellular processes and trafficking of proteins [35]. NADH-ubiquinone/plastoquinone (complex I) protein is an enzyme of the respiratory chain located in the mitochondrion and involved in proteasome core complex assembly, response to misfolded protein and ubiquitin-dependent protein catabolic processes (http://www.arabidopsis.org/servlets/TairObject?id=27598&type = locus). CD36 ortholog in C. elegans hypothetically acts as a receptor for microbe-derived ligands and is thought to be involved in host resistance to fungal pathogens [36]. Whether reduced activity of these genes in M. incognita is associated with failure of the nematode to infect resistant cultivar remains a question (S9 Table). In brief, transcriptome analysis of two alfalfa cultivars contrasting in resistance to RKN M. incognita, identified nearly a thousand DEGs that are presumably involved in basal defense responses (cv. Lahontan) and in resistance pathways (cv. Moapa). Comparison of DEGs between the cultivars revealed a number of transcripts potentially associated with resistance to nematode in cv. Moapa. Their prospective roles in resistance mechanisms were based on analysis of the specific expression levels of DEGs in common and distinct to each of the lines and also on the extensive functional annotations generated by blast, GO and MapMan tools. The most promising gene-candidates are presented in S11 Table. Conclusions The results of this study demonstrate that the host response to RKN M. incognita is significantly larger in the susceptible cultivar, with the number of DEGs in cv. Lahontan exceeding those from cv. Moapa by three-to-one margin. The same was true for the number of induced transcripts. Apparently, this is related to the fact that M. incognita established successful infection in cv. Lahontan while in the resistant line the parasitic interaction with the host was mostly aborted. Potenza and co-authors [13,14] reported that J2 nematodes were clumped into root tips of cv. Moapa as early as 72 hrs after inoculation but migrated upward into the developing vascular cylinder in the roots of cv. Lahontan. This observation may indicate that most of the changes in plant gene expression critical to the initiation of resistance pathways occurred before this stage. However, the authors [13] were able to detect differential expression in cv. Moapa for as long as two weeks after inoculation. Besides, in our experiment, non-clustered migrating nematodes were occasionally observed in roots of cv Moapa for as long 7 dpi (S3 Fig.), which in addition to the sporadic presence of susceptible genotypes and delayed root penetration by J2 nematodes, could be attributed to the possible mode of resistance different from the one suggested by Potenza and co-authors [13]. Accordingly, DEGs found in this work could be actively involved in conferring resistance against M. incognita. Additional transcription profiling at the single-cell level with individually-infected root exudates will provide further answers to these questions. The raw data and transcriptome assembly obtained in this study were deposited in the Sequence Read Archive, NCBI (BioProject ID PRJNA266116).

v3-fos

2016-05-12T22:15:10.714Z

2015-06-01T00:00:00.000Z

18850404

s2

Initial pH of medium affects organic acids production but do not affect phosphate solubilization The pH of the culture medium directly influences the growth of microorganisms and the chemical processes that they perform. The aim of this study was to assess the influence of the initial pH of the culture medium on the production of 11 low-molecular-weight organic acids and on the solubilization of calcium phosphate by bacteria in growth medium (NBRIP). The following strains isolated from cowpea nodules were studied: UFLA03-08 (Rhizobium tropici), UFLA03-09 (Acinetobacter sp.), UFLA03-10 (Paenibacillus kribbensis), UFLA03-106 (Paenibacillus kribbensis) and UFLA03-116 (Paenibacillus sp.). The strains UFLA03-08, UFLA03-09, UFLA03-10 and UFLA03-106 solubilized Ca3(PO4)2 in liquid medium regardless of the initial pH, although without a significant difference between the treatments. The production of organic acids by these strains was assessed for all of the initial pH values investigated, and differences between the treatments were observed. Strains UFLA03-09 and UFLA03-10 produced the same acids at different initial pH values in the culture medium. There was no correlation between phosphorus solubilized from Ca3(PO4)2 in NBRIP liquid medium and the concentration of total organic acids at the different initial pH values. Therefore, the initial pH of the culture medium influences the production of organic acids by the strains UFLA03-08, UFLA03-09, UFLA03-10 and UFLA03-106 but it does not affect calcium phosphate solubilization. Introduction Acidity has a direct effect on the activity of the soil microorganisms involved in a variety of processes, including organic matter decomposition, mineralization, immobilization, ammonification, nitrification, volatilization, biological nitrogen fixation and insoluble inorganic phosphate solubilization. Therefore, acidity is a chemical property of soils that plays a central role in agriculture. Biological, chemical and physical factors may interfere with the ability of soil microorganisms to solubilize insoluble inorganic phosphates. In many cases, acidification is the main mechanism involved in phosphate solubilization. A significant negative correlation between the pH of the culture medium and phosphate solubilization by several genera and species of microorganisms was demonstrated by diverse authors [(Illmer and Schinner, 1992) -r = -0.49, (Chen et al., 2006) -r = -0.80, and(Marra et al., 2011) -r = -0.89]. For instance, Arthrobacter sp. solubilized 519.7 mg P L -1 when the pH of the culture medium decreased from 6.8 to 4.9 (Illmer and Schinner, 1992). In contrast, several studies have shown phosphate solubilization without a significant negative correlation with culture medium pH. For example, Pseudomonas sp. solubilized 31.0 mg P L -1 with no alteration of the culture medium, which was at an initial pH of 6.0 (Hariprasad and Niranjana, 2009). Narsian et al. (1995) also reported lack of correlation between pH and Ca 3 (PO 4 ) 2 solubilization by Aspergillus aculeatus after a 7 day incubation. Phosphate solubilization depends not only on the decrease of the culture medium pH but also on other factors, such as exopolysaccharide production secreted by microorganisms. Under the same culture conditions, Arthrobacter sp. solubilized 111.7 mg P L -1 as the culture medium pH was lowered from 7.0 to 4.5, whereas Enterobacter sp. solubilized 632.6 mg P L -1 when the culture medium pH decreased from 7.0 to 4.3 which was due to the larger amount of exopolysaccharide produced by Enterobacter sp. Authors suggested that EPS with ability of phosphorus -holding may be a novel important factor in the microbial dissolution of tricalcium phosphate acting synergistically with organic acid (Yi et al., 2008). Besides promoting a decrease in the pH of the medium, low-molecular-weight organic acids also chelate metals in solution, which increases the phosphorus available to plants. The degree of chelation depends on the type of organic acid involved, the number and proximity of carboxyl groups, the type of metal and the pH of the solution (Jones, 1998). Only one study (Chaiharn and Lumyong, 2009) has assessed the influence of the initial pH of the growth medium on phosphate solubilization; however, these authors did not assess the production of organic acids. Marra et al. (2012) studied the solubilization ability of 82 strains in three types of phosphates (P, Al and Fe) both in solid (82 strains) and liquid (5 strains) media and verified that Ca phosphates are the most solubilized. They also found that there was a significant negative correlation between the pH of the medium and the amount of soluble phosphorus for CaHPO4 (r = -0.51**) and FePO 4 .2H 2 O (r = -0.28**), a relationship that was not observed for Al(H 2 PO 4 ) 3 . The highest solubilization of Ca-phosphate is due to its weaker chemical bound. Besides, Ca is a nutrient required in higher amounts than Fe. Al phosphate has the strongest bound and it is not a nutrient for microorganisms. Therefore, in this study, we sought to assess the influence of the initial pH of the culture medium on the production of low-molecular-weight organic acids and on the solubilization of calcium phosphate. medium with different pH values and incubated at 28°C for 10 days. At the end of this period, the presence or absence of growth was assessed. The study design was completely randomized and included three replicates. Solubilization of calcium phosphate and production of organic acids Two experiments were performed to verify the ability of the above strains to solubilize insoluble inorganic phosphate from calcium phosphate in National Botanical Research Institute's solid and liquid growth media (NBRIP) (Nautiyal, 1999) containing 10 g L -1 glucose, 5 g L -1 MgCl 2 .6H 2 O, 0.25 g L -1 MgSO 4 .7H 2 O, 0.2 g L -1 KCl and 0.1 g L -1 (NH 4 ) 2 SO 4 . NBRIP medium was supplemented with Ca 3 (PO 4 ) 2 to a final concentration of 1000 mg of phosphorus per L in the solid medium and 100 mg of phosphorus per L in the liquid medium; different initial pH values were adjusted: 5.0, 6.0 and 7.0. To produce and standardize inocula, the strains were inoculated into liquid medium 79 (Fred and Waksman, 1928) containing 0.5 g L -1 K 2 HPO 4 , 0.2 g L -1 MgSO 4 .7H 2 O, 0.1 g L -1 NaCl, 10.0 g L -1 mannitol and 0.4 g L -1 yeast extract, pH 6.8. Strains were incubated with shaking (110 rpm) at room temperature under aerobic conditions. Readings were performed periodically on a spectrophotometer at a wavelength of 560 nm until an optical density (OD) of 0.5 was reached, which was equal to approximately 10 8 cells per mL. A 0.85% saline solution was used to adjust cells to the desired density when the OD exceeded 0.5. For assessment in solid NBRIP medium, Petri dishes containing NBRIP medium at each initial pH condition were inoculated in quadruplicate with 20 mL aliquots of each culture (strain) at an OD of 0.5. The control treatment consisted of non-inoculated NBRIP medium. The culture dishes were incubated at 28°C, the diameter of the solubilization halo (translucent area surrounding colonies) was measured at the beginning of solubilization, i.e., at the 3 rd day and, after the 15 th day incubation, using a digital paquimeter, and the Solubilization Index (SI) expressed as halo diameter (mm) / colony diameter (mm) was calculated (Akintokun et al., 2007). The investigated strains were classified based on their SI as demonstrating low (SI < 2.00), intermediate (2.00 < SI < 4.00) and high (SI > 4.0) solubilization capacities. For assessment in liquid NBRIP medium, a 1 mL aliquot of culture medium 79 with an OD of 0.5 at 560 nm was inoculated into a 125 mL Erlenmeyer flask containing 50 mL of NBRIP medium at different initial pH values. The flasks were incubated at 28°C with shaking at 130 rpm for 10 days. Subsequently, the samples were centrifuged (19,187 g for 5 min), and the pH was measured, as well as the amount of soluble phosphorus in the supernatant using the phosphomolybdate method (Murphy and Riley, 1962). In addition, the organic acids produced in the medium were quantified. For each initial pH value, a non-inoculated control was assessed. The ability of each strain to solubilize phosphate was calculated as the difference between the concentration of soluble phosphorus in the culture medium of samples that had been inoculated with bacterial strains and that of the non-inoculated control treatment. High-performance liquid chromatography (HPLC) (Agilent HP Series 1100) was used to identify and quantify organic acids. Samples were collected, filtered through a 0.45 mm cellulose membrane and injected into a Supelcogel C-610H 9 mm chromatographic column measuring 30 cm x 7.8 mm. The eleven Merck® pro-analysis organic acids that have been reported in the literature as being involved in solubilization were used as analytical standards. The mobile phase was 0.1% H 3 PO 4 (pH 1.81) with a 0.5 mL min -1 flow rate and a 100 mL injection per sample. The method was according manufacturer (SUPELCO/SIGMA ALDRICH) of the column Supelcogel. The acquisition time of the chromatograms was estimated to be 30 min with 30 min intervals between runs. Detection was performed by UV at 210 nm with a diode array detector (DAD). The molecules identified and their typical retention times were as follows: oxalic acid (10.10 min), 2-ketogluconic acid (12.10 min), citric acid (12.40 min), gluconic acid (13.04 min), maleic acid (13.33 min), tartaric acid (13.45 min), malic acid (14.85 min), malonic acid (15.23 min), lactic acid (17.89 min), succinic acid (17.91 min) and propionic acid (25.08 min). The quantification of acids was performed using calibration curves of the standards. The experiment with NBRIP liquid medium was performed in independent assays for each initial pH value with a completely randomized design and two replicates. The results were evaluated by variance analysis using Sisvar (version 4.6) (Ferreira, 2008), and means were compared using the Scott-Knott test at 5%. Results All strains grew in the culture medium 79 at all of the initial pH values studied. In solid NBRIP medium, the strain UFLA 03-116 did not solubilize Ca 3 (PO 4 ) 2 at any of the initial pH values studied. The other strains did solubilize Ca 3 (PO 4 ) 2 at all pH values and exhibited low SI after a 15-day incubation. The only exception was the strain UFLA 03-08 (R. tropici), which demonstrated an intermediate SI at all of the investigated initial pH values (Table 1). In liquid NBRIP medium, the strain UFLA 03-116 (Paenibacillus sp.) exhibited the same behavior as in the solid medium and did not solubilize Ca 3 (PO 4 ) 2 at any initial pH value, and the pH of the medium did not change from its initial value (Figure 1). The strains UFLA 03-08 (R. tropici), UFLA 03-09 (Acinetobacter sp.), UFLA 03-10 (P. kribbensis) and UFLA 03-106 (P. kribbensis) solubilized Ca 3 (PO 4 ) 2 at all initial pH values in liquid NBRIP medium, with no difference being observed in the amount pH and organic acids in P solubilization 369 ( of soluble phosphorus among the different initial pH values of the medium. It is worth noting that more than 60% (as much as 77% in some cases) of insoluble inorganic phosphate (Ca 3 (PO 4 ) 2 ) was solubilized by these strains. For most strains, the initial pH of 5.0 did not change after 10-day incubation nor it differed from the control (Figure 1); the only exception was the strain UFLA 03-09 (Acinetobacter sp.) in which the pH decreased to less than 4.0. At initial pH values of 6.0 and 7.0, the pH decreased after a10-day incubation among all the strains where solubilization occurred. The greatest difference was found at the initial pH of 7.0, which decreased to less than 4.0 for the UFLA 03-09 strain. With respect to the identification, for the strain UFLA 03-08 (Rhizobium tropici), the highest total acid concentration (malic acid, 18.90 mmol L -1 ) was found at an initial pH of 7.0; for this strain, acid production varied with the initial pH of the culture medium. Organic acids identified at pH 5.0 and 6.0 were respectively: 2-ketogluconic (1.28 mmol L -1 ) and lactic/succinic acid (0.37 mmol L -1 ). Other peaks were not identified in all pH values (Figure 4 -Chromatograms G, H and I). For the strain UFLA 03-09 (Acinetobacter sp.), the only acid detected was the gluconic acid, at pH values of 5.0 (34.25 mmol L -1 ), 6.0 (30.64 mmol L -1 ) and 7.0 (37.34 mmol L -1 ), which indicates consistency in the production of acids independent of the initial pH of the growth medium. The UFLA 03-116 (Paenibacillus sp.) was the only strain that did not produce any organic acids under any cul-pH and organic acids in P solubilization 371 ture condition and, therefore, it exhibited the same behavior as the control treatment; the chromatograms show a peak at a retention time of 8.91 min, which differs from all the retention times exhibited by the acids in this study (Figure 4). It is worth noting that this peak was also present in all of the other treatments and does not interfere with the identification and quantification of the acids studied. Under these treatment conditions, citric acid, oxalic acid, maleic acid and malonic acid were not found. Discussion The pH of the culture medium directly influences the growth of microorganisms and the biochemical processes they perform. In many cases, acidification is the main mechanism involved in phosphate solubilization (Halder et al., 1990;Jha et al., 2009;Marra et al., 2011;Marra et al., 2012;Whitelaw, 2000). However, several studies have shown a lack of correlation between solubilized phosphorus and pH of the medium (Chaiharn and Lumyong, 2009;Xie, 2009). Therefore, a better understanding of the behavior of phosphate-solubilizing bacteria inoculated into culture media at different initial pH values may contribute to the production and management of inoculants that improve crop production. Our results showed that in both solid and liquid NBRIP medium, the initial pH did not affect the solubilizing activity of strain UFLA 03-116 (Paenibacillus sp.) 372 Marra et al. because it was not able to solubilize Ca 3 (PO 4 ) 2 under these conditions. These results reveal that the inability to solubilize phosphate under these conditions is intrinsic to this strain, because it grew on solid medium, which was visible in Petri dishes, and liquid medium, as was verified by the presence of bacterial biomass during the centrifugation process. Studies (Marra et al., 2012) performed with this same strain of Paenibacillus sp. in solid and liquid GELP medium (Sylvester-Bradley et al., 1982) at an initial pH of 7.0 also demonstrated its inability to solubilize CaHPO 4 , Al(H 2 PO 4 ) 3 and FePO 4 .2H 2 O. The only exception was for FePO 4 .2H 2 O in liquid medium; for this phosphate, more than 20% of the phosphorus was solubilized (Marra et al., 2012). An initial pH of 7.0 in GELP medium may contribute to the solubilization of FePO 4 .2H 2 O by strain UFLA 03-116 (Paenibacillus sp.). With respect to the strains that solubilized Ca 3 (PO 4 ) 2 , UFLA 03-08 (R. tropici) was the only one to have an intermediate SI, which occurred at all of the initial pH values studied, after a 15-day incubation at 28°C. These SI values were higher than those reported for Rhizobium species obtained from Crotalaria retusa and Crotalaria verrucosa inoculated onto solid Pikovskaya culture medium (Pikovskaya, 1948) at an initial pH of 7.0 (Sridevi et al., 2007), thereby demonstrating that pH does not interfere with solubilization by this strain. Conversely, the low SI exhibited by the strains UFLA 03-09, UFLA 03-10 and UFLA 03-106 on solid medium contrasts with that of liquid medium in which these strains solubilized significant quantities of phosphates. The strains UFLA 03-08 (R. tropici), UFLA 03-09 (Acinetobacter sp.), UFLA 03-10 (P. kribbensis) and UFLA 03-106 (P. kribbensis) solubilized Ca 3 (PO 4 ) 2 to a similar degree (more than 60%) at all of the initial pH values of NBRIP medium studied. UFLA 03-09 (Acinetobacter sp.) was the only strain that decreased the pH during all treatments, which may be related to its production of gluconic acid. Chaiharn and Lumyong (2009) found that after a 5-day incubation of Acinetobacter sp. in nutrient broth with an initial pH of 7.0 or 9.0, the pH of the medium decreased, but the pH increased to 6.17 in medium with an initial pH of 5.0; nevertheless, solubilization of calcium occurred. However, these authors did not assess the production of organic acids. Several authors have suggested that a decrease in pH due to the production of organic acids and the release of protons is a basic principle of phosphate solubilization, (Chen et al., 2006;Sperber, 1958;Whitelaw, 2000). However, the strains UFLA 03-08, UFLA 03-10 and UFLA 03-106 did not decrease the initial pH of 5.0 after 10-day incubation, thereby demonstrating that acidification is not the mechanism used to promote solubilization at this initial pH, even when organic acids are produced in different concentrations. In this case, the acids may be present in anionic forms and therefore do not function in medium acidifica-tion but, rather, in Ca 2+ chelation (Jones, 1998;Whitelaw, 2000). Moreover, the strain UFLA 03-08 at an initial pH of 6.0 exhibited efficient solubilization, with a decrease of pH but producing a low concentration of lactic/succinic acid (0.37 mmol L -1 ). This result indicates that other solubilization mechanisms are involved and that this strain utilizes different mechanisms when the pH of the medium varies. These mechanisms can be: proton exclusion (via cellular respiration and ammonium absorption as N source) (Illmer P and Schinner F, 1992), siderophores (Hamdali et al., 2008) and exopolisaccharide (EPS) production (Yi et al., 2008). The first two mechanisms were not evaluated in this paper, however, all these three strains produce large amounts of EPS that could act synergistically with acid production as suggested by Yi et al. (2008). Non-solubilizer strain UFLA 3-116 also produces large amounts of EPS however it did not produces organic acids. Conversely, medium acidification occurred at initial pH of 6.0 and 7.0, after a 10-day incubation, followed by the production of lactic/succinic and malic acids by the strain UFLA 03-08, 2-ketogluconic, tartaric and propionic acid by the strain UFLA 03-10, and tartaric and propionic acids by the UFLA 03-106. Propionic acid was produced to the greatest degree by the latter two strains which belong to the same species. The acids produced in larger amounts (propionic > gluconic > tartaric > malic) have pka varying from 4.79 (propionic) to 3.07 (tartaric) without showing any relationship with phosphate solubilization. On the other hand, strain UFLA03-09 (Acinetobacter sp.) which decreased the pH to lower level, only produced gluconic acid which has an intermediate pka (3.65). The strains UFLA 03-08, UFLA 03-09, UFLA 03-10 and UFLA 03-106 solubilized Ca 3 (PO 4 ) 2 at all three initial pH values studied. Brazilian soils usually exhibit acidic pH values, often varying between 5.0 and 6.5, and the practice of liming aims to reach pH 5.5-6.5. Therefore, these strains may increase and maintain the availability of phosphorus to plants across a wide variety of soil management practices. Besides contributing to solubilization, the production of certain acids by these strains may also serve as a readily accessible source of carbon for these microorganisms (Jones, 1998). The results show that: the initial pH of the culture medium influences the production of organic acids by the strains UFLA 03-08, UFLA 03-09, UFLA 03-10 and UFLA 03-106 but they do not promote the solubilization of calcium phosphate; thus medium acidification is not the mechanism by which the strains UFLA 03-08, UFLA 03-10 and UFLA 03-106 solubilize calcium phosphate when the initial pH at medium is 5.0 and that strains UFLA 03-09 and UFLA 03-10 produced the same acids when the culture medium exhibited different initial pH values. 374 Marra et al.

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2019-03-31T13:44:58.326Z

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Chemical Constituents of Essential Oil from the Leaves of Ipomoea batatas L. (Lam.) The essential oil, obtained by hydrodistillation in an all glass Clevenger apparatus, from the airdried leaves of Ipomoea batatas L. (Lam.) growing in Nigeria was analyzed by gas chromatography-flame ionization detector (GC-FID) and gas chromatography coupled with mass spectrometry (GC/MS). Forty-one constituents representing 93.5% of the oil were identified from the GC/MS spectra. Monoterpenes (22.0%), sesquiterpenes (46.5%) and diterpenes (22.5%) were the classes of compounds identified in the oil. The main constituents of the oil were abietadiene (8.9%), β-caryophyllene (8.8%), abieta-8,11,13-triene (7.1%), trans-(Z)-α-bergamotol (6.0%), cissabinene (5.5%) and spathulenol (5.3%). This is the first report on the chemical composition of essential oil of I. batatas growing in Nigeria. Aims: The aim of the research is to investigate the volatile constituents from I. batatas collected in Original Research Article Ogunmoye et al.; IRJPAC, 7(1): 42-48, 2015; Article no.IRJPAC.2015.053 43 Ibadan, Oyo State, Nigeria Study Design: Extraction of essential oil from the air-dried leaf samples of I. batatas and investigation of its chemical constituents. Place and Duration of Study: Mature leaves of I. batatas were collected from a location in Ibadan, Oyo State, Nigeria, in May 2012. Methodology: Air-dried and pulverized leaves were hydrodistilled in a Clevenger-type apparatus to obtained colourless volatile oil whose chemical constituents was analyzed by GC and GC/MS. Results: A total of Forty-one compounds accounting 93.5% of the oil were identified from the GC/MS and the major constituents were found to be abietadiene (8.9%), β-caryophyllene (8.8%), abieta-8,11,13-triene (7.1%), trans-(Z)-α-bergamotol (6.0%), cis-sabinene (5.5%) and spathulenol (5.3%). Conclusion: The present oil compositions were found to be different from the results previously reported from the essential oils of different cultivars grown in other parts of the world. INTRODUCTION The sweet potato, Ipomoea batatas (L.) Lam. from the family Convolvulaceae is an herbaceous perennial vine that has white and purple flowers, large nutritious storage roots and heart-shaped, lobed leaves [1]. It is consumed as vegetables, commonly eaten as root crops in tropical areas and used as a folk medicine in several countries of the world. It was reported that I. batatas is the single most important dietary herb that would replace fatty foods [1]. The sweet potato represents the seventh most important food crop in the world. Traditionally, decoctions of the roots and leaves of I. batatas are used in the treatment of urinary infections, fever, skin diseases, diabetes, curing boils and acnes. The sweet potato is harvested when the production of premium-sized roots is maximised. There are several cultivars of I. batatas. Ipomoea batatas is exceptionally rich in a variety of valuable protective nutrients [2][3][4][5]. It is a source of ascorbic acid, β-carotene, proteins and free sugars [6,7]. Extracts and compounds from I. batatas are known for their biological potentials. Anthocyanins from purple sweet potato have stronger binding ability with DNA [8] and possess antioxidative, antimutagenic and antiproliferative activities [9,10]. In addition, the anthocyanins also exhibited protective effects on damages of thymocytes caused by the Co 60 irradiation [11] and inhibitory effect on transplantation tumor of mice [12]. Extracts from the plant improve insulin sensitivity in insulinresistant rats [13] and displayed antibacterial [14][15][16], antidiabetic [17] and anti-neuroinflammatory [1] activities. The plant is a source of anti-diabetic flavones [18]. Aldose reductase inhibitors were characterized from the plant [19]. Extracts from I. batatas may be used as potential supportive treatment for thrombocytopenic disorders [20]. A review on pharmacological and phytochemical studies on I. batatas has been published [21]. In continuation of our research into the volatile constituents of Nigeria flora [31], we report in this paper the compounds identified in the essential oil of the leaves of I. batatas, which has not been published previously. Plant Material Fresh and mature leaves of I. batatas were collected from plants growing at a location in Ibadan, Oyo State, Nigeria, in May 2012. Botanical identification of the plant material was carried out at the Herbarium, Forestry Research Institute of Nigeria (FRIN), Ibadan, where a voucher specimen was deposited. Oil Isolation The air-dried and pulverized leaves of I. batatas (500 g) were subjected to hydrodistillation in a Clevenger-type glass apparatus for 3 h in accordance with the British Pharmacopoeia specification [32]. The oil collected was preserved in a sample tube and stored under refrigeration until moment of analysis. Gas Chromatography (GC) Analysis GC analysis of the oil was carried out on a Hewlett Packard HP 6820 Gas Chromatograph equipped with a FID detector and HP-5 column (60 m x 0.25 mm id), film thickness was 0.25 μm and the split ratio was 1:25. The oven temperature was programmed from 50°C (after 2 min) to 240°C at 5°C/min and the final temperature was held for 10 min. Injection and detector temperatures were maintained at 200°C and 240°C, respectively. Hydrogen was the carrier gas at a flow rate of 0.5 mL/s. An aliquot (0.5 µL of the diluted oil in hexane) was injected into the GC. Peaks were measured by electronic integration. A homologous series of n-alkanes were run under the same conditions for determination of retention indices. The relative amounts of individual components were calculated based on the percentage relative area (FID response) without using correction factors. Gas Chromatography-Mass Spectrometry (GC/MS) Analysis GC-MS analysis of the oil was performed on a Hewlett Packard Gas Chromatography HP 6890 interfaced with Hewlett Packard 5973 mass spectrometer system equipped with a HP 5-MS capillary column (30 m x 0.25 mm id, film thickness 0.25 µm). The oven temperature was programmed from 70-240°C at the rate of 5°C/min. The ion source was set at 240°C and electron ionization at 70eV. Helium was used as the carrier gas at a flow rate of 1 mL/min. Scanning range was 35 to 425 amu. Diluted oil in n-hexane (1.0 µL) was injected into the GC/MS. The constituents of essential oil were identified as previously described [31]. Previous analyses on essential oils of I. batatas from other parts of the world have reported with most concentrated on tubers. A comparison of the present result with previously analysed samples of I. batatas revealed some qualitative and quantitative variations. For example, phenyacetaldyde the main compound of I. batatas L. cv Ayamurasaki [34] was not identified in this sample while the content of nhexadecanoic Nigerian sample was also insignificant. In addition, linoleic acid and 4methyl-1-(2.3.4.5-tetrahydro-5-methyl[2.3'bifuran]-5-yl)-2-pentanone, the main constituents of I. batatas L. cv Beniazuma and I. batatas L. cv Simon [34] respectively are conspicuously absent in the present oil sample. A study has shown that the sweet potato weevil (Cylas formicarius elegantulus) could readily orient in the dark to the volatile phytochemicals emanating from the plant parts placed in their vicinity. The females are attracted by the volatiles emanating from the leaves and storage roots while the males are attracted by leaf volatiles but little attracted by those emanating from the storage roots. This suggested that there are qualitative and/or quantitative variations in the volatile compounds present in the different parts of I. batatas [33]. This may have been responsible for the observed compositional variations in the volatile oils of this plant from Nigeria and elsewhere. CONCLUSION The chemical compositions of essential oil from the leaves of I. batatas grown in Nigeria are being reported for the first time. It was found that the compositional pattern was different from previous studies on the essential oils from the tubers of different cultivars of this plant as well as those from other member of the genus. This may be attributable to the fact that different parts of the same plant contained different phytochemicals. In additional factors such as the ecological and climatic conditions as well nature and age of the plant, period of collection, handling procedures etc may contribute to the different nature of the compounds obtained from the essential oil.

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2019-05-29T13:14:50.699Z

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Substitution Effects of Coconut Milk with Soymilk on Sensory Acceptance and Shelf Life of ‘Nasi Lemak’ This work was carried out in collaboration between all authors. Authors MNL, ZN and AY designed the study, performed literature searches, and prepared the study protocol. Authors NAHM and PLK performed the study protocol, literature searches and statistical analysis. Author MNL wrote the first draft of the manuscript and authors ZN and AY contributed in improvement of the manuscript. All ABSTRACT Aims: ‘ Nasi lemak’ is one of the most favourite rice-based products for Malaysians. One of the main ingredients used to prepare ‘Nasi lemak’ is coconut milk. The aim of this study was to develop ‘Nasi lemak’ where coconut milk was substituted with soymilk to provide healthier choice for consumers. Study Design: In this study, five formulations of ‘nasi lemak’ with different percentages of ratio between coconut milk and soymilk (100:0, 75:25, 50:50, 25:75, 0:100) were studied. Place and Duration of Study: The experiments were conducted under controlled environment in the Food Service Laboratory and Food Microbiology Laboratory, University Malaysia of Terengganu, from July 2011 to April 2012. Methodology: The work scope of this study was to examine the effects of the coconut milk to soymilk substitution on sensory preference and shelf life of ‘nasi lemak’. In terms of shelf life study, Aerobic Plate Count, Yeast and Mould Count, and Bacillus cereus Count were determined throughout storage of samples for 24 hours at ambient temperature (28º±2ºC) and 7 days at chilling temperature (4º±2ºC). Results: Fresh ‘nasi lemak’ samples were subjected to preference test and it was found that up to 25% of substitution of coconut milk with soymilk, there was no significant difference ( P >0.05) found with control sample. For shelf life studies, there were no significant differences ( P >0.05) found for Aerobic Plate Count and Yeast and Mould count among samples stored at ambient temperature, except for B. cereus Count. However, significant differences ( P <0.05) were found in all three counts: Aerobic Plate Count, Yeast and Mould count and B. cereus Count among samples stored at chilling temperature. Conclusion: The sample with 75% coconut milk ratio with 25% soymilk is highly recommended to obtain good acceptance compared to control sample (100% coconut milk) with acceptable shelf life (21 hours) at ambient temperature and 6-days at chilling temperature. INTRODUCTION Malaysia is famous with variety of food. One of the most famous traditional foods in Malaysia is 'Nasi Lemak'. A serving of 'nasi lemak' consists of a plate of white rice cooked together with coconut milk and accompanied with 'sambal', chilli paste mixed with salted anchovies. The presence of coconut milk in 'nasi lemak' contributes to high calorie content due to its high fat content. Excessive intake of high-fat and high-calorie food is harmful to health [1]. Coconut milk, also known as 'santan' in Malaysia and Indonesia and 'gata' in the Philippines, is defined as the liquid product obtained by grating the solid endosperm of a coconut fruit, with or without addition of water. Coconut milk is usually used as an ingredient in various traditional recipes [2]. The main components of coconut milk apart from water are fat and protein. The high amount of fat and protein contained in it makes coconut milk prone to microbial spoilage [3]. As there is increasing demand for healthier and safer product, the effect of substitution of coconut milk with soymilk in 'nasi lemak' is an alternative way to reduce its calorie content. Substitution of coconut milk with soymilk in 'nasi lemak' may affect the sensory acceptance, microbiological quality and shelf life of this product. Shelf life is an important parameter to evaluate the safety of a food product during storage. Therefore, the objective of this study was to determine effect substitution of coconut milk with soymilk on sensory acceptance and microbiological shelf life of 'nasi lemak'. Sample Preparation and Experimental Design Materials used in this study were rice, soybean, fresh coconut milk, salt, ginger, banana leaves, and pandan leaves. All these materials were purchased from local shops around Kuala Terengganu. Soymilk was prepared freshly from soybean and used to substitute coconut milk to cook the rice following normal procedure for preparation of 'nasi lemak'. 'Nasi lemak' was prepared in a controlled and aseptic environment at the Food Service Laboratory, University Malaysia of Terengganu. Different ratios between coconut milk and soymilk were employed as follows: Sample A, 100:0 (control), Sample B, 75:25, Sample C, 50:50, Sample D, 25:75, and Sample E, 0:100 (Table 1). Where there was a ratio between coconut milk and soymilk in production of a sample as shown in Table 1, some amount of coconut milk was replaced with soymilk following the formulation and the mixture of both ingredients was used in the production of 'nasi lemak' in similar way used in producing the control sample which only used coconut milk. Preparation of 'Nasi Lemak' 'Nasi lemak' was prepared using the modified method from [4]. Firstly, 100 g of rice was cleaned and rinsed. Then, the rice was soaked in 250 ml of water with ratio of 1:2.5 overnight. After an overnight soaking, the water was drained off. A steamer was prepared and the bottom of the steamer was covered with banana leaves with approximate size of 20 cm x 20 cm and the steamer was then heated up. Next, the rice was placed on the banana leaves. After that, three pieces of pandan leaves and ½ inch ginger (15 g) were added inside the steamer. Then, one teaspoon of salt was sprinkled on the surface of rice and the rice was steamed for 10 minutes. After 10 minutes, the rice was removed from the steamer when it was half cooked. The halfcooked rice was added with 75 ml of mixture of water and thick coconut milk (15 ml water + 60 ml thin coconut milk) in a separated bowl by using a fork until the water was thoroughly absorbed (about 3-4 minutes). Then, one teaspoon of salt was added into the cooked rice and was mixed properly. The rice was next steamed for 15 minutes. Then, 37.5 ml of thick coconut milk and salt were added to the rice and mixed until they were fully absorbed. Again, the rice was steamed for another 15 minutes or until the rice was fully cooked. The products were kept in separate sterile and microwavable polypropylene rectangle cases with clear colour (11.8 cm (L) x 17.2 cm (W) x 6.5 cm (H) for shelf life study. Preparation of Soymilk Soymilk was prepared by using the method modified from [5]. Firstly, dry soybean was cleaned and the weight was measured. After that, soybean was soaked in water with the ratio of 1:3 for about 8 to 10 hours. Next, soybean was grinded with water in the ratio of 1:10 for thick soymilk and 1:11 for thin soymilk by using a blender. Slurry produced was then filtered through a muslin cloth, followed by pasteurization at boiling temperature for about 20 min. Then, soymilk was allowed to cool and kept in the chiller for storage. Temperature and time used in the process were controlled as they are important to avoid under or over cooking of the product. Sensory Preference Test Sensory preference test was carried out by 35 untrained panelists on 5 attributes: colour, texture (mouth-feel), odor, taste and overall preference by using ranking method. Samples were ranked by panelists according to their preference for each attributes. All samples were labeled with random three-digit codes and presented in a randomize arrangement. Data collected was calculated using Least Significant Difference test (LSD). Microbiological Analysis 25 g of 'nasi lemak' was weighed and placed in a sterile stomacher bag. The sample was then homogenized in 225 ml of peptone water making serial 10-fold dilutions. These serial dilutions that contained samples were further analyzed. In order to enumerate the microbial count of samples, 0.1 ml portions of appropriate dilutions were transferred onto triplicate plates. A sterile L-spreader was used to spread the diluents on the surface of prepared media ( Statistical Analysis Microbiological data were presented as growth curves of CFU/g vs. time and mean values and standard deviation of microbial counts (log 10 CFU/g) obtained were used for statistical analysis using one-way ANOVA and followed by Tukey test. Significant difference was determined at P<0.05. The software used for statistical analysis was Minitab (Version 14.0). Table 2 shows that in terms of colour, there were no significant differences (P>0.05) among samples whose coconut milk had been replaced with soymilk up to 75% substitution. Sample produced with 100% soymilk had the lowest preference due to its yellowish color that was less preferred. Sensory Preference of 'Nasi Lemak' For odor, up to 50% of substitution of coconut milk with soymilk was found to not give significant effect (P>0.05) on samples' odor preference. Meanwhile for texture, substitution of coconut milk with soymilk up to 75% gave no significant differences (P>0.05) in samples' preferences. The least preferred sample for texture was the one produced with 100% soymilk. Soymilk has higher protein content and denatured protein has more hydrophobic unfolding proteins. Thus, sample with more soymilk had harder texture. Different pattern of preferences could be seen in taste where only sample with 25% substitution of coconut milk with soymilk that was not significantly different (P>0.05) with control sample. A similar trend was also observed in overall preference where only the sample with 25% soymilk was not significantly different (P>0.05) with control sample. Higher level of substitution gave more beany flavour to 'nasi lemak' produced, thus samples with higher levels of substitution were less preferred. Aerobic Plate Count of 'Nasi Lemak' Aerobic Plate Count [APC] is used to indicate level of microorganisms in a product [7]. Obtaining an estimate number of microorganisms in a food product will aid in evaluating sanitary practices during processing and handling, as well as determining potential sources of microbial contamination. In a study conducted by [8], it was reported that cooked rice could be stored in refrigerating temperature for 6 to 7 days and about 6 months if stored frozen. For raw white rice, if it is stored in tightly closed container, it was reported to be able to be stored at room temperature and used within one year while brown rice and wild rice have shorter shelf life of 6 months only [8]. For cooked rice, the safety level can be determined based on regulations applied on cooked, ready-to-eat foods which was Table 4 shows that there was significant differences (P<0.05) in APC of sample C compared to other samples after 6 days of storage at chilling temperature. While performing microbial analyses, samples were also observed for their physical characteristics. All samples still looked good until 7 days of storage at chilling temperature; however, the samples' APCs had already reached the unsatisfactory level for human consumption. This results show that physical observation only is not sufficient to determine safety of a product. Yeast and Mould Count of 'Nasi Lemak' Yeast and mould can invade and grow on virtually any type of food at any time [6]. Determination of Yeast and Mould Count is used to detect the presence of yeast and mould in a sample. Specifically, mould can grow in foods that have high acidity and low moisture [11]. Meanwhile, yeast is commonly found on plants, grains, fruits, and other foods containing sugar and it can cause food to spoil but it lacks the risk of foodborne illnesses. Yeast and mould have slower growth in comparison to bacteria and they were often out-competed [12]. However, many moulds can grow well in refrigerated temperature and becomes the common cause of spoilage in refrigerated foods [11]. Fig. 3 shows yeast and mould count [YMC] for all samples stored for 24 hours at ambient temperature, where they were all within the satisfactory level of lower than 1.0 x 10 5 CFU/g, which is considered as is the end of safe shelf life for yeast and mould count in ready-to-eat (RTE) products [13]. Samples E and D had higher YMC compared to samples A, B, and C since they contained more soymilk than coconut milk. Table 5 reveals that there are no significant differences (P>0.05) of yeast and mould count between all samples. Fig. 4 shows the YMC values of 'nasi lemak' samples stored at chilling temperature (4±1ºC). Sample D was the first sample to reach the unsatisfactory level of 10 5 CFU/g after 3 days while sample C managed to stay within the satisfactory level for YMC even after 6 days of storage. It can be observed from Table 6 that there were significant differences (P<0.05) in YMC of 'nasi lemak' samples after 6 days of storage at chilling temperature. It was deducted that spoilage of yeast and mould of 'nasi lemak' was most probably due to its ingredients such as coconut and spices [14]. Bacillus cereus Count of 'Nasi Lemak' Rice is arguably the most important foodstuff associated with B. cereus food-poisoning. Because of its particular cultivation conditions in rice paddies where B. cereus comprises about 10% of the soil microflora, raw rice is generally contaminated to varying degrees with B. cereus [15]. Research done by [11] mentioned that rice is a well-recognized source of B. cereus as most samples contain the organism but usually at low levels. A report by [16] stated that cooked rice was the first product to be recognized as a cause of food poisoning through contamination with B. cereus. Growth and toxin production by psychrotrophic strains can be prevented by storage temperatures of below 4ºC and pH above than 5 [17]. B. cereus can cause two distinct forms of foodborne disease: the emetic and diarrhea syndromes. The Food Standard Guidelines of Australia/New Zealand has determined that B. cereus count of 10 4 CFU/g and above indicates potentially hazardous level [9]. CONCLUSION In conclusion, up to 25% substitution of coconut milk with soymilk in 'nasi lemak' successfully provided the sensory characteristics close to control sample that are preferred by panelists. In terms of microbiological shelf life, there were no significant differences (P>0.05) of aerobic plate count and yeast and mould count among samples when stored at ambient temperature, except for B. cereus count. However significant differences (P<0.05) were observed when samples were stored at chilled temperature. This study strongly indicates that substitution of coconut milk to soymilk had significantly affected the shelf life of 'nasi lemak' at chilled temperature. Overall, the sample B with 75% coconut milk ratio with 25% soymilk is highly recommended to obtain good sensory acceptance comparable to control sample (100% coconut milk) with acceptable shelf life (21 hours) at ambient temperature and 6-days at chilling temperature.

v3-fos

2018-12-18T08:41:44.374Z

2015-09-29T00:00:00.000Z

58922794

s2

Population Dynamics of Rhizobacteria and Its Potency as a Biological Control Agent to Control Fusarium Disease in the Nursery of Agarwood (Aquailaria Malaccensis Lamrk) Agarwood is a resin product produced by particular trees and has a certain high comercial value. In Central Bangka Regency, agarwood is the main commodity of forest. The research was aimed to determine the dynamic population of rhizobacteria and its potential as a biological control agent to control Fusarium disease in the nursery of agarwood (Aquailaria malaccensis Lamrk). The research was carried out by using exploration and identification methods. Sixty nine bacterial isolates were obtained from 20 samples. The samples taken were from Pangkalan Baru and Koba districts. After selection process, 49 bacterial isolates were tested for the capacity of inhibition. Results showed that 37.50 % of the bacterial isolates indicated a strong inhibition capacity, meanwhile 58.33% indicated a moderate and only 4.70% possessed a weak inhibition. Pseudomonas fluorescens, P. aerogi-nosa, P. malthophilia and Klebsiella pnemoniae were identified from the selected isolates. These bacteria were potentially able to protect plants against Fusarium disease and to promote plant growth. This research needed to be continued at the field level in order to know the real effects on plant. INTRODUCTION Agarwood is a forest product which has a high economical value compared to other forest products, therefore it has potential to develop. In the wild nature, the production of agarwood is less than 5%. If agarwood had been formed, the amount of it was usually less than 10% of the biomass of the infected tree. Because of the valuable price, exploitation of natural agarwood was conducted without proper consideration of its sustainability. As a result, population of agarwood species declined rapidly, so that this species was included in Appendix II CITES. As a consequence, in formal trade, agarwood should be produced from cultivation, not from nature. The cultivation of agarwood had problem such as pests and diseases especially in the seedling. Murdan (2008) reported that there are seven plant pathogenic funguses associated with a rotten root of agarwood and one isolate was identified as Fusarium sp.. Leaves of agarwood fell continuously. It caused a bare crown, rotten root, and finally caused plant die. Those were the symptoms of Fusarium disease. The disease attacked agarwood from the beginning of seedbed to the two years old of plant (with diameter of stem > 2 cm). Fusarium sp. was one of the pathogens found in most of the plants and was causing a lot of economic loss. Murdan (2008) reported that Agarwood Development Center in Senaru village, West Lombok covering an area of 225.7 hectars, had beenbeen attacked by Fusarium disease and the loss reached 9.75% (or equivalent to Rp 274,015,599). Nowadays, there have been some ways to control pest and disease biologically. One of biological agents used are endophytic bacteria and rhizobacteria inside and around the plant itself. Natural bioactive compounds are produced by endophytic microbes potential for application in the fields of health, agriculture and industry (Joseph and Priya, 2011). Endophytic bacteria species diversity reflects many possible ways to reduce plant pathogens by produced antibiotic compounds (Bacon and Hinton, 2007). The way of endophytic bacteria work as biological control are to produce a mixture of anti-microbial materials, to compete a space and nutrients, to compete a micro nutrients such as iron and siderophores production, and to cause the host plant becomes resistant. Based on the explanation above this research was significant to be conducted, since the information on science and technology of agarwood pest and disease and also the controlling was still limited. The objective of this research was to assess the population dynamics of rhizosphere bacteria of agarwood (Aquailaria malaccensis Lamrk) and its potency as a Bioagents against pathogenic (Fusarium sp.) in the nursery. MATERIALS AND METHODS The research was conducted in the District of Pangkalan Baru and Koba in Central Bangka regency, Laboratory of the Ministry of Health Center Palembang, and Phytopathology Laboratory of the Department of Plant Pests and Diseases Faculty of Agriculture, Sriwijaya University Inderalaya South Sumatra, from February to August 2014. Exploration and identification methods were used to conduct this research. Isolation of Endophytic and Rhizobacteria Isolation of endophytic rhizobacteria was carried out by collecting of soil rhizosphere and root of agarwood from 20 sites soil rhizosphere and root of agarwood in the Garden Growers and Nursery of Horticulture and Forestry Department, Central Bangka regency. The procedure followed the method of isolation of endophytic bacteria which had been modified. The roots of agarwood was cut and washed with water, then dried. 1 g of roots were taken and the surface sterilized successively with 70% alcohol (30 seconds), then dipped in a solution of 2% NaOCl (for 2 minutes), and then dipped in sterile water. Before grinding, root of agarwood onis was applied on petridish containing TSA media (as a control). If bacteria grew on the control group, it was necessary to identify the bacteria that would grow as a marker of bacterial endophytic bacteria using the controls group. Roots were transferred into a mortal and crushed and added to 9 ml of sterile aquadesh. 1 ml of root extract was inserted into a test tube containing 9 ml of sterile aquadesh, then shaken until homogeneous, the next 1 ml of the extract was taken and diluted in series to 10-4 dilution. Next 0.1 ml of extract from the series of dilution 10-3 and 10-4 was put in sterile petridish that had been filled by media TSA. Rhizobacteria was isolated by means of soil around the roots of plants that had been dried, taken as 1 g put in 9 ml of sterile distilled water, then mixed until homogeneous. 1 ml of the extract was put into a test tube containing 9 ml of sterile distilled water, then shaked until homogeneous and 1 ml was transferred to the next tube, and did it continually until there was dilution series 10 1 -10 4 . 0.1 ml of extract dilution series 10 3 and 10 4 inserted into the filled sterile petridish TSA medium and then spread evenly in petridish. The extract that had been deployed in petridish, incubated for 3 days, then counted the number of colonies of bacteria growing. Isolation of Fusarium sp. Pathogens were obtained from the isolation of the pathogen on the roots and stems of the infected agarwood from planting directly method. The pieces of tissue which had a size 5 mm x 5 mm soaked in 1% NaOCl for 1 minute, then rinsed with sterile water and dried on sterile filter paper. The tissue pieces were then placed on plates containing potato dextrose agar medium (PDA) and incubated at room temperature for 48 hours. Colonies of fungus that grows were identified. The results showed that the isolated fungus were identified as Fusarium sp., then they were purified and used in further tests. Antagonist Test A total of 49 isolated endophytic bacteria and antagonists tested rhizobacteria against Fusarium sp. were obtained. Antagonists testing by in vitro was conducted usinga dual-culture technique. Identification of Bacteria Bacteria with potential biological agent were identified based on the strong inhibition zone (after the dual-culture technique), and they were identified by biochemical tests: motility test, acid and gas production test of some carbohydrates tests, gelatin hydrolysis test, casein hydrolysis test, starch hydrolysistest, nitrate and nitrite reduction tests, indole and H 2 S gas production tests, oxidase test, catalase test, methylred (MR) test and vogespraskauer (VP) test (Cowan, 1985). Parameters observed in this study were a population of bacteria, inhibitory power against Fusarium sp, the biochemical bacteria reaction obtained for identification. Population of Endophyitic Bacteria and Rhizobacteria The population densities of indigenous endophytic bacteria and rhizobacteria in agarwood roots and soil between the plants were varied. The isolated agarwood from 20 samples from the district of Pangkalan Baru and Koba were obtained 69 bacteria. Most of bacterial population presented in the roots, and they were about 209 colonies from Pangkalan Baru districts compared to the root from Mesu. The number of bacteria colonies obtained from the root were more than bacteria obtained from the soil, even in some petri dishes there were no bacteria grown. The number of bacteria colonies obtained from Pangkalan Baru district were more than bacteria obtained from Koba district (Table 1 and Table 2). It had been predicted, since the demographic circumstances and the way of farming in Pangkalan Baru district was different with Koba. Population density of bacteria from the soil was less than the density of the bacteria population derived from the root, and it was the same as that reported McInroy and Kloepper (1995) who stated that the population of endophytic bacteria was commonly found in the roots and ramifications (Misaghi and Donndelinger, 1990;Bell et al., 1995). Endophytic bacteria population density was very dependent on the type of selected plant tissue and roots that seem highest and lowest of the trunk and acropetal decline. Root was considered as a preferrable place for bacteria to enter the plant, and in particular it explained the high number of bacteria in roots in the early stages of growth. The root system seemed to be more supportive of this habitatas if it was related to water availability and temperature changes (Sessitsch et al., 2004;He et al., 2009;Sarr et al., 2010). But factors such as crop rotation, organic matter, temperature, rainfall, soil physical properties and chemical constituents might have an effect on bacteria populations Sessitsch et al., 2004) and some of these factors might have an influence in these research. In vitro antagonism of Endophytic Bacteria and Rhizobacteria Towards Fungal Pathogens (Fusarium sp.) A total of 49 isolated bacteria were tested with antagonist fungus Fusarium sp. Level antagonist indicated by the variation of zone inhibition such as weak (<1 cm), moderate (1 to 2 cm) and strong (>2 cm) (Rocha et al., 2009) (Table 3). and each of bacterial isolated power had a different inhibitors (Figure 1). After selection, 49 isolates were tested for inhibitation power. Results showed that 37.50% of the bacterial isolates indicated a strong inhibition capacity, 58.33% indicated a moderate and only 4.70% possessed a weak inhibition. In this study the antagonistic test used Nutrient Agar (NA) because it had been predicted that by using this medium the fungus and bacteria would grow optimally, furthermore the selection of the growing medium could provide a high impact on the inhibition shown (Yang et al., 2011). This results might be affected by medium used for antibiosis in vitro assay. Antagonism test of bacteria towards pathogenic fungi by using invitro method provided a fast way to select initial candidate biological control based on antibiosis. Identification of Bacteria Based on the results of the antagonism test there were 11 antagonistic isolated bacteria that had a strong inhibition against Fusarium sp. After identification by biochemical tests (Table 4), it was known that bacteria were Pseudomonas fluorescens, P.aeroginosa, P.malthophilia and Klebsiella pnemoniae. Pseudomonas group had been studied for biological control of P. flourescens primarily as an inducer of plant a. b. From these results known that P. Fluorescens was the most common of the 11 isolates that had the greatest percentage of inhibition, four isolates. The suppression of pathogenic fungi Fusarium sp by bacteria P. flourescens occured because bacteria were able to remove antibiotics such as pyoverdine, pyoluteorin, 2,4 diacetylphloroglucinol and monoacetylphloroglucinol that could inhibit the growth of pathogens (Blanco and Bakker, 2007). Besides, P. flourescens could also inhibite the development of the disease by nutrient competition of iron Fe (III) and carbon element, HCN production, stimulate phytoalexin accumulation so that the plant became resistant, colonized roots and stimulated plant growth (Defago et al., 1990;Notz et al., 1990;Widodo et al., 1993). P. maltophilia produced siderophores in a form of maltophilin, produced extra cellular protease enzyme that could control Pythium ultimum in sugar beet rhizosphere of plants and able to induce resistance of onion toward the bacterial leaf blight (Jakobi et al., 1996;Dunne et al., 1997;Ernita et al., 2010). The success of biological control of plant diseases was determined by the mechanism of inhibition of biological agents. The mechanism of inhibition was commonly found in biological agents such as siderophores, antibiosis, competition of mycoparasitism, PGPR, impactresistance, enzymesandtoxins (Soesanto, 2008). Besides the group of Pseudomonas, there was another bacteria that had been identified. It was Kleibsella pneumoniaea diazotrof endophytic bacteria. Diazotrof endophytic bacteria generally did not cause a disease, proliferates in plant tissues, however it did not form an endosymbiont inside cells of living plant. Diazotrof endophytic bacteria normally lived in the intercellular spaces or xylem vessels of roots, stems, leaves, and seed surface (James et al., 2000). The colony of diazotrof endophytic bacteria inplant tissue could exploit the carbon substrate supplied by plants without competing with other microbes. Some diazotrof endophytic bacteria was not only able to tie up nitrogen but also secrete acid indole-3acetic (Ladha et al., 1997). Klebsiella sp isolated from tomato plants could produce IAA and also siderophores, hydrogencyanide (HCN) and salicylic acid (Nandhini et al., 2012). Azospirilium brasilense, Azotobacter chroococcum and K. pneumoniae could inhibit F.oxysporum f.sp lycopersicy, Rhizoctonia solani and Pythium sp. that attacked cucumber plants in vitro (Hassouna et al., 1998). CONCLUSION Endophytic bacteria and rhizobacteria in Indonesia forest especially agarwood (A. malaccensis) had a potency as abiological control agents that was useful to protect from pathogenic Fusarium sp. A total of 69 isolated bacteria were obtained from Pangkalan Baru and Koba District in Central Bangka, and After selection, 49 isolates were tested for inhibitation power. Results showed that 37.50 % of the bacterial isolates indicated a strong inhibition capacity, 58.33% indicated a moderate and only 4.70% possessed a weak inhibition. Pseudomonas fluorescens, P. aeroginosa, P. malthophilia and Klebsiella pnemoniae were identified from the selested isolates. These bacteria were potentially able to protect plants against Fusarium disease and promote plant growth. ACKNOWLEDGEMENT The deepest gratitude is delivered to the Government of Central Bangka regency for supporting all funding and permission in doing parts of research in nursery garden of agarwood in Central Bangka Regency.

v3-fos

2018-12-10T22:42:08.132Z

2015-11-10T00:00:00.000Z

55721674

s2

Phytochemical screening, antioxidant and anticholinesterase effects of Alangium salvifolium (L.F) Wang root extracts Alangium salvifolium wang is a medicinal plant of the Alanginaceae family which was used as a traditional medicine to cure or prevent a variety of ailments. The aim of the study was to investigate and compare the phytochemical profiles, antioxidant and anticholinesterase effects of ethanol (EASR), dichloromethane (DASR), chloroform (CASR) and aqueous (AASR) extracts of A. salvifolium root. Phytochemical screening was done by using qualitative methods whereas total phenol content (TPC), total flavonoid content (TFC) and total flavonol content (TFlC) were determined by Folin-Ciocalteau reagent, aluminium trichloride and sodium acetate solution methods, respectively. Antioxidant activities were assessed by DPPH radical scavenging, ferric reducing antioxidant power (FRAP) and total antioxidant content (TAC) assay. Ellman's assay was applied to investigate acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzyme inhibitory effect. Preliminary phytochemical screening revealed the presence of valuable phytochemicals with significantly (P*<0.05, P**<0.01, P***<0.001) different content of TPC, TFC and TFlC. CASR, among the extracts, had shown the highest TPC (492.38±22.34 mg/g gallic acid), followed by TFC (276.25±17.23 mg/g quercetin) and TFlC (332.92±7.07 mg/g quercetin). Moreover, maximum antioxidant potential, including DPPH radical scavenging (IC50: 11.26±1.29 μg/ml), FRAP (EC50: 26.64±2.17 μg/ml) and TAC (639.55±10.51 mg/g ascorbic acid) was found in the CASR. Donepezil, a standard drug, showed maximum inhibitory effect of AChE (IC50: 7.94±1.12 μg/ml) and BChE (IC50:12.58±2.15 μg/ml). CASR followed by DASR had potent inhibitory effects while AASR had mild and EASR practically had no inhibitory effects of the enzymes. The present study has demonstrated that the root extracts of the A. salvifolium have moderate to potent antioxidant and enzyme inhibitory effects. INTRODUCTION Free radical damage and oxidative stress are considered as important causative factors for generation as well as exacerbation of various ailments like cancer, diabetes, asthma, and the pathogenesis of alzheimer's disease (AD) (Asmat et al., 2015). Oxidative stress, a potential source of damage to DNA, lipids, sugars and proteins, causes an imbalance between the intracellular production of free radicals/reactive oxygen species (ROS) and antioxidant defense mechanisms, resulting in cellular injury (Gjumrakch et al., 2008). The brain consumes a large proportion of the inhaled oxygen, and therefore produces a comparatively large quantity of free radical by-products (Yongxin et al., 2013). However, less quantity of the reactive oxygen (ROS) species are the precondition to keep the integrity of the neuronal cells and subsequently their normal functioning, since the elevated level of the radicals can lead to neuronal cell death (Yongxin et al., 2013). In contrast, antioxidants, being the defensive agents against the oxidative stress, have multiple functions in biological systems, including maintenance of cell integrity and cell signaling pathways (Kumar et al., 2008). One principal cellular function of antioxidants is to prevent damage caused by the ROS. Various studies have proved that an antioxidant may scavenge a highly reactive free radical or may inactivate it by donating a proton atom or by accepting an electron from the radical, and eventually prevents the free radicalinduced diseases (Jiaojiao et al., 2012). Alzheimer, the most common among the neurodegenerative disorders and dementia, is a major challenge of the modern era, and is a slowly progressive disease of the brain that is characterized by the impairment of memory (Rahmat et al., 2012). For normal functioning of brain, sufficient level of acetylcholine (Ach) is necessary which is essential for proper neurotransmission. Acetylcholinesterase (AChE) enzyme catalyzes hydrolysis reaction of the Ach and butyrylcholinesterase (BChE) potentiates the catalyzing activity of the AChE, resulting in a decreased level of Ach in the brain (Zeb et al., 2014). This condition leads to neurodegeneration and subsequently cognition. So, inhibition of AChE and BChE may be the most effective way of protecting the Ach to prevent or to improve dementia. Alangium salvifolium wang belongs to the family of Alanginaceae. Ankola and Alangi are its common name in India, and Stone Mango in English. It is a small deciduous thorny tree or shrub (Uthiraselvam et al., 2012) which is distributed in tropical and subtropical region such as Bangladesh, India, China Phillipines, Africa, Srilanka and Indochina (Ronok et al., 2013). An array of ailments including diabetes, jaundice, gastric disorders, protozoal diseases, rheumatic pain, burning sensation, haemorrhages, lung cancer, poisonings, leprosy and many inflammatory patches have been treated by using various parts of the plant (Meera et al., 2013). Many bioactive phytochemicals such as several flavanoids, phenolic compounds, irridoid glycosides and oxyoglucosides have been isolated by phytochemical screening of it (Gopinath, 2013). Literature review of the plant indicates the presence of coumarins, triterpenoids, and some potent alkaloids in it (Savithramma et al., 2012). The aim of the present study was to evaluate antioxidant and anticholineesterase effects of various extracts of the A. salvifolium root. Plant For the investigation, A. salvifolium wang root was collected from Rajshahi, Bangladesh between January and June, 2013 and identified by an expert of the Bangladesh National Herbarium, Dhaka, where a voucher specimen number was retained with an accession no. 40214. The collected plant part was cleaned, dried for one week and pulverized into a coarse powder using a suitable grinder. Powdered material was stored in an airtight container and kept in a cool, dark, and dry place until further analysis was taken. Extract preparation Approximately 500 g of powdered root was placed separately in four clean and flat-bottomed glass containers and soaked in ethanol, dichloromethane, chloroform and distilled water. All the containers with their contents were sealed and kept for 7 days. Then extraction was carried out using ultrasonic sound bath accompanied by sonication (40 min). The entire mixture then underwent a coarse filtration by a piece of clean, white cotton material. Then the extract was filtered through Whatman filter paper and concentrated by using a rotatory evaporator at reduced pressure. The gummy extracts were then dried by using an electric oven, and finally obtained EASR (12.25 g), DASR (9.5 g), CASR (7.5 g) and AASR (14.17 g). The dried extracts were separately stored in air tight containers until completion of the analysis. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Germay. All other reagents and solvents used for the study were of highest purity grade and commercially available. Phytochemical screening Phytochemical screening of the extracts was done by applying some previously established methods. Alkaloids, saponins, terpenoids and steroids were detected by applying Harborne (Harborne, 1973) method. Flavonoids and tannins were examined by applying methods of Sofowara (Sofowara, 1993). Reducing sugar and resins were evaluated by following methods of Dipali (Dipali et al., 2013). Coumarins, anthraquinones, cardiac glycosides and phlobatannins were detected by applying the methods of Trease and Evans (Trease and Evans., 1989). Determination of total phenolic content (TPC) TPC of the extracts was determined by using the Folin-Ciocalteau method with slight modification (Gao et al., 2000). Briefly, the extracts and standard gallic acid solution (1 ml) was mixed with 2.58 ml of Folin-Ciocalteu's phenol reagent. After 3 min, 0.3 ml of saturated sodium carbonate solution was added to the mixture and incubated at room temperature (25°C) for 20 min. Then, absorbance of each sample was measured at 760 nm with a spectrophotometer. TPC of the extracts was calculated from the regression equation (r 2 = 0.958) of the standard gallic acid and the results were expressed as milligram per gram of gallic acid equivalent of the dried extracts. Determination of total flavonoid content (TFC) 1 ml extract in methanol (200 mg/ml) was mixed with 1 ml aluminium trichloride in ethanol (20 mg/ml, and a drop of acetic acid), and then the mixture was diluted by the addition of ethanol up to its 25 ml volume. Blank samples were prepared by adding all the reagents with equal volume used in the sample, except the extract. The absorbance of the solution was read at 415 nm after 40 min of incubation at room temperature. Using the same procedure for absorbance of quercetin, standard compound of flavonoid was read and TFC of the extracts was calculated from the standard curve (r 2 = 0.902) of the quercetin (12.5 to 200 mg/ml). Total flavonoid content was expressed as mg/g of quercetin equivalent (Kumaran and Karunakaran, 2007). Estimation of total flavonol content (TFlC) TFlC was determined by applying a method previously described by Mbaebie et al. with slight modification (Mbaebie et al., 2012). According to the method, 1 ml of the extracts (200 µg/ml) was taken separately in different test tubes. 2 ml ethanol solution of AlCl 3 and 3 ml of (50 g/l) sodium acetate solution were added in the test tubes. After gently mixing, all the test tubes were allowed to stand for 2.5 h at 20°C temperature. Then, absorbance was determined by using a spectrophotometer at a wavelength of 440 nm. Quercetin was used as standard flavonol compound. Following the aforementioned procedure, absorbance of the quercetin was taken at various concentrations (25 to 400 μg/ml) of series dilution. TFlC of the extracts was calculated from regression equation (r 2 = 0.951) of the standard quercetin and the results were expressed as milligram per gram of quercetin equivalent of the dried extracts. Determination of total antioxidant content (TAC) TAC of the extracts was evaluated by phosphomolybdenum complex method with slight modification, which was described by Prieto et al. (1999). Briefly, a reagent solution was prepared having 0.6M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate in distilled water. 1 ml of each extract was combined with the reagent solution in separate test tubes. After shaking gently, the test tubes were incubated for 90 min at 95°C temperature. Then after cooling at room temperature, absorbance was measured at 695 nm wavelength using a spectrophotometer. Similarly, ascorbic acid, a standard antioxidant, was run through the process at different concentration gradient (25 to 400 μg/ml). Using this absorbance value, a standard calibration curve and a regression equation (r 2 = 0.964) was derived, from which TAC of each of the extracts was determined and expressed as mg/g of ascorbic acid equivalent of the dried extracts. Determination of 1, 1-dipheny-l-2-picrylhydrazyl (DPPH) radical scavenging activity The DPPH free radical scavenging activity was measured by an established method described by Braca et al. (2002). Briefly, 0.004% w/v of DPPH radical solution was prepared in methanol and then 900 μl of this solution was mixed with 100 μl of extract or standard ascorbic acid solution (12.5 to 200 μg/ml) and kept in a dark place for thirty minutes. Then, absorbance was measured at 517 nm. Scavenging capacity of DPPH radicals (% Inhibition) was measured by the following formula and finally the 50% inhibition concentration (IC 50 ) was calculated using MS-Excell software. Where A 0 = Absorbance of control group, A s = Absorbance of sample. Ferric reducing antioxidant power (FRAP) assay The Fe 3+ reducing power was determined by the method of Oyaizu (1986) with slight modifications. Shortly, 1 ml of extract or standard ascorbic acid solution was taken in a test tube and mixed with 2.5 ml of phosphate buffer solution (0.2 M, pH 6.6). Then 2.5 ml of potassium ferricyanide (1%) was added and incubated at 50°C for 30 min. After that, 2.5 ml of trichloroacetic acid (10%) was added and centrifuged at 4000 rpm for 10 min. Finally, 2.5 ml of the supernatant was mixed with 2.5 ml of distilled water and 0.1 ml of FeCl 3 (0.1%) solution followed by incubation at 35°C for 10 min. The absorbance was measured at 700 nm and the reducing power of the extracts was compared with the standard ascorbic acid. From standard calibration curve, median effective concentration (EC 50 ) was calculated. The EC 50 value (µg/ml) is the effective concentration giving an absorbance of 0.5. Anticholinesterase (AChE and BChE) assays AChE from Electric eel and BChE from equine serum were used to explore the enzymes inhibitory potential of A. salvifolium root extracts by using Ellman's assay (Classics et al., 1961). The assay is based on the hydrolysis of acetylthiocholine iodide or butyrylthiocholine iodide by the respective enzymes and the formation of 5-thio-2-nitrobenzoate anion followed by complexation with DTNB to give a yellow colour compound which is detected with spectrophotometer beside the reaction time. Preparation of solutions A phosphate buffer solution (0.1 M and 8.0 ± 0.1 pH) was prepared by adding K 2 HPO 4 (17.4 g/L) and KH 2 PO 4 (13.6 g/L) in distilled water. Various concentrations (25,50,100,200,400, 800 μg/ml) of the extracts and standard drug Donepezil were prepared by series dilution. AChE (518 U/mg solid) and BChE (7 to 16 U/mg) were diluted by adding the freshly prepared buffer solution up to obtain 0.03 and 0.01 U/ml concentration of the enzymes, respectively. Solutions of DTNB (0.0002273 M), ATChI and BTChI (0.0005 M) were prepared in distilled water and were kept in eppendorp caps in the refrigerator at 8°C temperature. Spectroscopic analysis For these assays, 5 μl of AChE/BChE enzymes were taken in different cuvette followed by addition of 205 μl sample (extracts/standard solution) and 5 μl DTNB reagent solutions. The solution mixture in each cuvette was mixed gently and maintained at 30°C for 15 min using water bath with subsequent addition of 5 μl substrate solution (ATChI in AChE containing cuvettee and BTChI in BChE containing cuvettee). Absorbance was read against a blank solution by using a UV-Visible spectrophotometer. The absorbance of each solution along with the reaction time was taken for four minutes at 30°C. The enzyme activity and enzyme inhibition by control and tested samples were calculated from the rate of absorbance change with time (V = ΔAbs / Δt) as follows: Enzyme inhibition (%) = 100 -percent enzyme activity. Enzyme activity (%) = 100 × V/V max . Where, V is the enzyme activity in the presence of standard drug or extracts and V max is the enzyme activity in the absence of extracts or standard drug. 50% inhibition concentration (IC 50 ) values were calculated by using MS-Excel software. Determination of correlation (r 2 ) between antioxidant activities and phytochemical assay MS-excel program was used to determine the correlations between antioxidant activities and phytochemical contents. IC 50 values of DPPH, EC 50 values of FRAP and TAC were put against TPC, TFC and TFlC values of the extracts. In each set, pearson correlation (r 2 value) was determined from the regression equation. Statistical analysis All the data were presented as the mean value of triplicate experiment (n=3) along with standard deviation (Mean±SD). P* < 0.05, P** < 0.01 and P*** < 0.001 were considered as significance level. ANOVA, followed by dunnett's test was done in SPSS version 15.0 and 95% confidence of interval was calculated from it. IC 50 and EC 50 values were calculated by using the MS-excel program.TPC, TFC and TFlC were calculated from regression equation of each standard sample by using the program (MS-excel). All the figures were prepared by using Graph Pad Prism software, version 5.0. Preliminary phytochemical screening Preliminary phytochemical screening of the extracts revealed the important bioactive metabolites which are presented in Table 1. Total phenol content (TPC) All the extracts showed phenolic content with significant (P**<0.01, P***<0.001) difference among them which are summarized in Figure 1A. DASR, among the extracts, showed the highest phenolic content followed by CASR. DPPH free radical scavenging activity All the extracts inhibited DPPH radicals at concentration gradient manner (more concentration more inhibition). (Table 2). Ferric reducing power assay Reducing power of all the extracts and the standard compound ascorbic acid was increased with the gradual increase of concentration. (Table 2). Total antioxidant content (TAC) The phosphomolybdate method, another quantitative method of antioxidant effect measurement, is based on the reduction of molybdenum (VI) to molybdenum (V) which takes place for the presence of antioxidant compound in the extracts. In the present study, all experimented samples had good TAC but in significantly (P* < 0.05, P** < 0.01 and P*** < 0.001) different extent. CASR had the highest (639.55 ± 10.51) while EASR had the lowest TAC (114.11 ± 12.83). The order of TAC among the extracts was CASR > DASR > AASR > EASR ( Figure 1D). (Table 3). Correlation between antioxidant effects and phytochemicals The correlation analysis was performed to investigate the Data are expressed as mean ± standard deviation (n = 3). P*< 0.05, P**<0.01 and P***<0.001 are considered as significant difference of IC 50 /EC 50 value compared with the highest value. relationship between the phytochemicals and antioxidant activity of the extracts. Among the phytochemicals, TFlC showed strong positive correlation with DPPH (r 2 = 0.913), FRAP (r 2 = 0.803) and TAC (r 2 = 0.782). TFC had well positive correlation with TAC (r 2 = 0.764) while weak correlation with DPPH and FRAP effects. TPC showed weak correlation with FRAP and TAC but moderate correlation with the DPPH test (Table 4). DISCUSSION Oxidative stress plays a vital role for generation and progression of AD, where nerve cells or cellular components are oxidized by some free radicals that are considered as powerful oxidizing agents. Among these, the ROS ( • O 2 -, • OH, H 2 O 2 , O 3 ) are very potential to induce lipid peroxidation and subsequently cell death. These are generated mostly by mitochondrial oxidation and moderately by the influence of environmental pollutants, smoking and harmful radiations (Lobo et al., 2010). We have a self protective mechanism against the radicals, namely antioxidant defense system, composed of some enzymatic antioxidants, main function of which is to protect our body from the oxidative stress. Here, antioxidants, enzymatic or non enzymatic, show their Data are expressed as mean ± standard deviation (n = 3). P * < 0.05, P ** <0.01 and P *** <0.001 are considered as significant difference of IC 50 value compared with the highest value. (Laura et al., 2012). The coordinate action of antioxidant system is very critical for the detoxification of the radicals. Superoxide dismutase acts on highly reactive superoxide radical ( • O 2 -) and converts it to less reactive H 2 O 2 radical. Then catalase and glutathione peroxidase converts the H 2 O 2 to water, and thus brain tissues are protected from the reactive radicals (Lin et al., 2008). However, in the case of stress condition, defensive power of the natural antioxidant system declines sharply, since the brain cells consume large proportion of the inhaled oxygen which consequently generates increased number of free radicals due to high metabolic rate in it. Moreover, ascorbate and transient metals, largely present in the nerve tissues, acts as prooxidant and potentiates the oxidative damage of the nerve cells due to their high content of polyunsaturated fatty acids (Laura et al., 2012). So, when free radicals exceeds their normal threshold level, oxidative stress proceeds abundantly, and the cells fail to function effectively, and consequently cellular degeneration takes place which is a way of the AD progression. In this condition, antioxidant supplement is essential to combat with the radicals, and to protect the brain from the cell degeneration (Varcin et al., 2012). AD is developed by numerous pathogenic factors such as formation of abnormal compound, namely amyloid-β peptide (Aβ) and intracellular neurofibrillary tangles (NFTs), reduction of acetylcholine level and exacerbation of oxidative stress (Iwaki and Namoto, 2014). Acetylcholine, an organic molecule, acts as a neurotransmitter, and is associated with neuronal networking in central and peripheral nervous systems. Naturally, it is produced in some of our brain cells which are called cholinergic neurons. After a specific life span, ACh goes to break down by the AChE and BChE enzymes. In case of normal healthy people, the rate of synthesis and cleavage of the ACh remain steady to maintain its normal level. In this case, the AChE is 1.5-fold to 60-fold more active than that of BChE. But, in the case of AD, enzyme performance shifts towards the BChE, where its activity increases up to 120%. In contrast, AChE loses its effectiveness by 10 to 15% (Faiyaz et al., 2013). This abnormality, increased break down rate of Ach, leads to decrease the availability of the ACh than its normal physiological scale. Furthermore, the reduced level of ACh adversely affects the physiological functions of the brain. In addition, AChE and BChE potentiates neuronal degeneration by forming some protein complexes such as: neurofibrillary tangles (NFT) and neuritic plaques (NP) which are aggregates of hyperphosphorylated tau protein and extracellular neurotoxic deposits of Aβ, respectively (Dominik and Kamila, 2012). AChE/BChE bind with Aβ and a protein called ApoE protein, resulting in the formation of a highly stable complex (AChE/BChE-Ab-ApoE complex) in cerebrospinal fluid (CSF) of the brain. This stable complex directly interacts with ACh receptors and therefore, interferes with their signal transductions and potentiates ultrafast hydrolysis of ACh (Swetha et al., 2013). Researchers, for example, from postmorterm studies of AD patients, have found strongly reduced number of ACh receptors and loss of basal forebrain and cortical cholinergic neurons (Taiwo et al., 2010). Therefore, inhibition of AChE and BChE is the most effective therapeutic approach to treat the symptoms of AD. Consequently, cholinesterase inhibitors are the only approved drugs for treating patients with mild to moderately severe Alzheimer's disease (Faiyaz et al., 2013). Many phytochemicals have been reported to have satisfactory antioxidant and anticholinesterase effects. Among these phenolics and flavonoids, potent antioxidative compounds act as free radical scavengers (Fadwa et al., 2012). Majority of the phytochemicals having potent AChE and BChE inhibitory effects, are alkaloids followed by terpinoids, steroids, flavonoids, glycosides, saponins and essential oils (Seyed et al., 2014). Since most of the natural or synthetic products, having enzyme inhibitory effects are known to contain nitrogen atom, the promising effect of the medicinal plants could be due to their high alkaloidal contents (Seyed et al., 2014). Alangium salvifolium is rich with biologically active phytochemicals where various types of alkaloids have been isolated and identified. Among these alangimaridine, meyhyl-1H pyrimidine-2, 4-dione, alangine A and B, alangicine, markindine, lamarckinine and emetine are important. Besides, phytochemical screening of it revealed the presence of flavonoids, phenolics, glycosides etc (Ashalatha and Gopinath, 2013;Ronok et al., 2013;Savithramma et al., 2013). So, these compounds may be considered for the antioxidant effect and enzyme (AChE and BChE) inhibitory activities of the extracts. Conclusion A. salvifolium wang is extensively used as folk medicine. The present study showed that the plant is important for its phytochemical constituents. It has significant amount of phenolics, flavonoid and flavonol. Root extracts of the plant have shown moderate to potent antioxidant potential. These are also effective to inhibit AChE and BChE enzymes. So the plant is effective to protect from alzheimer disease. However, further analysis is necessary to isolate the key compounds and to find out the actual mechanism of action.

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2015-03-07T18:39:34.000Z

2015-01-13T00:00:00.000Z

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Bovine respiratory syncytial virus and bovine coronavirus in Swedish organic and conventional dairy herds Background Infections with bovine respiratory syncytial virus (BRSV) and bovine coronavirus (BoCV) are endemic to the cattle populations in most countries, causing respiratory and/or enteric disease. It has been demonstrated that herds can remain free from these infections for several years also in high prevalence areas. Organically managed (OM) dairy herds have been shown to have lower seroprevalence of both viruses compared to conventionally managed (CM) herds. The objective of this study was to challenge the hypothesis of a lower occurrence of BRSV and BoCV in OM compared to CM dairy herds. In November 2011, May 2012 and May 2013 milk samples from four homebred primiparous cows were collected in 75 to 65 OM and 69 to 62 CM herds. The antibody status regarding BRSV and BoCV was analysed with commercial indirect ELISAs. Herds were classified as positive if at least one individual sample was positive. Results The prevalence of positive herds ranged from 73.4% to 82.3% for BRSV and from 76.8% to 85.3% for BoCV among OM and CM herds, over the three sampling occasions. There was no statistically significant difference between OM and CM herds at any sampling occasion. The incidence risk of newly infected herds did not differ statistically between OM and CM herds at any sampling occasion, neither for BRSV nor for BoCV. The incidence of herds turning sero-negative between samplings corresponded to the incidence of newly infected. Bulk tank milk (BTM) samples were also sampled in the herds and analysed. Several herds were negative on individual samples but positive in BTM. Herd-level data on production, health and reproduction were retrieved from VÄXA Sweden and the study herds were representative of the source population. Conclusion There was no difference in prevalence of or incidence risk for BRSV or BoCV between Swedish OM and CM herds. Because the incidence of herds becoming seropositive was balanced by herds becoming seronegative it should be possible to lower the prevalence of these two infections among Swedish dairy cattle herds if biosecurity is improved. Electronic supplementary material The online version of this article (doi:10.1186/s13028-014-0091-x) contains supplementary material, which is available to authorized users. Background Infections with bovine respiratory syncytial virus (BRSV) [1][2][3][4] and bovine coronavirus (BoCV) [2,5] are endemic in the cattle populations in most countries. It has been demonstrated that a cattle herd can remain free from these infections for several years [6], even when located in high prevalence areas [7] and in close proximity to herds experiencing an (BRSV) outbreak [8]. Herds may become antibody negative to any of these infections within a few years provided the virus is not re-introduced into the herd [6,8]. BRSV commonly cause respiratory disease, particularly in calves. Disease can be caused by BRSV only or in combination with other viruses (e.g. BoCV) or secondary bacterial infection [9][10][11]. BoCV also causes enteric disease, in particular calf diarrhoea [12], and is the causative agent of Winter Dysentery, outbreak of diarrhoea, in adults [13]. There have been reports of BoCV as the single agent in outbreaks of respiratory disease as well [14,15]. In addition to impaired animal welfare due to illness, these infections may cause losses to production by reduced weight gain [16,17], reduced milk yield [4,18], increased bulk tank [19] and individual [18] milk somatic cell counts. After infection with BRSV or BoCV animals will remain seropositive for several years. This was demonstrated by Bidokti et al. [20] who found herd where the older cows were sero-positive while the younger cows were sero-negative, i.e. there had been no virus circulating for several years. Maternal antibodies remain detectable for approximately 6 months [13,21], i.e. a never-infected heifer will be seronegative at the time of first calving. Both milk and blood samples can be used to assess the serological status of cattle [22]. When the herd's status is based on a bulk tank milk (BTM) sample, which is convenient e. g. for screening a population, the result will reflect the long term, i.e. up to the life-span of the oldest cows, history of the herd. However, if primiparous homebred cows are sampled, the results will give a more accurate description of the recent, i.e. the life-span of the tested cows, history of the herd. Although these viruses may spread during the warmer seasons, seroconversion with or without an outbreak of clinical disease is more frequent during the housing season (autumn and winter) [6,23,24]. There is however, still a knowledge gap concerning what the most important routes for virus transmission between herds are. A few studies from the Nordic countries have studied risk factors for herds to be and to become seropositive to BRSV and BoCV. Risk factors at herd level have included a short distance to nearest herd, not providing boots to visitors, large herd size and a high density of cattle in the area [5,7,25,26]. A recent study found that organically managed (OM) dairy herds had significantly lower seroprevalence of both BoCV and BRSV compared to conventionally managed (CM) herds. However, the study could not explain the reason for the differences in the two production systems [20]. Although the difference was statistically significant, the study was made with a relatively small sample of herds. It would be beneficial for the organic as well as the conventional dairy production if these results were validated and studied further. Moreover, the Swedish dairy industry has over the last decade undergone rapid structural changes with increasing herd sizes and decreasing herd numbers. Simultaneously, the proportion of dairy cows under OM management has increased from 6% in 2005 to 13% in 2011 [27,28]. This also calls for a new assessment of disease occurrence in the two management systems. The objective of this study was therefore to challenge the hypothesis of a lower occurrence of BRSV and BoCV in OM compared to CM dairy herds and, specifically, to compare the herd prevalence and incidence. Methods This was a prospective longitudinal observational study including OM and CM dairy herds. The unit of interest was herd. Study population The sampling frame was all dairy herds with a yearly average herd size of at least 50 cows and enrolled in the Swedish Official Milk Recording Scheme. The inclusion criterion of a yearly average herd-size of at least 50 cows was chosen to exclude small herds which do not represent the "future" herd. Geographically, all counties except for most southern Skåne were included. Skåne was excluded because it was known that there are very few BRSV and BoCV negative herds in this region. OM herds were defined as herds with a dairy production certified according to the standards by the association KRAV (www.krav.se), the Swedish member of the International Federation of Organic Agriculture Movements. The desired size of the study group was 100 OM and 100 CM herds which was as many as the project would be able to manage. The number of invited herds was based on previous experiences of about 30% willingness among Swedish dairy farmers to participate in similar observational studies. A simple random sample of 400 CM herds from the 1800 in the sampling frame, and all eligible OM herds (n = 244) were sent a written invitation to the study in May 2011. In total, 75 (31%) and 69 (17%) of farmers with OM and CM herds, respectively, agreed to participate in the project. Participants gave a written permission to access the herd's data from the milk recording scheme. Data collection and management Milk samples and analysis Study herds were sent instructions, material and protocol for sampling in November 2011, May 2012, and May 2013. These occasions were chosen to include as many stall periods as possible during the project. At each sampling occasion, milk from four homebred primiparous cows and BTM were sampled into test tubes with 1.5 mg of the preservative agent Bronopol. Samples were returned to the National Veterinary Institute by prepaid mail where they were stored at −20°C until analysed. The antibody status regarding BRSV and BoCV was analysed with the commercial indirect ELISAs (Svanovir BRSV-Ab and Svanovir BCV-Ab, Boeringer Ingelheim Svanova, Uppsala, Sweden). The optical density (OD) of samples was corrected by subtraction of negative control OD, and the percent positivity (PP) value was calculated as the corrected OD divided by the corrected OD for positive controls and multiplied by 100. The samples from individuals were classified as negative if PP < 10, following the manufacturer's guidelines, and bulk tank samples if PP < 5. The sensitivity and specificity of the ELISA test kit was 94% and 100%, respectively, for BRSV and 84.6% and 100%, respectively, for BoCV, according to the manufacturer. After each completed sampling occasion, the farmers were sent information on their herd's serological status and information about basic biosecurity measures. A lottery ticket (value approx. 3 euro) was enclosed to the letter. Statistical analyses Samples from individuals Antibody status as measured in individual milk samples was used as an indicator of the herd's recent infection status regarding the two viruses. Herds were defined as antibody positive if at least one of four samples from individual cows was positive. A stricter definition of positive herd where all four individual samples had to be positive was also applied. To assess any difference in antibody status between OM and CM herds, the prevalences of positive herds at each sampling occasion among OM and CM herds, respectively, were calculated with exact binomial confidence intervals using the package "binom" for R [29]. Data management and analysis was performed in R version 3.0.1 [30]. Further, the incidence risk with exact binomial confidence intervals was calculated for the samplings in 2012 and 2013, for OM and CM herds, respectively. The period at risk was from the last sampling occasion, i.e. 6 months and 12 months, respectively. Bulk tank milk samples To explore if there were differences in antibody level in the bulk tank milk between the CM and OM herds, the results from the BTM samples (PP values) were categorised into six groups; PP < 5, 5 ≤ PP < 10, 10 ≤ PP < 30, 30 ≤ PP < 60, 60 ≤ PP < 100, and 100 ≤ PP. The frequencies of OM and CM herds in each group were tabulated for each year and virus. Herd data Data on herd production, health and reproduction were retrieved from the Swedish Official Milk Recording Scheme database managed by VÄXA Sweden for all selected herds, i.e. including also the invited but nonparticipating herds. The information included averages of routinely collected parameters for the 12 months before the start of the study. Herd parameters were described and tested for difference between the OM and CM herds that participated in 2011 i.e. entered the study, using a two-sided Wilcoxon rank-sum test or Fischer exact test. To study how the study group characteristics were affected from dropout herds leaving the study, herd parameters were described for the subsample of OM and CM study herds that completed the last sampling in 2013. To examine the representativeness of the study herds, herd parameters were also described for all invited OM and CM that did not participate in the study. Results The number of study herds per year was 144, 132, and 127 in 2011, 2012 and 2013, respectively (Table 1) Samples from individuals Based on the test results from sampled individuals the prevalence of BRSV positive herds ranged from 73.4% to 82.3% over three sampling occasions among OM and CM herds (Table 1). In 2011, the prevalence of positive herds was lower in CM herds, and in 2012 and 2013 in OM herds. For BoCV the prevalence estimates ranged from 76.8% to 85.3% positive herds over three sampling occasions among OM and CM herds (Table 1). In 2011 and 2012 the prevalence of positive herds was lower in CM herds and in 2013 in OM herds. The confidence intervals of OM and CM herds' prevalences were overlapping all years and it was therefore concluded that there was no statistical difference in the prevalence of BRSV or BoCV between OM and CM herds. The prevalence of BRSV-positive herds decreased to 50.7-71.0% and for BoCV to 64.1-78.7% with the stricter the criteria of four out of four positive individual samples for a herd to be classified as positive at the sampling occasion ( Table 1). The incidence risk of newly infected herds, i.e. herds that went from negative to having at least one of four primiparous cows with a positive test result, did not differ statistically between OM and CM herds in any study year, neither for BRSV nor for BoCV (Table 1). There were in total 12 herds that were antibody-negative to both viruses in 2011 (8.3%), and 11 herds in both 2012 (8.3%) and 2013 (8.6%) ( Table 1). Bulk tank milk samples Not more than 7, 3 and 4 herds were BRSV negative and 5, 6, and 3 herds were BoCV negative in 2011, 2012 and 2013, respectively ( Table 2). All these herds negative in BTM were also negative based on the individual samples. Further, for both viruses, in a high proportion of all herds with BTM PP < 30, all individual samples were negative. And, on the contrary, in a high proportion of herds with BTM PP ≥ 100, all individual samples were positive ( Table 2). Herd data At study start the OM study herds were larger, had higher BTM somatic cell counts, lower incidence of veterinary treated disease events but also a lower production compared to the CM study herds (See Additional file 1: Table S1). Herd data for herds entering the study and herds completing the study were judged as comparable (See Additional file 1: Table S1). The characteristics of the herds entering the study were judged as comparable to the invited herds not entering the study (See Additional file 1: Table S1). Discussion In this study, there was no difference in incidence or prevalence of BRSV or BoCV antibody positivity between OM and CM herds. This disagrees with previous results from Sweden where the mean seroprevalence of antibodies in individual cows for BRSV and BoCV was found to be lower in OM dairy herds compared to CM [20]. Ohlson et al. [25] could not report any differences between OM and CM herds with respect to BRSV and BoCV seropositivity, which agrees with the results in the present study. However, that study included only a small sample of OM herds. The number of Swedish OM dairy cows increased from 22,321 in 2005, when the herds in Bidohkti et al. [20] were recruited, compared to 44,133 in 2011 while during the same time period, the total number of dairy cows decreased from 393,263 to 346,495 [27,28]. A possible explanation to the different results in the current study compared to [20] could be that the characteristics of the farmers with OM and their herds as a group have changed from 2005 to 2011. Such a change of the "typical organic farmer" over time and in relation to market demand and political decisions, e.g. how incentives for organic farming might have developed from ideological for early converters to more financial for later, was discussed for Denmark [31]. Interestingly, the total number of OM herds going from positive to negative was higher compared to CM herds, but the opposite was true for herds going from negative to positive status (Table 1). This could be hypothesised to be a result of better implementation of the biosecurity advice to participants by the farmers with OM. Although OM herds were overrepresented in the study group, the overall prevalence estimates, as well as for OM and CM separately, are in concordance with previous studies where herd prevalence of BRSV in BTM samples was 41-51% in northern Sweden and 84-89% in the most southern parts [3], and 85% in a study with seven southern and northern counties [25]. For BoCV herd prevalence was reported as 70-74% to 95-100% in BTM samples depending on region [5] and 81% in a study with southern and northern counties [25]. The national prevalence of these two infections seems not to have changed a lot from the late 1990-ies. It would be interesting to further explore how changes of routines or characteristics of dairy herds during a bit more than a decade might be associated with the prevalence of the two infections. For example, if the greatly improved biosecurity in many farms might have been offset by poor routines in others during the last decade. The current sampling frame included all counties of Sweden but the Skåne county which from previous research was known to have a herd prevalence near 100% and therefore considered as of less interest in this study. In a screening of bulk tank milk samples in 2013, including 95% of all Swedish dairy herds, the herd prevalence in Skåne was 99% (personal communication A Ohlson). Every year several herds became antibody negative, and in other words gained BRSV and/or BoCV free status, but the incidence of number of herds becoming positive equalled out the number of herds clearing the infection. There were also a considerable number of herds with negative primiparous cows but positive BTM, i.e. herds in which there had been no virus circulating for at least 2-3 years. Apparently, there are good chances for a herd to free from these infections if good biosecurity is practiced and the virus is not reintroduced. This result agree with a Norwegian survey where the incidence of herds becoming BRSV positive (at least one antibody positive animal) and negative after six months was 33% and 42%, respectively [8]. A high turnover of positive herds was also found in a Swedish study over several years [6]. If introduction of BRSV and BoCV to a herd can be eliminated or kept at lowest possible level, it would be possible to significantly reduce the prevalence of these infections in the Swedish dairy population. Many of the herds with positive BTM samples, but with a low PP value, i.e. <30 had only negative individuals. This indicates that there had been no virus circulating in the herd the last 2 to 3 years (Table 2) but was rather due to antibodies from older cows who experienced the infections several years ago. This finding agrees with Ohlson et al. [6] where all sampled primiparous cows in herds with a BTM sample PP value <30 were negative. We would therefore recommend sampling milk from young homebred cows over BTM samples in prevalence studies/screenings, because of the advantage that herds where there has been no recent active infection can be identified. To save analysis expenditure individuals' samples, in addition, can be pooled for analysis without significant loss of sensitivity or specificity at herd level [22]. Also, the prevalence of positive herds decreased when "positive" was defined as four of four positive individuals (10 ≤ PP) but the difference in prevalence using this stricter definition was not significant (Table 1). This supports that BRSV and BoCV virus have efficiently spread in the herd after introduction and results in a subsequent high within-herd seroprevalence. With a herd size of 70, the test characteristics of the two ELISAs used, and a cut-off point for positive herd of one positive out of four tested individuals, the HSe for BRSV is 93% and 99% at a within-herd prevalence of 50% and 70% respectively. For BoCV the HSe is 90% and 98% at a within-herd prevalence of 50% and 70%, respectively. HSp is 100% for both tests as calculated with the web based tool [32] using the hypergeometric distribution. The herd sensitivity of the serological test can be increased by increasing the number of sampled individuals per herd; however, with a high within herd prevalence this becomes less influential. A limitation of the study is the less than perfect sensitivity of the two ELISA tests used. Because the aim was to compare OM and CM herds, the true prevalence or incidence risk was not estimated. Any misclassification bias due to test characteristics should be the same in the two groups. Further, the different lengths of period at risk for the incidence estimates at the second and third sampling occasions was not optimal, because it made comparisons between sampling occasions impossible. This, however, does not influence the comparison of OM and CM herds at the same sampling occasion. However, this comparison could have been improved with a larger number of study herds. For example, the number of herds going from positive to negative or the opposite was now low and the confidence intervals of estimates wide. It would also have been beneficial with a longer total study period, thus including more than two housing periods, and more frequent sampling to explore how herds' status change over time with better precision. We consider the OM and CM study herds as representative of the OM and CM dairy herds in Sweden, because their production and health parameters did not differ substantially from the invited but non-participating herds (Additional file 1: Table S1). However, the study herds could still be biased towards more engaged and skilful farmers compared to non-participants and a higher response rate would have been desirable to minimize this risk. This is particularly valid for the CM herds, where the response rate was 17% compared to 31% for the OM herds. This could mean that the farmers with OM more accurately represent the OM population, while a possibly stronger bias towards a stratum of "interested, progressive and better" farmers with CM could be envisioned. The invited CM herds were a random sample meaning the herds entering the study represented 4% of the eligible herds, while the invited OM herds were a census, thus the participating herds represented 31% of the eligible herds. Because the study included three sampling occasions the possible increase in response rate after reminders, e.g. by telephoning non-responders, was weighted against the risk of losing these additional participants to the next sampling occasion. The inclusion criterion of a yearly average herdsize of at least 50 cows meant that 1,960 of the 4,022 dairy herds enrolled in milk recording in 2011 were not eligible for the study and the results should only be extrapolated to the larger herds. We judged that the study herds that participated to the last sampling occasion were comparable to study herds that participated at the start, i.e. herds leaving the study did not change the characteristics of the study group (Additional file 1: Table S1). Conclusions There was no difference in prevalence of or incidence risk for BRSV or BoCV between organically and conventionally managed dairy herds in Sweden. The incidence risk of herds becoming seropositive was balanced by herds becoming seronegative, but with improved biosecurity it should be possible to lower the prevalence of these two infections among Swedish dairy cattle herds. Additional file Additional file 1: Table S1. Herd characteristics and production and health parameters at the time for the start of the study for organically managed (OM) and conventionally managed (CM) herds entering the study, the study herds completing the full study period and invited but non-participating herds.

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2016-05-04T20:20:58.661Z

2015-01-26T00:00:00.000Z

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Colonization and Diversity of AM Fungi by Morphological Analysis on Medicinal Plants in Southeast China The arbuscular mycorrhizal (AM) fungal distributions in the rhizosphere of 20 medicinal plants species in Zhangzhou, southeast China, were studied. The results showed 66 species of 8 genera of AM fungi were identified, of which 38 belonged to Glomus, 12 to Acaulospora, 9 to Scutellospora, 2 to Gigaspora, 2 to Funneliformis, 1 to Septoglomus, 1 to Rhizophagus, and 1 to Archaeospora. Glomus was the dominant genera and G. melanosporum, Acaulospora scrobiculata, G. etunicatum, Funneliformis mosseae, and G. rubiforme were the prevalent species. The highest colonization (100%) was recorded in Desmodium pulchellum (L.) Benth. while the lowest (8.0%) was in Acorus tatarinowii Schott. The AM fungi spore density ranged from 270 to 2860 per 100 g soil (average 1005), and the species richness ranged from 3 to 14 (average 9.7) per soil sample. Shannon-Wiener index ranged from 0.52 to 2 (average 1.45). In the present study, the colonization had a highly negative correlation with available K and electrical conductivity. Species richness correlated positively with electrical conductivity and organic matter. Shannon-Wiener index had a highly significant negative correlation with pH. This study provides a valuable germplasm and theoretical basis for AM fungal biotechnology on medicinal standardization planting. Introduction Arbuscular mycorrhizal (AM) fungi, the most ubiquitous symbiosis in nature, are a kind of these soil microbes. Reports suggest that estimated 80% of plant species forms mycorrhizas [1]. In general, AM fungi and the host plants are reciprocal symbionts. The symbiosis improves plants the nutrient uptake and provides protection from pathogens, while the AM fungi receive carbohydrates [2][3][4]. All over the world, 80% of the rural population in developing countries utilizes locally medicinal plants for primary healthcare. And in China, the use of different parts of medicinal plants to cure specific illness has been popular from ancient time. In Zhangzhou, southeast China, the typical humid subtropical monsoon climate contributes to the growth of more than 700 kinds of lush medicinal plants and creates unique ecological conditions for species diversity and distribution of AM fungi. The distribution of AM fungi associated with medicinal plants has been reported. In a survey on AM association with three different endangered species of Leptadenia reticulata, Mitragyna parvifolia, and Withania coagulans, high diversity of AMF was observed, and Glomus constrictum, Glomus fasciculatum, Glomus geosporum, Glomus intraradices, Glomus mosseae, and Glomus rubiforme were the most dominant species [5]. Similarly, 34 AM fungal species were identified from 36 medicinal plant species [6]. Approximately 15 fungal species from 10 genera were isolated from the collected soils in medicinal plant species, lemon balm (Melissa officinalis L.), sage (Salvia officinalis L.), and lavender (Lavandula angustifolia Mill.) [7]. About 50 species of medicinal plants from 19 families have been studied in the association with AM fungi [8]. However, not enough has been focused on the mycorrhizal association with medicinal plants. Generally, AM fungi species in different ecosystems are affected by edaphic factors, so it is necessary to investigate the spatial distribution and colonization of AM fungi related to the medicinal plants [9][10][11][12][13]. Hence, the present study is attempted to investigate Study Sites. The city of Zhangzhou, Fujian province, a subtropical region, is located on 23 ∘ 08 -25 ∘ 06 N and 116 ∘ 53 -118 ∘ 09 E. The mean annual temperature is 21 ∘ C with yearly precipitation of 1000-1700 mm and annual sunshine of 2000-2300 hours. Frost-free periods add up to more than 330 days with cool summer and warm winter. The medicinal plants in this study were collected from Xiaoxi town (24 ∘ 44 N, 118 ∘ 17 E), which was cinnamon soil from farmland, and Guoqiang village (24 ∘ 35 N, 117 ∘ 56 E), which was cinnamon soil from woodland, in Zhangzhou. Sample Collection. The plants grew under natural environmental conditions. Six healthy individuals per plant species of medicinal plants (Table 1) were randomly selected for the collection of rhizospheric soil and root samples; 180 soil and root samples were collected from Xiaoxi town and Guoqiang village in October 2011. For each plant, three random soil cores at the depth of 0-30 cm about 1000 g were established by contacting from the 6 duplicate plants. Approximately 20 plants species and 120 soil samples were collected in total. The subsamples were air-dried for 2 weeks and stored in sealed plastic bags at 4 ∘ C for the following analysis. Estimation of AM Colonization. The mixed soil and roots samples of each plant species were packed in polyethylene bags, labeled and brought to the laboratory. The soil samples were air-dried at room temperature. Roots were washed to remove soil particles, preserved with FAA. For colonization measurement, roots were cleared in 10% (w/v) KOH and placed in a water bath (90 ∘ C) for 20-30 min. The cooled root samples were then washed with water and stained with 0.5% (w/v) acid fuchsin. Fifty root fragments for each sample (ca. 1 cm long) were mounted on slides in a polyvinyl alcohol solution [14] and examined for the presence of AM structures at 100-400x magnification with an Olympus BX50 microscope for the presence of AM structures. The percentage of root colonization was calculated using the following formula: Number of arbuscular mycorrhiza positive segments Total number of segments studied AM Fungus Spore Quantification and Identification. Three aliquots of soil (20 g) were obtained for every plant species. AM fungal spores were extracted from the soil samples by wet sieving and sucrose density gradient centrifugation [15]. Spores were counted under a dissecting microscope, and spore densities (SD) were expressed as the number of spores per 100 g of soil. The isolated spores were mounted in polyvinyl lactoglycerol (PVLG). Morphological identification of spores up to species level was based on spore size, color, thickness of the wall layers, and the subtending hyphae by the identification manual [16] and the website of the International collection of vesicular and AM fungi (http://invam.wvu.edu/). Soil Analysis. Soil samples were air-dried and sieved through 2 mm grid. Three rhizospheric soil samples (≤2 mm fraction) for each medicinal plant were analyzed for their pH, electrical conductivity (EC), organic matter (OM) content, available N (N), available P (P), and available K (K). Soil pH was measured in soil water suspension 1 : 2 (w/v) by pH meter (PHS-3C, Shanghai Lida Instrument Factory). EC was measured at room temperature in soil suspension (1 : 5 w/v) using conductivity meter (DDS-11C, Shanghai Hong Yi instrument company). OM content was determined by the Walkley-Black acid digestion method. P (extracted with 0.03 M NH 4 F-0.02 M HCl) was measured by molybdenum blue colorimetry, K by an ammonium acetate method using a flame photometer, and N by the alkaline hydrolysis diffusion method [17]. Diversity Studies. Ecological measures of diversity, including spore density (SD), species richness (SR), isolation frequency (IF), Shannon-Wiener index ( ), and evenness ( ), were used to describe the structure of AM fungi communities [18,19]. Diversity studies were carried out from Zhangzhou separately for abundance and diversity of AM fungal species. Spore density was defined as the number of AM fungi spores and sporocarps in 100 g soil; species richness was measured as the number of AM fungi species present in soil sample; isolation frequency (IF) = (number of samples in which the species or genus was observed/total samples) × 100%. Species diversity was assessed by the Shannon-Weiner index as follows: = − ∑ =1 ( ln ); species evenness is calculated by the following formula: = / max where max = − ln , = total number of species in the community (richness). is the relative abundance of each identified species per sampling site and is calculated by the following formula: = / , where is the spore numbers of a species and is the total number of identified spore samples. max is the maximal and calculated by the following formula: = ln , where is the total number of identified species per sampling site. Statistical Analysis. The analysis of Pearson correlation coefficient, variance (ANOVA), and principal component were all carried out with SPSS Bass 18.0 (SPSS Inc., USA). The Pearson correlation coefficient was employed to determine the relationships between AM colonization, SD, SR, IF, , , and soil parameters. Differences in soil parameters, colonization, SD, SR, IF, , and were tested using one-way ANOVA and means were compared by least significant difference at 5% level. Soil Parameters. Results of the rhizospheric soil parameters of the 20 medicinal plants harvested at both sites are summarized in Table 2. The soil P ranged from 10.46 mg kg −1 to 979.94 mg kg −1 , the soil K from 28.64 mg kg −1 to 184.81 mg kg −1 , and the soil N from 14.93 mg kg −1 to 111.48 mg kg −1 . The OM ranged from 5.49 g kg −1 to 14.44 g kg −1 . Furthermore, the soil was acidic as the pH ranged from 4.60 to 7.78. EC was 28.15 s cm −1 to 259.75 s cm −1 . AM Colonization, Diversity Index, and Diversity of AM Fungi. Colonization rate, SD, SR, , and of AM fungi in the rhizosphere of 20 medicinal plants species are presented in Table 3. The percentage of root colonization ranged from 8% to 100% with an average of 58.99%. The highest colonization was observed in Desmodium pulchellum (L.) Benth. and lowest in Acorus tatarinowii Schott. The SD in association with the 20 medicinal plant species ranged from 270 to 2860 spores per 100 g soil, with an average of 1005 spores per 100 g soil. The highest SD was observed in the rhizospheric soil of Lophatherum gracile Brongn. and significantly different with in Leonurus heterophyllus Sweet f. The highest SR (14) was recorded in Acorus tatarinowii Schott., while the lowest (3) appeared in Leonurus heterophyllus Sweet f., with a mean of 9.68. The maximum occurred in Acorus tatarinowii Schott. The results showed that 66 species of 8 genera of AM fungi were isolated and identified, of which 38 belonged to 4 The Scientific World Journal Glomus, 12 to Acaulospora, 9 to Scutellospora, 2 to Gigaspora, 2 to Funneliformis, 1 to Septoglomus, 1 to Rhizophagus, and 1 to Archaeospora (Table 4). Based on IF, Glomus melanosporum, Acaulospora scrobiculata, Glomus etunicatum, Funneliformis mosseae, and Glomus rubiforme were the prevalent AM fungi in decreasing order (Table 4). Generally, AM fungi with an IV greater than 50% were defined as dominant species. So Glomus melanosporum was the prevalent AM fungi with the highest IF (100%). Glomus was the dominant genus with an IF (100%), followed by Acaulospora, IF (95%). Correlation Analysis. As the soil characteristics may play a key role in the ecological distribution of AM fungi, P, K, N, OM, pH, and EC of the soil samples were investigated. In the present study, the colonization was negatively correlated with AK and EC, but positively correlated with OM. Spore density was positively correlated with OM. The same correlation was found between SR and N, EC and OM. was negatively correlated with pH, whereas was negatively correlated with SD (Table 5). Discussion In the present study, the composition and diversity of the AM fungi composition were described based on morphological species. The results indicated that Glomus was the dominant genus, followed by Acaulospora. Acaulospora and Glomus species usually produce more spores than Gigaspora and Scutellospora species in the same environment [20,21]. This may be explained by the difference in development. Acaulospora and Glomus species are thought to require less time to produce spores than Gigaspora and Scutellospora species. Furthermore, members of the Gigasporaceae typically establish an extensive mycelium in soil and produce fewer spores than those of the Acaulosporaceae and Glomaceae [22,23]. The results showed a strong symbiotic relationship between 20 medicinal plants and AM fungi, but significant differences were observed in the different plant species. As the studies have shown nonrandom differences in distribution among different AM fungi species and genera in the field, it is also likely that the preferences of different AM fungi for different host plants in our study might be reflected at the species or family level [24,25]. All 20 medicinal plants were infected by AM fungi, but the degree of colonization and the spore density varied among plant species. This may due to differences in the ability of AM species to sporulate [26]. The host plants used in the trap cultures may also have been an important factor influencing mycorrhizal development, spore formation, and distribution of AM fungi [27]. Many AM species which infect the roots of plants but do not sporulate in the soil may have remained undetected in the present study [28]. Further studies using molecular tools could solve this situation by allowing identification of AM fungi that colonize the roots but remain unsporulating. AM fungal SD, SR have been positively correlated with OM. OM could enhance spore production [29], extra radical proliferation of hyphae [30], and improve AM colonization [31]. In addition, AM fungal hyphae grew best in soils with a high amount of OM [32]. Soil pH in our study was negatively correlated with AM fungal . Soil pH could affect sporulation, spore germination [33], hyphal growth and root colonization [34], and reproduction and community structure of AM fungi [35]. The range of pH from 5.5 to 6.5 has been found to favour Glomus to sporulate more abundantly in acid soils [33]. In the present study, SR was positively correlated with EC. High EC could directly affect the solutes on osmotic potential and delay or prevent all or any of the spore germination phases by dissolved salts in the soil solution. As solution concentrations increased, maximum percent germination and germination rate declined. Effects of salinity on photosynthesis are known to differ between plant species and also between plants at different stages of development [35]. 6 The Scientific World Journal The AM colonization and diversity of medicinal plants in southeast China were investigated in the present study. From the research, we could conclude that the biodiversity of AM fungi was abundant, though Glomus was the dominant genus. The degree of colonization and the spore density varied markedly among plant species. Considering the potential application of AM fungi on medicinal plants, it seems that more attention should be paid to the predominant AM fungi during the process of their cultivation, especially mycorrhizal performance (e.g., improving growth, increasing secondary metabolite production). Conflict of Interests Mingyuan Wang and Pan Jiang declared that there is no competing interest regarding the publication of this paper.

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2019-04-01T13:09:12.958Z

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Imbibition profile in polyethylene glycol 6000 osmotic solution and physiological potential of soybean seeds The objective of this work was to study the imbibition profile in water or in polyethylene glycol 6000 osmotic solution and the effect of the osmoconditioning on the germination and vigor of seeds of six soybean cultivars Confiança, UFV-16, Splendor, Garantia, UFVS 2005 and UFV-18. The cultivars were grown in the field at Viçosa, Minas Gerais, in a randomized complete block design, the seeds were harvested at the R8 stage and 15 and 30 days later. Seed samples of each cultivar per harvest time and replication were soaked in distilled water (control) or osmoconditioned in -0.8 MPa PEG 6000 solution at 20 °C, for 96 h, in the presence of 0.2% Captan fungicide. Vigor and viability of the seeds were evaluated by the first and final counting in the germination test on paper rolls and speed of seedling emergence on sand seedbed. The imbibition speed and the humidity of the osmoconditioned seeds of all six cultivars and three harvest times were lower than of those seeds soaked in water. The germination and vigor of osmoconditioned seeds were higher for all cultivars at all harvest times, mainly with 30 days harvest delay, indicating the conditioning efficacy to increase the germination of weathered seeds. Introduction Seed quality characteristics such as dry matter weight and physiological potential are maximal at the physiological maturity, decreasing from this point depending on the environmental condition prior to the harvest and the processes of seed production. Harvest must occur as soon as possible after the seed reaches its physiological maturity, since rainy periods may cause irrecoverable damage to the quality of the seeds (Sediyama et al., 1972). The tolerance level for seed deterioration in the field varies depending on cultivar and the environment, however high temperature and precipitation are more important than the period of time that the seed remains in the field after the physiological maturity. Unfavorable weather conditions have led to the production and rejection of soybean seed lots that do not meet the quality standards, among them the minimum of 80 percent germination rate (Peske & Meneghello, 2013). Imbibition is the absorption of a fluid by a solid or colloid that results in swelling and it is the first stage of a sequence of events for the retake of the embryo development and growth in the germination process. Seeds generally present a lower water potential than the substrate in which they germinate, which causes a fast entrance of water into the cotyledons, death of the superficial cells and, in certain situations, damage due to the leakage of solutes and resulting on decrease of the seedlings emergence (Matthews & Powell, 1986). The speed how the water is absorbed seems to be decisive for the success of germination. Furthermore, osmoconditioning with PEG or NaCl has also been identified to be an effective way to reduce physiological and biochemical damage induced by imbibitions at chilling stress (Posmyk et al., 2001). During osmoconditioning, many genes are induced, which are beneficial for plant to survive the subsequent chilling stress (Farooq et al., 2010). Soybean seeds harvested at three times from the R8 stage, when 95% of the pods have reached their mature colour, showed greater percentage of imbibition and lower vigor, germination and index of resistance to seed coat shrinking with harvest delay after the 21 st day after physiological maturity (R8) (Rocha et al., 1984). The rate of water absorption by the seeds increased with the harvest delay, indicating greater permeability of the membranes caused by the deterioration process. Reduction of problems caused by fast water uptake by osmoconditioning the seed, which consists in its controlled hydration, that activates the pre-germinative metabolic processes (Nascimento, 2005), but stops just before the root protrusion (Bradford, 1990), has been reported. This process improves germination and seed vigor, besides allowing a faster and more uniform seedling emergence (Del Giúdice et al., 1998;Nascimento, 2005;Khalil et al., 2010;Yadav et al., 2011). The objective in this work was to evaluate the imbibition in water or in osmotic solution of polyethylene glycol 6000 -PEG 6000, and the effect of the osmoconditioning on the germination and vigor of seeds of six soybean cultivars, harvested at three different times. Material and Methods Soybean seeds were multiplied at the Prof. Diogo Alves de Mello Experimental Field and analyzed at the Soybean Breeding and Seed Research laboratories of the Plant Sciences Department at the main campus of the Federal University of Viçosa, Minas Gerais. Seeds of six soybean cultivars of different maturity periods -Confiança (semi-early), UFV-16 (medium), Splendor (medium), Garantia (semi-late), UFVS 2005 (late) and UFV-18 (late) -were produced in the agricultural year of 2005/2006, in a randomized complete block experimental design with four replications. Each plot had 12 rows of plants with five meters length and a spacing of 50 cm between them. The soil was prepared for planting with one ploughing and two disking; the fertilization, the cultural techniques and the phytosanitary control were carried out according to recommendations for the crop (Embrapa Soja, 2011). The daily climatic data of rain precipitation and minimum, average and maximum temperatures were registered during the period of seed development and harvesting ( Figure 1). Four rows of plants of each cultivar were harvested at the R8 reproductive stage (when 95% of pods have the typical coloration of mature pods), and three rows each at 15 and 30 days after this maturation stage, to distinguish the seed vigor levels. The plants were threshed with a stationary machine and the seeds were submitted to sun drying, until they presented 11 to 12% of moisture content on wet basis. The seeds were packed in cotton cloth bags and kept in a chamber at 10 °C and 70% of air relative humidity until they were evaluated in a laboratory, when they were cleaned and size uniformized with sieve number 13 (6.5 mm of diameter). The PG 6000 concentration, for the achievement of the osmotic potential of the conditioning solution of -0.8MPa at the temperature of 20 °C, was 251.028 g L -1 of demineralised water, according to the equation of Michel & Kaufmann (1973): Ψos (atm) = (1.18 x 10 -2 )C -(1.18 x 10 -4 )C 2 + (2.67 x 10-4)CT + (8.39 x 107)C 2 T, in which: Ψos (atm) = osmotic potential; C = concentration (g/L); T = temperature (°C); and 0.1MPa = 1atm. After the osmoconditioning period, the seeds were superficially washed under tap water to remove PEG 6000, than were dried at room temperature for 48 hours to the initial seed moisture content (10-11%) and were stored until the beginning of the assays. The seed moisture content, measured on samples dried at 105 ± 1 °C for 24 h (Brasil, 2009), was determined after 0, 2, 4, 6,8,10,12,24,48,72,96 and 120 h of imbibition in distilled water or in PEG 6000 solution. The last humidity evaluation was carried out when about 50% of the seeds showing radical protrusion (about 1 mm), which corresponded to the period of 48 h for those imbibed in distilled water. A sample of 100 seeds per cultivar, period of harvest and replication in the field was put into a gerbox, transparent 11 x 11 x 3.5 cm acrylic boxes with lid, with four sheets of germitest paper towel soaked with 30 mL of distilled water or with the same amount of PEG 6000 solution, with the osmotic potential adjusted at -0.8 MPa, in the presence of 0.2% of Captan fungicide. The gerbox with the seeds were placed in a Biochemical Oxygen Demand (B.O.D.) chamber at 20 ± 1 °C (Del Giúdice et al., 1998), set at 12 h photofase. The germination, with the final counting on the eighth day of the test, according to the Rules for Seed Analysis (Brasil, 2009), was determined for seeds of each cultivar, harvest time and replication in the field, for those which were not conditioned and those which were conditioned in PEG 6000 solution during 96 h (Del Giúdice et al., 1998), the results were presented in percentage of normal seedlings. The seedling emergence test on sand seedbed were carried out in a greenhouse on samples of 50 seeds per cultivar, harvest time, conditioning and field replication sown on plastic trays with sand substrate, the air temperature in the greenhouse ranged from 14 to 34 °C. Daily countings of emerged seedlings were performed up to the 15 th day after sowing to allow the estimation of the seedling emergence speed index, according to Maguire (1962). The statistical model was the split plot design, with cultivars in the plots and harvest times in the subplots, in a randomized complete block experiment, with four replications. The data were submitted to the analyses of variance and regression, and the averages of the qualitative factor were compared with the Tukey´s test, at 5% of probability, when the F test was significant. The regression models were chosen according to the regression coefficient significance by the t test at 5% of probability and also selecting those more suited to the biological phenomenon to be described. The percentage data of the germination test were transformed to arcsine √x/100 for the statistical analysis and the means were de-transformed for table presentation. The data processing was carried out with the SAS software (Delwiche & Slaughter, 2013). Results and Discussion The imbibition profiles in water and in PEG 6000 solution of the seeds from the six soybean cultivars, Confiança (semiearly), UFV-16 (medium), Splendor (medium), Garantia (semi-late), UFVS 2005 (late) and UFV-18 (late), were similar in all three harvest times (Figures 2, 3 and 4). The lower imbibition speed and moisture content of the seeds in PEG 6000 solution demonstrates the effectiveness of this product in restraining water absorption, as reported for conditioned seeds of scarlet eggplant (Gomes et al., 2012); of wheat seeds in osmopriming solution (Jafar et al., 2012) and carrots in PEG 6000 solution at -1.2 MPa at 20 °C (Pereira et al., 2009). The initial moisture content of soybean seeds varied from 11 to 13% (Figures 2, 3 and 4), with faster water absorption in the first 12 h, but with lower imbibition intensity in the PEG 6000 solution. The seed moisture content on paper soaked with distilled water was 54% after 12 h (Figures 2, 3 and 4), higher than those in PEG 6000 solution, which, after achieving 37%, had an extension of the phase II of hydration. It occurs when the pre-germinative repair-mechanism of the macromolecules and other cellular structures takes place (Varier et al., 2010). The fast imbibition in the germination process is called phase I. It is a consequence of the reduced matrix potential of dry seeds and can reach up to -100MPa, which explains their fast hydration even in osmotic solutions. The phase II is characterized by a dramatic reduction in the hydration speed and the respiratory intensity, which depend on the water potential of the substrate and the seed species. The primary root protrusion characterizes the phase III of imbibition (Bewley, 1997). The duration of priming should be taken into account, since the effects of priming can be altered by the duration of treatment, as already observed for carrot (Lopes et al., 2011) and scarlet eggplant (Gomes et al., 2012) whose conditioning reduced germination and seed performance under the conditions evaluated. The moisture content of soybean seeds at the end of the osmotic treatment in PEG 6000 solution for 96 hours was, in average, 44% (Figures 2, 3 and 4). For osmotic conditioning, carrot seed imbibition is recommended in the PEG 6000 solutions at -1.0 and -1.2 PMa until moisture contents are obtained between 40% and 45% in moistened paper and 45 to 50% in aerated solution (Pereira et al., 2009). The final seed moisture content in distilled water was 60% (Figures 2, 3 and 4), corresponding to the beginning of phase III, when 50% of the seeds showed root with about 1 mm. Sweet pepper seeds conditioned in water also had a progressive increase in the degree of moisture in the first 12 h of imbibition, reaching 55% (Posse et al., 2001). The phase III of seed imbibition in distilled water or in PEG 6000 solution was achieved after 48 and 100 h, respectively, as reported for soybean seeds of the UFV-10 (Uberaba), IAC-8, Doko RC and Savana cultivars (Del Giúdice et al., 1998). The best adjustments of the regression curves, for the seeds imbibed in water and in the PEG 6000 solution, were achieved with quadratic and cubic root equations. The imbibition behaviour of soybean seeds is in accordance with the mentioned three phases pattern, characterized by a phase of fast water absorption (phase I), followed by a stationary phase (phase II), and finishing with the increase in the absorption rate, which coincides with the root protrusion and the seedling growth (phase III) (Bewley, 1997). The speed of water uptake by seed tissues is decisive for the germination success, but fast imbibition may damage them, when in contact with pure water (Matthews & Powell, 1986). The damage caused by fast seed imbibition may occur due to the reduction in the integrity of the cell membranes, with loss of essential nutrients, increase of microorganism activity, leaking of solutes or low oxygen availability, leading to the anaerobic respiratory process (Armstrong & McDonald, 1992). The cultivar, the harvest time and the osmotic conditioning affected the percentage of normal seedlings in the first and final countings of the germination test, and the speed of seedling emergence on sandbed (Table 1). No significant interactions were observed, therefore, the harvest time and the osmotic 1/ Percentage values of germination were transformed to arcsine √x/100 for statistical analysis. **,* F significant at 1 and 5% probability, respectively. Table 1. Analysis of variance of the first (FC) and final counting in the germination test (GT) and seedling emergence speed index on sand seedbed (ESI) of the seeds of six soybean cultivars harvested at three different times, osmoconditioned or not with PEG 6000. Viçosa, Minas Gerais, 2007 1/ conditioning additively affected the germination and vigor of the seeds of all soybean cultivars. Higher germination averages (Table 2) were observed for the seeds of Confiança (97%), Splendor (96%), UFVS 2005 (95%) and UFV-16 (93%) cultivars; and lower for the seeds of Garantia (85%) and UFV-18 (90%) cultivars. Correspondingly, the same rank was observed in the first counting results, seedling emergence speed index, it was observed higher speed for Confiança (7.450) and lower for Garantia (6.534) seeds. The germination was higher at the R8 and R8+15 harvest times than at the R8+30, which is in accordance with the decrease of seed germination with the harvest delay (Sediyama et al., 1972). The maximal longevity potential of the soybean seeds is attained close to the full maturity stage after which the seed humidity content naturally declines to 14-15%. For best quality of soybean seeds, it is recommended to harvest between 12 and 15% water (Embrapa Soja, 2011). Conditioning the seeds with PEG 6000 improved the germination (96%) when compared to nonconditioned seeds (89%) ( Table 2). The same results were obtained for seed vigor, evaluated by the first count in the germination test (96 for conditioned seeds against 86% for nonconditioned) and the seedling emergence speed index on sand seedbed (7.506 against 6.638). Priming in PEG 6000 for four days increased the percentage and speed of germination and seedling emergence of carrot (Pereira et al., 2009). Aymen et al. (2012 also found beneficial effects of priming when evaluating the growth and yield of safflower under saline condition. However, the greatest positive effect of the conditioning on the germination was observed at the R8+30 harvest, which demonstrates the efficacy of the treatment with PEG 6000, as reported for soybean seeds of the cultivars UFV-10 (Uberaba), IAC-8, Doko RC and Savana (Del Giúdice et al., 1998). Comparing the embibition profiles of the cultivars ( Figures 2, 3 and 4) and the physiological quality of their seeds (Table 2), there is no evidence of relationship between imbibition profile in water or in PEG 6000 and the different seed quality of the six cultivars. The imbibition speed and the humidity of the osmoconditioned seeds of all six cultivars and three harvest times were lower than of those seeds soaked in water. The germination and vigor of osmoconditioned seeds were higher for all cultivars at all harvest times, mainly with 30 days harvest delay, indicating the conditioning efficacy to increase the germination of weathered seeds. 1/ Means of cultivars followed by the same lower case letter in the column or by the same capital letter in the horizontal line, do not differ by the Tukey´s test at 5% probability. Percentage data were transformed to arcsine √x/100 for analysis and, later, the averages were de-transformed for presentation. Coefficients of variation: Error a (FC, GT and ESI) = 24.33, 20.24 and 19.97%; Error b (FC, GT and ESI) = 14.02, 11.99 and 11.58%; Error c (FC, GT and ESI) = 13.79, 11.15 and 8.81%. Table 2. Estimated means of the first (FC) and final counting (GT) in the germination test and seedling emergence speed index on sand seedbed (ESI) of the seeds of six soybean cultivars harvested at three different times, osmoconditioned or not with PEG 6000. Viçosa, Minas Gerais, 2007 1/

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2016-03-14T22:51:50.573Z

2015-11-01T00:00:00.000Z

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Global Profiling of Various Metabolites in Platycodon grandiflorum by UPLC-QTOF/MS In this study, a method of metabolite profiling based on UPLC-QTOF/MS was developed to analyze Platycodon grandiflorum. In the optimal UPLC, various metabolites, including major platycosides, were separated well in 15 min. The metabolite extraction protocols were also optimized by selecting a solvent for use in the study, the ratio of solvent to sample and sonication time. This method was used to profile two different parts of P. grandiflorum, i.e., the roots of P. grandiflorum (PR) and the stems and leaves of P. grandiflorum (PS), in the positive and negative ion modes. As a result, PR and PS showed qualitatively and quantitatively different metabolite profiles. Furthermore, their metabolite compositions differed according to individual plant samples. These results indicate that the UPLC-QTOF/MS-based profiling method is a good tool to analyze various metabolites in P. grandiflorum. This metabolomics approach can also be applied to evaluate the overall quality of P. grandiflorum, as well as to discriminate the cultivars for the medicinal plant industry. Introduction Platycodon grandiflorum, a perennial herb grown widely in Northeast Asia, contains triterpenoid saponins, carbohydrates, fibers, etc. [1,2]. P. grandiflorum has been used as a food material and a traditional medicine. Platycodin radix, the root of P. grandiflorum, has various benefits for health, and its biological usefulness has been reviewed [3]. Interest in platycodin saponins, the main components of P. grandiflorum, has increased recently due to their novel pharmacological activities, including anti-inflammatory, anti-oxidant, anti-lipidemic, anti-obesity, anti-cancer activities and their ability to improve insulin resistance [4][5][6][7][8][9][10][11]. Furthermore, in a previous study, various components were isolated from the flower of P. grandiflorum, and their biological activities were monitored [12]. Thus, it is critical to study not only Platycodin radix, but also different parts of P. grandiflorum in order to screen it for useful components. Recently, metabolomics approaches have been used to assess the metabolite contents of individual plant species [13]. For plant metabolomics, well-constructed analytical platforms are necessary [14,15]. Nuclear magnetic resonance spectroscopy and gas chromatography or liquid chromatography (LC) coupled with mass spectrometry (MS) have been widely used to analyze plant metabolites [16][17][18][19][20]. In particular, metabolite profiling by LC/MS is applicable to phenotype and can discriminate individual plant species [21][22][23]. In this study, we constructed a profiling method based on LC/MS to analyze the metabolites of P. grandiflorum. Metabolite extraction protocols and LC/MS conditions were optimized to profile two different parts ((1) P. grandiflorum roots (PR) and (2) P. grandiflorum stems and leaves (PS)). Construction of LC-MS Conditions to Profile Platycodon grandiflorum Metabolites For the high-throughput and sensitive analysis of various metabolites in P. grandiflorum, it is necessary to construct a robust method of profiling. The UPLC system with its small particle size column enables the fast and effective separation of various molecules. Furthermore, QTOF/MS is a good tool for a full mass scan with high resolution. Thus, in this study, UPLC-QTOF/MS was applied to profile P. grandiflorum metabolites. First, we used seven standards (i.e., platycoside E, platycodin D3, platycodin D2, platycodin D, polygalacin D, platycogenic acid A and platycodigenin) to optimize the LC-MS conditions. In the negative mode of electrospray ionization (ESI), seven compounds were mainly detected as [M´H]´and [M + COOH]´ions (Table 1). These standards were separated well and eluted for 15 min ( Figure 1) at a flow rate of 450 µL/min by using an ACQUITY BEH C18 column (2.1 mmˆ100 mm, 1.7 µm particle size). Second, two extracts of PR and PS were analyzed in both positive and negative ion modes. As a result, chromatographic data from the positive mode showed poor efficiency of ionization (data not shown). Thus, we carried out the profiling of PR and PS in the negative mode only. Various metabolites of PR and PS were also separated well in 15 min ( Figure 1). The gradient elution program consisted of a first linear gradient from steady (A/B: 90/10) to solvent (A/B: 80/20) for 3 min; followed by a second linear gradient to solvent (A/B: 77/23) for 8 min; a third linear gradient to solvent (A/B: 5/95) for 9 min; and a forth linear gradient to solvent (A/B: 90/10) for 1 min. The column was equilibrated at 10% Solvent B for 4 min before reuse. The total run time was 25 min for each analysis. Third, we validated the performance of the P. grandiflorum metabolites' profiling method. For this, we drew the standard curves of seven standards and calculated their linearity range and correlation. The limits of detection (LODs) of each isolated compound are also listed ( Table 2). Optimization of Extraction Protocols for Platycodon grandiflorum Metabolites Next, we optimized the protocols to extract metabolites, including various isolated compounds from two different parts (PR and PS). There are several factors involved in the optimization of extraction protocols. First, it is critical to select suitable solvents for the extraction of various metabolites with different polarities. A single solvent is insufficient to dissolve the wide range of compounds, and several solvents, such as water, ethanol (EtOH) and methanol (MeOH), have been widely used to extract plant metabolites. To find the most effective solvent for extraction, we tried to compare six different solvents (50% EtOH, 70% EtOH, 80% EtOH, 50% MeOH, 70% MeOH and 80% MeOH) while other factors, such as solvent amount (20 mL) and sonication time (30 min), were kept constant. As a result, 70% EtOH exhibited the largest number of peaks and the highest intensity of several compounds. Second, the ratio of solvent to sample is also a critical factor in the metabolites' extraction due to the limited solubility of samples. Thus, we compared four different amounts (10, 20, 30 and 40 mL) of solvent, 70% EtOH, to extract metabolites from the 50 mg of samples while the sonication time (30 min) was kept constant. Among them, 40 mL provided the largest amount of extracts. Third, different sonication times (15,30,45,60 and 75 min) were tested in the use of 40 mL of 70% EtOH, and as a result, it was found that at least 60 min of sonication were required to extract many metabolites. Finally, we optimized the solvent used (70% EtOH), the ratio of solvent to sample (40 mL:50 mg) and the sonication time (60 min) for the effective extraction of metabolites from P. grandiflorum. Analysis of Various Metabolites in the Stem, Leaf and Roots of Platycodon grandiflorum We applied the proposed method based on UPLC-QTOF/MS to profile various metabolites in PR and PS. Three species of P. grandiflorum were used to obtain three PRs and three PSs. We extracted the metabolites from the three PRs and three PSs, respectively, analyzed each extract three times (n = 3) and then processed each set of data using the UNIFI TM software (Version 1.7.1; Waters Corp.). As a result, various metabolites, including several platycosides, were identified based on the library of UNIFI software containing information for the molecule's name and formula [24]. From the processed data, compounds identified repeatedly (n = 3) having high mass accuracy (ppm <˘5) are listed in Tables 3 and 4 respectively. In the peak assignment, it is critical to estimate retention time (RT) (RT tolerance: 0.2 min) and mass accuracy. In the samples of PR and PS, four compounds, such as platycoside E, platycodin D, platycodin D2 and platycodin D3, were analyzed with small RT shifts compared to the standard analysis. We also describe the Venn diagram of metabolites analyzed in three PRs and three PSs to show how the metabolic compositions of each plant species (PR-1, -2 and -3 or PS-1, -2 and -3) were different ( Figure 2). Two parts of P. grandiflorum (PR and PS) showed qualitatively and quantitatively different metabolite profiles. Furthermore, the metabolite profiles differed according to individual samples. In particular, several platycosides were abundantly different in the three PRs and three PSs, respectively ( Figure 3). For example, comparing to other PR species, PR-1 has 1.5-times more abundant platycodin D3, and PR-2 has two-times less abundant platycodin A. Compared to other PS species, PS-3 also has 2.5-times more abundant platycodin A. Optimization of Extraction Protocols for Platycodon grandiflorum Metabolites Next, we optimized the protocols to extract metabolites, including various isolated compounds from two different parts (PR and PS). There are several factors involved in the optimization of extraction protocols. First, it is critical to select suitable solvents for the extraction of various metabolites with different polarities. A single solvent is insufficient to dissolve the wide range of compounds, and several solvents, such as water, ethanol (EtOH) and methanol (MeOH), have been widely used to extract plant metabolites. To find the most effective solvent for extraction, we tried to compare six different solvents (50% EtOH, 70% EtOH, 80% EtOH, 50% MeOH, 70% MeOH and 80% MeOH) while other factors, such as solvent amount (20 mL) and sonication time (30 min), were kept constant. As a result, 70% EtOH exhibited the largest number of peaks and the highest intensity of several compounds. Second, the ratio of solvent to sample is also a critical factor in the metabolites' extraction due to the limited solubility of samples. Thus, we compared four different amounts (10, 20, 30 and 40 mL) of solvent, 70% EtOH, to extract metabolites from the 50 mg of samples while the sonication time (30 min) was kept constant. Among them, 40 mL provided the largest amount of extracts. Third, different sonication times (15,30,45,60 and 75 min) were tested in the use of 40 mL of 70% EtOH, and as a result, it was found that at least 60 min of sonication were required to extract many metabolites. Finally, we optimized the solvent used (70% EtOH), the ratio of solvent to sample (40 mL:50 mg) and the sonication time (60 min) for the effective extraction of metabolites from P. grandiflorum. Analysis of Various Metabolites in the Stem, Leaf and Roots of Platycodon grandiflorum We applied the proposed method based on UPLC-QTOF/MS to profile various metabolites in PR and PS. Three species of P. grandiflorum were used to obtain three PRs and three PSs. We extracted the metabolites from the three PRs and three PSs, respectively, analyzed each extract three times (n = 3) and then processed each set of data using the UNIFI TM software (Version 1.7.1; Waters Corp.). As a result, various metabolites, including several platycosides, were identified based on the library of UNIFI software containing information for the molecule's name and formula [24]. From the processed data, compounds identified repeatedly (n = 3) having high mass accuracy (ppm < ±5) are listed in Tables 3 and 4, respectively. In the peak assignment, it is critical to estimate retention time (RT) (RT tolerance: 0.2 min) and mass accuracy. In the samples of PR and PS, four compounds, such as platycoside E, platycodin D, platycodin D2 and platycodin D3, were analyzed with small RT shifts compared to the standard analysis. We also describe the Venn diagram of metabolites analyzed in three PRs and three PSs to show how the metabolic compositions of each plant species (PR-1, -2 and -3 or PS-1, -2 and -3) were different ( Figure 2). Two parts of P. grandiflorum (PR and PS) showed qualitatively and quantitatively different metabolite profiles. Furthermore, the metabolite profiles differed according to individual samples. In particular, several platycosides were abundantly different in the three PRs and three PSs, respectively (Figure 3). For example, comparing to other PR species, PR-1 has 1.5-times more abundant platycodin D3, and PR-2 has two-times less abundant platycodin A. Compared to other PS species, PS-3 also has 2.5-times more abundant platycodin A. Platycodon grandiflorum Samples The samples of Platycodon grandiflorum (roots, stems and leaves) were purchased from a Daegu Herbal Market in Daegu Gyeongbuk Province, Korea, in 2014. Voucher specimen (NIHHS150128) was deposited at the herbarium of the Department of Herbal Crop Research, National Institute of Horticultural and Herbal Science, Rural Development Administration, Eumseong, Korea. Standard Constituents and Reagents HPLC-grade acetonitrile, methanol and water were obtained from Merck (Darmstadt, Germany). Formic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA). Standard compounds were isolated and purified from Platycodon grandiflorum roots by a series of chromatography procedures in our laboratory, and their structures were elucidated by a comparison of the spectroscopic data (MS, 1 H-NMR and 13 C-NMR) with the literature data: platycoside E [25], platycodin D3 [26], platycodin D [27], platycodin D2 [26], polygalacin D [26], platycogenic acid A [2] and platycodigenin [28]. The purity of the isolated compounds was determined to be more than 98% by normalization of the peak areas detected by HPLC analysis. Sample Preparation Each sample was dried at 40 °C in a forced-air convection-drying oven for 48 h after washing, and then weighed. The main and lateral roots were used in experiments after removing the rhizomes and fine roots. The roots were ground (<0.5 mm) using a mixer (Hanil, Seoul, Korea) and thoroughly mixed, after which the subsamples were homogenized further using a Retsch MM400 mixer mill (Retsch GmbH, Haan, Germany) for the analyses. Fine powder was weighed (50 mg), suspended in 40 mL of 70% (v/v) ethanol and ultrasonically extracted for 1 h at 50 °C. The extract was filtered and evaporated by using a solvent evaporator, Genevac Ez-2 (Genevac Ltd, Suffolk, UK), and the residue (5 mg) was dissolved in the 1 mL of 70% methanol. The solution was filtered through a syringe filter (0.22 µm) and injected directly into the UPLC system. UPLC-QTOF-MS Analysis UPLC was performed using a Waters ACQUITY H-Class UPLC (Waters Corp, Milford, MA, USA). Chromatographic separations were performed on an ACQUITY BEH C18 column (2.1 mm × 100 mm, 1.7 µm). The column oven was maintained at 40 °C, and the mobile phases consisted of Solvent A (5% acetonitrile + 0.1% formic acid (v/v)) and Solvent B (95% acetonitrile + 0.1% formic acid (v/v)). The flow rate was 450 µL/min, and the injection volume was 2 µL for each run. Next, MS analysis was performed using a Waters Xevo G2-S QTOF MS (Waters Corp.) operating in positive and negative ion mode. The mass spectrometers performed alternative high-and low-energy scans, known as the MS E acquisition mode. The operating parameters were set as follows: cone voltage, Platycodon grandiflorum Samples The samples of Platycodon grandiflorum (roots, stems and leaves) were purchased from a Daegu Herbal Market in Daegu Gyeongbuk Province, Korea, in 2014. Voucher specimen (NIHHS150128) was deposited at the herbarium of the Department of Herbal Crop Research, National Institute of Horticultural and Herbal Science, Rural Development Administration, Eumseong, Korea. Standard Constituents and Reagents HPLC-grade acetonitrile, methanol and water were obtained from Merck (Darmstadt, Germany). Formic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA). Standard compounds were isolated and purified from Platycodon grandiflorum roots by a series of chromatography procedures in our laboratory, and their structures were elucidated by a comparison of the spectroscopic data (MS, 1 H-NMR and 13 C-NMR) with the literature data: platycoside E [25], platycodin D3 [26], platycodin D [27], platycodin D2 [26], polygalacin D [26], platycogenic acid A [2] and platycodigenin [28]. The purity of the isolated compounds was determined to be more than 98% by normalization of the peak areas detected by HPLC analysis. Sample Preparation Each sample was dried at 40˝C in a forced-air convection-drying oven for 48 h after washing, and then weighed. The main and lateral roots were used in experiments after removing the rhizomes and fine roots. The roots were ground (<0.5 mm) using a mixer (Hanil, Seoul, Korea) and thoroughly mixed, after which the subsamples were homogenized further using a Retsch MM400 mixer mill (Retsch GmbH, Haan, Germany) for the analyses. Fine powder was weighed (50 mg), suspended in 40 mL of 70% (v/v) ethanol and ultrasonically extracted for 1 h at 50˝C. The extract was filtered and evaporated by using a solvent evaporator, Genevac Ez-2 (Genevac Ltd, Suffolk, UK), and the residue (5 mg) was dissolved in the 1 mL of 70% methanol. The solution was filtered through a syringe filter (0.22 µm) and injected directly into the UPLC system. UPLC-QTOF-MS Analysis UPLC was performed using a Waters ACQUITY H-Class UPLC (Waters Corp, Milford, MA, USA). Chromatographic separations were performed on an ACQUITY BEH C18 column (2.1 mm1 00 mm, 1.7 µm). The column oven was maintained at 40˝C, and the mobile phases consisted of Solvent A (5% acetonitrile + 0.1% formic acid (v/v)) and Solvent B (95% acetonitrile + 0.1% formic acid (v/v)). The flow rate was 450 µL/min, and the injection volume was 2 µL for each run. Next, MS analysis was performed using a Waters Xevo G2-S QTOF MS (Waters Corp.) operating in positive and negative ion mode. The mass spectrometers performed alternative high-and low-energy scans, known as the MS E acquisition mode. The operating parameters were set as follows: cone voltage, 40 V; capillary, 3.0 kV; source temperature, 120˝C; desolvation temperature, 300˝C; cone gas flow, 30 L/h; and desolvation gas flow, 600 L/h. Accurate mass measurements were obtained by means of an automated calibration delivery system, which contains the internal reference (Leucine, m/z 556.276 (ESI+), m/z 554.262(ESI-)). Data were collected between 100 and 2000 m/z. In the quantitative analysis of each metabolite, the height of peaks was used to measure the intensity. Conclusions A profiling method based on UPLC-QTOF/MS was developed to analyze various metabolites contained in P. grandiflorum. UPLC separation conditions were optimized by using seven isolated compounds. The protocols for extracting P. grandiflorum metabolites were optimized as follows: solvent used, 70% EtOH; the ratio of solvent to sample, 40 mL:50 mg; and sonication time, 60 min. We applied this method to profile two different parts of P. grandiflorum (PR and PS), in what was a first attempt to characterize various metabolites in PR and PS, respectively. In the negative ion modes, PR and PS showed qualitatively and quantitatively different metabolite profiles. The metabolite compositions also differed according to the each species. These results indicated that the UPLC-QTOF/MS-based profiling method has potential as a tool to analyze various metabolites in P. grandiflorum. Hence, this study is of great significance regarding evaluations of the overall quality of P. grandiflorum in pharmacological and clinical investigations of drug products. Furthermore, this metabolomics approach could be applied to discriminate cultivars for use in the agricultural and pharmacological industries.

v3-fos

2018-04-03T05:41:56.669Z

2015-03-14T00:00:00.000Z

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Selective pruning in pineapple plants as means to reduce heterogeneity in fruit quality Heterogeneity in fruit quality (size and taste) is a major problem in pineapple production chains. The possibilities were investigated of reducing the heterogeneity in pineapple in the field by pruning slips on selected plants, in order to promote the fruit growth on these plants. Slips are side shoots that develop just below the pineapple fruit during fruit development. Two on-farm experiments were carried out in commercial fields in Benin with a cultivar locally known as Sugarloaf, to determine (a) the effect of slip pruning on fruit quality; (b) whether the effect of slip pruning depends on the pruning time; and (c) whether slip pruning from the plants with the smallest infructescences results in more uniformity in fruit quality. A split-plot design was used with pruning time (2 or 3 months after inflorescence emergence) as main factor and fraction of pruned plants (no plants pruned (control); pruning on the one-third plants with the smallest infructescences; pruning on the two-thirds plants with the smallest infructescences; pruning on all plants) as sub-factor. Fruit quality characteristics measured at harvest were the fruit (infructescence + crown) weight and length, the infructescence weight and length, the crown weight and length, the ratio crown length: infructescence length, the total soluble solids, the juice pH and the flesh translucency. Results indicated that pruning of slips of any fraction of the plants at 2 or 3 months after inflorescence emergence did not lead to a consistent improvement in quality or uniformity. Consequently it is not recommended to farmers in Benin to prune the slips. Introduction In developing countries, many producers -especially the smallholder producers-face difficulties in entering the international market because of the high quality standards and the need to supply high and regular quantities of product (Murphy 2012). Nowadays, the uniformity in product quality also has become an important criterion. As a proof of that, the Codex Alimentarius, an organization focusing on the establishment of food quality and safety rules for export products to which most developing countries belong, elaborated a set of export criteria for individual food quality attributes as well as for acceptable product heterogeneity (Codex Alimentarius 2005). A recent study on pineapple [Ananas comosus (L.) Merrill] supply chains in Benin revealed that heterogeneity in quality attributes such as fruit weight, taste, firmness and flesh translucency was a constraint to the success of the chain (Fassinou Hotegni et al. 2014a). Heterogeneity in quality is caused by many factors including the environmental conditions and cultural practices underlying its production (Luning and Marcelis 2006). It then becomes important to find ways to reduce heterogeneity in fruit quality by designing crop management strategies yielding a more uniform product quality at harvest. The most important pineapple cultivar in Benin is the sweet cultivar locally known as Sugarloaf -but possibly equal to cv. Pérola-grown by 97% of the pineapple growers (Fassinou Hotegni et al. 2014a). In this cultivar type and several other types like cv. Singapore Spanish grown in, e.g., South Asia, three development phases exist: the vegetative phase (from planting to flowering induction); the generative phase (from flower initiation to fruit maturity), and the propagative phase in which new shoots are produced (begins at the generative phase and continues after the fruit has been harvested) and which partly overlaps with the generative phase. Cultural practices to control heterogeneity are carried out mainly before flowering induction. Heterogeneity in pineapple fruit quality (fruit weight and length attributes) at harvest is clearly associated with heterogeneity in plant vigor at flowering induction time (Fassinou Hotegni et al. 2014b), and consequently cultural practices achieving uniform plant development before flower induction (like planting material grading by size at planting time) have received considerable attention , Py et al. 1987). Yet smallholder systems still show a high heterogeneity in quality . The present research focused on the potential of practices applied after flowering induction to reduce the heterogeneity in pineapple fruit quality, i.e., in total fruit (infructescence + crown) weight and length, infructescence weight and length, crown weight and length, the ratio crown length: infructescence length, the total soluble solids (TSS) in the pineapple juice, the juice pH, and the flesh translucency. Due to the overlap between the generative phase and the propagative phase the generative phase is not only characterized by development and growth of the fruit; also new shoots develop during that phase, such as slips (produced on the peduncle at the base of the fruit), hapas (produced above ground from the stem at the junction of the stem and the peduncle), suckers (side shoots originating on the stem) (Hepton 2003) and the crown. These vegetative organs can be used as propagules for planting a next crop. The most common shoots produced in the Pérola pineapple type and cv. Singapore Spanish are the slips and the crown. The slips are initiated just after the end of the initiation of the florets (Kerns et al. 1936). Studies on the effect of removing slips -called pruning or thinning-on the fruit size gave contradictory results. Wee and Ng (1970) removed all slips in cv. Singapore Spanish in excess to two slips that were kept on the plants and found no significant effect of slip pruning on fruit weight and fruit length. Similar results were also found by De Lima et al. (2001) on cv. Pérola. Norman (1976 removed the slips in Sugarloaf pineapple when the fruits started to develop and found that slip pruning increased fruit weight but had no effect on TSS concentration in the fruit juice. Recent glasshouse studies on cv. Smooth Cayenne suggested that slips could be an important source of assimilates for fruit growth and maintenance (Marler 2011) which again suggests that slip removal may affect fruit quality. Such inconsistent results emphasize the need to improve the understanding of the effect of slip pruning on fruit quality. Since the production of the slips overlaps with fruit development and growth, slips may compete with the fruit for assimilates available in the plant especially at an earlier stage of their development when they are not yet capable of producing their own assimilates. Thus, earlier slip pruning may have more positive effects on average fruit quality than later pruning. It was shown in pineapple that the least developed plants at flower induction produce lighter fruit than well-developed plants (Fassinou Hotegni et al. 2014b). We therefore assume that a higher uniformity in fruit weight and length might be achieved by early pruning of the slips of the least developed plants. A practical criterion for farmers to identify the least developed plants after flower induction would be the length of the developing infructescence. The objectives of this paper are to determine (1) the effect of slip pruning on the fruit quality, namely fruit (infructescence + crown) weight and length, infructescence weight and length, crown weight and length, the ratio crown length: infructescence length, the TSS in the pineapple juice, the juice pH and the flesh translucency; (2) whether the effect of slip pruning depends on the pruning time; and (3) if slip pruning from the plants with the smallest infructescences results in more uniformity in fruit quality. Experimental sites and set up Two on-farm experiments (Expt 1 and Expt 2) were conducted in two commercial pineapple farms (Farm A and Farm B) in the Atlantic department in the south of Benin between October 2010 and August 2012. Different producers of a cultivar locally known as Sugarloaf, but possibly equal to cv. Pérola, were selected per experiment based on (a) the age of their pineapple crop being close to the common artificial flowering induction time and (b) whether they applied the common practices described by Fassinou Hotegni et al. (2012) for this cultivar, as suggested by Mutsaers et al. (1997) for on-farm studies. Cultivar Sugarloaf was selected because (1) it is grown by 97% of the pineapple producers in the department (Fassinou Hotegni et al. 2014a) and (2) it produces numerous slips during the generative phase (Fassinou Hotegni et al. 2014b, Norman 1976. Information on the farms and cultural practices from planting until harvest time is presented in Table 1; mean monthly temperature and total monthly rainfall amount during the experimentation period are depicted in Figure 1. In each experiment, a split-plot design was used with slip pruning time [2 and 3 months after inflorescence emergence (MIE); Figure 2] as the main factor and fraction of plants per plot selected for pruning of slips (none, one-third, two-thirds, all) as the split factor. The pruning time 2 MIE was selected because at that time the slips and fruit were developed well enough to allow pruning without damaging the growing fruit ( Figure 2B). The pruning time 3 MIE was selected because at that time the slips were well-developed when compared with their size at 2 MIE ( Figure 2C). Plants for slip pruning (i.e., fraction of plants pruned) were the oneor two-third(s) plants (per plot) with the smallest infructescences (in length) at the time of pruning. The infructescence length was selected because it is a practical and easy criterion for farmers to identify the least developed plants and because a pre-experiment (not reported here) had shown that the infructescence length at 2 MIE was correlated with fruit weight and infructescence length at harvest (r = 0.63 and 0.64 respectively). Each experiment had four replicated blocks. Each net plot consisted of 60 plants arranged in 6 lines of 10 plants each; the number of plants selected for pruning was fixed at 0, 20, 40 or all 60 per net plot. Each net plot was surrounded by at least 2 guard rows and 2 guard plants in a row. The pineapple fruits were harvested following farmers' practice which was at the moment when the skin color had started to change from green to yellow in at least 25% of the plants in a net plot (i.e., 15 out of 60 plants). All fruits per plot were harvested on that day and were individually processed. Collected data Data were collected on all individual plants per net plot before pruning and at harvest. Before pruning, the infructescence length and the number of slips per plant were recorded. At harvest time, only data on fruit quality attributes were collected. Fruit quality attributes included some listed in the Codex Alimentarius such as the ratio crown length: infructescence length, the TSS in the pineapple juice and the flesh translucency, and some such as fruit and infructescence weight (mentioned in the Codex as size attributes) and juice pH (affecting the taste of the fruit) of high importance for pineapple consumers in some countries such as Benin, Nigeria, Burkina Faso and Niger (Fassinou Hotegni et al. 2014a), and some such as fruit, crown and/or infructescence lengths and weights underlying the fruit weight, the ratio crown length: infructescence length, or needed for their assessment. Data on fruit quality collection followed the procedures described by Fassinou Hotegni et al. (2014b), with TSS being measured in the pineapple juice in°Brix using a hand refractometer and the juice pH using a hand-held pH meter. Flesh translucency was based on the percentage of fruit flesh that was translucent; it was visually estimated on a cut half pineapple following the method of Paull and Reyes (1996). Data analysis Data were analyzed using GenStat for Windows 15th Edition (VSN International 2012). The initial status of the plants at pruning time was described in two ways. First, the proportion of plants with slips and the total number of slips produced per plant were calculated per plot and checked for being similar across treatments. A two-way ANOVA for a split-plot design was used; the proportion of plants with slips was transformed using arcsine transformation on the square root of the proportions before the analysis. Second, sextiles were calculated per plot. Plants were ranked according to infructescence length from the smallest to the highest values per plot and then allocated to six classes. The number of plants with slips was counted per class. Plants (n = 960) from all treatments at one pruning time were combined per sextile and graphs were plotted to evaluate how the proportion of plants without slips and the number of slips per plant in plants with slips varied in the sextiles at each pruning time. Because not all plants had produced slips, two data sets were created for evaluating fruit quality attributes: (1) a data set based on all plants per plot (with or without slips at pruning time) and (2) a data set based on plants with slips at pruning time. A two-way ANOVA for a split plot design was performed on each data set to test the effect of pruning time and fraction of plants pruned on the average quality of the fruit quality attributes and on fruit quality heterogeneity. Flesh translucency data were transformed using square root transformation ffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi x þ 0:5 p À Á before analysis (Bartlett 1936, Gonzalez 2009). Fruit quality heterogeneity was calculated per plot using the coefficient of variation (CV), i.e., the measure of the variability in the value in a population relative to the mean, for the two data sets: all plants and plants with slips at pruning time. When the F value was significant, LSD was used to separate means of average quality or CV in quality. Initial status of the plants at pruning time The pruning time, the fraction of plants pruned and their interaction were confirmed to have no effect on the proportion of plants with slips and the number of slips at pruning (Table 2). This shows that plants with and without slips were evenly distributed across the plots at the moment the treatments started. In Expt 1, there were fewer plants without slips than in Expt 2 ( Figure 3); in addition, the total number of slips produced in Expt 1 was higher than that produced in Expt 2. Infructescence length at pruning ranged from 5.5-20 cm in Expt 1 and from 6.6-19.0 cm in Expt. 2 when pruning was carried out at 2 MIE; at 3 MIE, infructescence length ranged from 8.5-24.5 cm in Expt 1 and 8.5-21.5 cm in Expt 2 (data not shown). When plants per plot were arranged into sextiles based on their infructescence length at pruning time, plants in lower sextiles -in which fractions most of the plants that had to be pruned fellless likely had produced slips (Figure 3)and if so, the number of slips was lower (Figure 4). This meant that a possible effect of pruning on fruit quality was diluted by the plants that could not be pruned because they did not have slips. Therefore, data were split into two sets: (1) a data set based on all plants per plot (with or without slips at pruning time) and (2) a data set based on the plants with slips at pruning time. Infructescence length at pruning in the latter data set ranged from 6.0-20 cm in Expt 1 and 8.0-19.0 cm in Expt 2 when pruned at 2 MIE, and from 8.5-24.5 cm in Expt 1 and from 8.5-21.5 cm in Expt 2 when plants were pruned at 3 MIE. The data set based on all plants per plot is useful for showing the relevance of pruning for commercial practice and the data set based on the plants with slips for understanding the effect of slip pruning per se. Effects of fraction of plants pruned and pruning time on fruit quality In both data sets -data on all plants per plot and data based on the plants with slips at pruning time-the interaction between pruning time and fraction of plants pruned was not significant for any of the quality attributes and main effects were only incidentally significant (Table 3). In both data sets, the fraction of plants pruned had no significant effect on average quality, except on juice pH in Expt 1 (Table 3), where pruning of the twothirds plants with the smallest infructescences led to higher juice pH than no pruning or pruning all plants (Table 4). This trend in juice pH was not found in Expt 2. In both data sets, pruning time had no significant effect on the average fruit quality attributes, except on crown weight in Expt 1 (Table 3) where pruning at 2 MIE resulted in heavier crowns than pruning at 3 MIE (Table 4). In Expt 2, effects on crown weight were not significant. Results also indicated that there were only small differences across experiments as shown by the grand means for the two data sets in Table 4, with slightly higher fruit and infructescence weights and lower crown weights in Expt 1 than in Expt 2, and lower fruit, infructescence and crown lengths, a lower ratio crown length: infructescence length and lower TSS in Expt 1 than in Expt 2. The juice pH was slightly higher in Expt 1 than in Expt 2 ( Table 4). Effects of fraction of plants pruned and pruning time on the heterogeneity in fruit quality In the data set on all plants, interaction between the pruning time and fraction of plants pruned was not significant for variation -measured as CV-in any of the quality attributes whereas main effects were only incidentally significant (Table 3). The fraction of plants pruned had only a significant effect on variation in crown length in Expt 1; fruits from plots where no slips were pruned, showed the lowest CV in crown length, although not significantly different from fruits from plots in which slips were pruned from all plants (Table 4). In Expt 2 this was not found. An effect of pruning time was only significant for variation in fruit weight in Expt 1 (Table 3) where plants pruned at 2 MIE had significantly higher CVs in fruit weight than plants pruned at 3 MIE (Table 4). This effect was not significant in Expt 2. For the plants that had slips at pruning time, interaction between pruning time and fraction of plants pruned was significant for variation in fruit and infructescence weight in Expt 2 ( Table 3); pruning of the twothirds plants with the smallest infructescences at 3 MIE reduced significantly the CV in fruit and infructescence weight when compared with no pruning, but this was not found when pruning at 2 MIE. For variation in the other quality attributes, no main effects of the fraction of plants pruned were significant (Table 3). Comparing the two pruning times, interaction in Expt 2 indicated a significantly lower CV in fruit and infructescence weight when pruning 3 MIE compared with pruning at 2 MIE only when two-thirds of the plants were pruned. A main effect of the pruning time on the variation in other quality attributes was significant for fruit length in Expt 1 (Table 3) where pruning at 3 MIE gave lower variation in fruit length compared with pruning at 2 MIE. This was not found in Expt 2. Results also indicated that differences in CV across experiments were in general very small, as revealed by the grand means of the CVs in Table 4. The CVs in the two experiments for the two data sets were the same for the crown weight and fruit length. For other quality attributes except the flesh translucency, differences in CVs were very small ranging from 1-3% (Table 4). For the flesh translucency, the CV was higher in Expt 1 than in Expt 2. Infructescence length and slip production Infructescence length is an easy criterion for farmers to differentiate between plants. Our results showed that plants with higher infructescence length (in the higher sextiles) at pruning were more likely to have produced slips at pruning time ( Figure 3) and produced more slips than plants with lower infructescence length (Figure 4). The higher number of slips will be related to more assimilates available in these plants and/or a better nutrient status (cf. Swete Kelly 1993). This also suggests that plants in Expt 1 -with slightly higher fruit and infructescence weight-had a better nutritional status than those in Expt 2 at the moment of pruning, what is in line with findings by Fassinou Hotegni et al. (2014b) who showed a positive association between the plant vigor at flowering induction and the slip number, fruit weight and infructescence weight at the moment of fruit harvest. Effects of pruning on fruit quality and variation in fruit quality In both data sets, the fraction of plants pruned and pruning time had no consistent effects on fruit quality nor on variation in fruit quality (Tables 3 and 4). The lack of any consistent effect on average quality was surprising because slip development overlaps with fruit development and it was obvious that competition for available assimilates or nutrients within a plant might take place between the developing slips and the fruit as is the case in many crops producing fruits and side shoots, e.g. in tomato (Heuvelink 1997) and tangelo (Morales et al. 2000). Also the size of the side shoots to be removed at pruning time ( Figure 2) and their number (Figure 4) were substantial. Our results agree with the results obtained with cv. Pérola in Brazil for which slip pruning did not affect any fruit variable other than crown weight (De Lima et al. 2001). In the study by De Lima et al. (2001), slips were pruned at 90 days after flowering induction; in our study, slips were pruned based on the development stage of the plant. The lack of effect of slip pruning on average fruit quality was also confirmed by Fassinou Hotegni et al. (in press) who investigated if the effect of pruning was different for plants having a different infructescence length at the moment of pruning. The few significant effects shown by 9 out of the 240 P-values (Table 3) were always small (Table 4) and never consistently significant in both experiments (Table 3); they therefore most likely might have occurred by chance. The lack of an effect of pruning on quality of cv. Sugarloaf was additionally confirmed by the fact that the P-values in the data set containing only plants with slips were not clearly lower than the P-values in the data set including all plants. Lack of effect of pruning on the average fruit quality attributes might be caused by slips becoming autotrophic at a very early stage of their development and by slips being only initiated when the plant is likely to support their growth. Kerns et al. (1936) working with Cayenne pineapple cultivar, found that during the generative phase, the infructescence is completely formed before the slips are initiated. Since the fruit is a stronger sink than other developing sinks , it would tend to take more assimilates from the plant than the other sinks. In these conditions, the slips, at the earlier stage of their development, i.e., when they appear like a bud at the upper part of the peduncle, would also take assimilates from the plants but not in a way to limit the assimilates needed for the fruit development and growth. When the slips turn from the bud stage to the leaf production stage, they certainly start producing their own assimilates for their development and growth, hence they become autotrophic. This view agrees with absence of slips or the lower number of slips produced in less vigorous plants (Figures 3 and 4); it suggests that the Sugarloaf pineapple plant adjusts the number of slips so that their need for assimilates at an early stage of development does not compromise the needs for assimilates of the fruit. The lack of a consistent significant effect of pruning on the variation in fruit quality attributes might be a direct consequence of the lack of effect of pruning on individual fruit quality. The differences in pineapple quality between experiments (Table 4) may be related to differences in cultural practices (Table 1) and the weather conditions ( Figure 1). The higher fruit and infructescence weights and smaller crown weight and lengths in Expt 1 could be related to a later moment of flowering induction (Fassinou Hotegni et al. 2014b). The lower TSS and higher pH in Exp. 1 could be related to the cooler temperatures in the last month before harvest (Paull and Chen 2003). Implications Pruning of slips, either in selected plants or across all plants, did not lead to a consistent significant improvement in the average quality of the harvested pineapple fruits nor in the variation in quality compared with no pruning. Practical implications of the results are that it is not recommended to farmers to prune slips. Further studies should be done to determine how the Sugarloaf pineapple plant adjusts the available assimilates at flowering induction to the number of the side shoots to be produced. Abbreviations CV: Coefficient of variation; MIE: Months after inflorescence emergence; TSS: Total soluble solids. Competing interests The authors declare that they have no competing interests. : Values followed by the same letters in the same columns for each quality attribute, are not significantly different based on LSD (0.05). Lines in regular font type indicate the grand means for each quality attribute. Individual treatment means are shown in italic font type for quality attributes in which the effects of pruning time, pruning treatment or their interaction were significant in one of the data sets or experiments. Values in bold indicate the means in which effects were significant.

v3-fos

2016-05-12T22:15:10.714Z

2015-03-04T00:00:00.000Z

5044054

s2

Mapping Isoflavone QTL with Main, Epistatic and QTL × Environment Effects in Recombinant Inbred Lines of Soybean Soybean (Glycine max (L.) Merr.) isoflavone is important for human health and plant defense system. To identify novel quantitative trait loci (QTL) and epistatic QTL underlying isoflavone content in soybean, F5:6, F5:7 and F5:8 populations of 130 recombinant inbred (RI) lines, derived from the cross of soybean cultivar ‘Zhong Dou 27′ (high isoflavone) and ‘Jiu Nong 20′ (low isoflavone), were analyzed with 95 new SSR markers. A new linkage map including 194 SSR markers and covering 2,312 cM with mean distance of about 12 cM between markers was constructed. Thirty four QTL for both individual and total seed isoflavone contents of soybean were identified. Six, seven, ten and eleven QTL were associated with daidzein (DZ), glycitein (GC), genistein (GT) and total isoflavone (TI), respectively. Of them 23 QTL were newly identified. The qTIF_1 between Satt423 and Satt569 shared the same marker Satt569 with qDZF_2, qGTF_1 and qTIF_2. The qGTD2_1 between Satt186 and Satt226 was detected in four environments and explained 3.41%-10.98% of the phenotypic variation. The qGTA2_1, overlapped with qGCA2_1 and detected in four environments, was close to the previously identified major QTL for GT, which were responsible for large a effects. QTL (qDZF_2, qGTF_1 and qTIF_2) between Satt144-Satt569 were either clustered or pleiotropic. The qGCM_1, qGTM_1 and qTIM_1 between Satt540-Sat_244 explained 2.02%–9.12% of the phenotypic variation over six environments. Moreover, the qGCE_1 overlapped with qGTE_1 and qTIE_1, the qTIH_2 overlapped with qGTH_1, qGCI_1 overlapped with qDZI_1, qTIL_1 overlapped with qGTL_1, and qTIO_1 overlapped with qGTO_1. In this study, some of unstable QTL were detected in different environments, which were due to weak expression of QTL, QTL by environment interaction in the opposite direction to a effects, and/or epistasis. The markers identified in multi-environments in this study could be applied in the selection of soybean cultivars for higher isoflavone content and in the map-based gene cloning. Traditional methods of genetic improvement of quantitative traits were mainly dependent on phenotypic information [24], which was readily affected by environmental factors. Molecular markers offered a faster and more accurate approach for breeding, because selection could be based on genotype rather than solely on phenotype. The use of molecular markers in the selection of important agronomic traits, or marker-assisted selection (MAS) could improve the efficiency of traditional plant breeding [25][26][27]. However, the use of MAS requires knowledge of reliable marker-trait associations that are relatively stable over multiple environments, because constant QTL over multiple environments might contribute to a consistent phenotype beneath changing conditions. An individual QTL is described as 'major' or 'minor' is based on the proportion of the phenotypic variation explained by a QTL (based on the R 2 value). Major QTL will account for a relatively large amount of R 2 value (R 2 > 10%), while minor QTL will usually account for a relatively small amount of R 2 value (R 2 < 10%) [28]. Sometimes, major QTL may refer to QTL that is stable across environments whereas minor QTL may refer to QTL that is environmentally sensitive [28]. Because soybean seed isoflavone accumulation is mainly dominated by minor-effect QTL that are often influenced by environment, discovering a stable QTL has been hindered. Therefore, it is imperative to identify some stable loci associated with isoflavone content in different environments. To boost QTL detection, appropriate crosses need to be selected to generate sufficient genetic variations in genomic level [29]. Although a linkage map is not strictly required for MAS, a dense marker genetic map greatly facilitates strong marker-gene correlations by permitting the utilization of improved QTL mapping approaches [30]. Epitasis is also thought to contribute to isoflavone variation [17]. The result of some studies indicated that significant QTL epistatic interactions influenced isoflavone content in soybean seed [17,32]. Hence, the isoflavone content in soybean seed is considered as complex quantitative trait because their levels are highly variable and regulated by multiple genetic and environmental factors [1,[19][20][21][22][23]. Zeng et al. [31] identified fifteen QTL underlying seed isoflavone contents of soybean based on a RI line population derived from a cross between 'Zhong Dou 27 0 and 'Jiu Nong 20 0 through a genetic linkage map including 99 SSR markers. In the present work, 95 additional SSR markers were added to the map of Zeng et al. [31] to identify novel QTL with main, epistatic or QTL × environment effects that associated with seed isoflavone contents of soybean. The screening of simple sequence repeats (SSR) markers Total DNA of the parents and each RI line were isolated from dried leaf tissues by CTAB method [40]. A total of 500 additional SSR markers, covering the whole genome of soybean (available at http://www.soybease.org), were used to detect polymorphisms. The PCR reactions were 94°C for 2 min, followed by 35 cycles of 30 s at 94°C, 30 s at 52°C, 30 s at 72°C and 5 min at 72°C after the last cycle. The amplified PCR products were mixed with loading buffer and denatured for 5 min at 94°C and then kept on ice for 5 min. The denatured PCR products were separated on 6% (w/v) denaturing polyacrylamide gel and visualized by silver staining [41]. The polymorphic SSR markers were integrated into the map constructed by Zeng et al. [31]. QTL analyses Polymorphic markers were identified and mapped on the 20 linkage groups by Mapmaker 3.0b with the Kosambi mapping function [42]. WinQTLCart2.1 [43] was used to detect QTL between marker intervals by 1,000 permutations at significance (P 0.05). The genetic linkage map was constructed using Mapchart 2.1 [44]. QTL genetic effects including additive, additive × additive epistatic effects and their environmental interaction effects were analyzed according to the method of Wang et al. [45]. The nomenclature of the QTL included four parts following the recommendations of the Soybean Germplasm Coordination Committee. For example, qDZI_1, q, DZ, I and 1 represent QTL, trait (daidzein, DZ), linkage group name and QTL order in the linkage group, respectively. GGE Bioplot methodology [46] was employed to analyze the interaction between QTL and environments, based on the formula: T ij − T j / S j = λ 1 z i1 τ j1 + λ 2 z i2 τ j2 + ε ij , where T ij was the mean value of QTL i for environment j; T j was the mean value of environment j over all QTL, S j was the standard deviation of environment j among QTL mean; z i1 and z i2 were the PC1 (first principle component) and PC2 (second principle component) scores respectively, for QTL mean i; τ j1 and τ j2 were the PC1 and PC2 scores respectively, for environment j; and ε ij was the residual of the model associated with QTL i, challenged with environment j. Linkage analyses In this study, a total of 500 new SSR markers were used to detect polymorphisms between the two parents, 95 of which were integrated into the linkage map constructed by Zeng et al [31]. This linkage map included 194 SSR markers and covered 2,312.16 cM with mean distance of 11.92 cM between markers. Identification of QTL for total and individual isoflavone contents In this study, the identification of QTL was based on multi-environmental phenotypic data of Zeng et al. [31]. Thirty four QTL underlying individual and total isoflavone contents were identified on thirteen LGs in seven environments over three years (Table 1, Fig. 1). Among them, six, seven, ten and eleven QTL were associated with DZ, GC, GT and TI, respectively. Three QTL (qDZF_2, qGTF_1, qTIF_2) associated with DZ, GT and TI were located on Gm13 (LG F) between Satt144-Satt569. They explained 1.87%-17.03% of the phenotypic variation in seven environments over three years. Another three QTL associated with GC, GT and TI were located on GM07 (LG M) between Satt540-Sat_244. They explained 2.02%-9.12% of the phenotypic variation across seven environments. Among the 34 QTL, 23 QTL were newly identified. Most of them were detected in one or two environments and explained 1.43%-17.05% of the phenotypic variation. The qTIF_1 in the interval between Satt423 and Satt569 shared the same marker Satt569 with qDZF_2, qGTF_1 and qTIF_2. The qGTD2_1 between Satt186 and Satt226 was detected in four environments and explained 3.41%-10.98% of the phenotypic variation. The qGTA2_1 was detected in four environments, which shared the same marker interval of Sat_040-Satt233 with qGCA2_1. Interestingly, six pairs of QTL overlapped with each other and shared the same marker interval (Table 1, Fig. 1). For example, the qGCE_1 overlapped with qGTE_1 and qTIE_1 in the interval between Sat_112 and Sat_380, the qGTO_1 overlapped with qTIO_1 in the interval between Sat_221 and Satt241, the qDZI_2 overlapped with qGCI_1 in the interval between Satt330 and Satt239, the qGCA2_1 overlapped with GTA2_1 in the interval between Sat_040 and Satt233, the qGTH_1 overlapped with qTIH_2 in the interval between Sat_334 and Satt253 and the qGTL_1 overlapped with qTIL_1 in the interval between Sat_113 and Sat_320. QTL × environment interaction Six, seven, ten and eleven QTL associated with DZ, GC, GT and TI respectively, had additive main effect (a) and/or additive × environment interaction effect (ae) at certain environments ( Table 2). Two QTL (qDZI_2, qDZF_2) associated with DZ, two QTL (qGCE_1, qGCM_1) associated with GC, four QTL (qGTD2_1, qGTF_1, qGTM_1, qGTA2_1) associated with GT and four QTL (qTIE_1, qTIL_1, qTIM_1, qTIF_2) associated with TI, contributed to the allele that increased individual and total isoflavone through significant a effects. Three QTL (qDZC2_1, qDZI_1, qDZK_1) associated with DZ, one QTL (qGCI_1) associated with GC, two QTL (qGTL_1, qGTF_2) associated with GT and three QTL (qTIA2_1, qTID2_1, qTIO_1) associated with TI, contributed to the allele that decreased individual and total isoflavone through significant a effects, respectively. The impact of QTL ae effects was different across seven environments and three years. four QTL (qTIF_1, qTIH_1, qTIH_2, qTIK_1) associated with TI, had only significant ae effects rather than significant a effects. Other QTL in seven environments had both significant a effects and significant ae effects. Epistatic analyses of QTL across multi-environments Six, seven, six and nine epistatic pairwise QTL were associated with DZ, GC, GT and TI content, respectively, in different environments (Table 3). Of them, three, one, three and three epistatic pairs of QTL positively increased DZ, GC, GT and TI content through significant aa effects in different environments, respectively. One, one, one and two epistatic pairs of QTL decreased DZ, GC, GT and TI content, respectively, through significant aa effects in different environments ( Table 3). The epistasis × environment interaction effect (aae) was an important component of QTL × environment (QE) interaction effects. One QTL and one pairs of QTL were found with only epistatic effects (aa), which was associated with DZ and TI content. Two, four, two and four pairs of QTL were found with only epistatic effects (aa), which was associated with DZ, GC, GT and TI content, respectively. Stability evaluation of QTL associated with individual and total isoflavone contents across mutli-environments GGE Biplot analysis [46] of seven novel main QTL for individual and total isoflavone contents against seven environments showed that these QTL explained 59% of the total variation of seed isoflavone (Fig. 2). The performance of QTL at each environment was evaluated. When QTL (qTIL_1, qGTD2_1, qDZI_2, qGCE_1 and qTID2_1) were set as the corner QTL, seven different environments fell in the sector in which the QTL qGTD2_1 was the best QTL for two environments (at Harbin in 2005 and at Hulan in 2006, Fig. 2). The qTIL_1 was the best QTL at Harbin in 2006 and at Suihua in 2007, and the qTID2_1 was the best one at Hulan in 2007. Discussion Soybean isoflavones have multiple uses in food, medicine, cosmetics and animal husbandry [47]. Improving seed isoflavone content, therefore, should be appeared to be a useful target of soybean breeding. MAS based on genotype selection rather than solely on phenotype provided an outstanding perspective in soybean breeding [30]. 'Zhong Dou 27 0 was proved to have high-isoflavone content (3,791 μg/g isoflavone in seed) among seven environments in our previous report [31]. Meng et al. [34] identified two QTL resisting to soybean aphid through isoflavone-mediated antibiosis in soybean cultivar 'Zhong Dou 27 0 . These two QTL were significantly associated with high isoflavone content with positive effects derived from 'Zhong Dou 27 0 , providing potential for MAS to improve the resistance of cultivar to aphid along with the increase of isoflavone content. This germplasm should be given more attention to reveal the underlying genetic mechanism. In our previous study, a numbers of QTL associated with seed isoflavone were identified in 'Zhong Dou 27 0 using 99 SSR markers. In the present study, additional ninety-five SSR markers were added to the existed linkage map of Zeng et al. [31]. The low level of phenotypic variation evaluated for these Table 3. Additive × additive epistatic effect and their environmental interaction effect of QTL associated with individual and total isoflavone at RIL population. QTL in this study (<10%) was indicative of the quantitative nature of individual and total isoflavone, which was similar to the other studies [14][15][16][17][31][32][33][34][35][36]. qDZF_2, qGTF_1 and qTIF_2 between Satt144-Satt569, and qGCM_1, qGTM_1 and qTIM_1 between Satt540-Sat_244, were identified in multiple environments (Fig. 1). These QTL detected by Satt540 and QTL detected by Satt144 on LG M and on LG F in this study were the same or similar to that of our previous studies [31][32][33][34]39], which provided a valuable resource for MAS to develop soybean varieties with high seed isoflavone content. Previously, two major QTL consistently affected isoflavone content across multiple environments were mapped on Gm05 (LG A1) and Gm08 (LG A2) by Gutierrez et al. [30] and Yang et al. [35], respectively. Here, we mapped three new QTL (qTIA2_1 located in Sct_067-Satt470; qGCA2_1 and qGTA2_1 located in Sat_040-Satt233) associated with individual and total isoflavone. The qGTA2_1 was detected in four environments and explained 3.51%-11.58% of the phenotypic variation. This QTL was near to the major locus identified by Yang et al. [35] that controlled the same trait GT, suggesting that qGTA2_1 might be an enzyme-related locus. The qGCA2_1 and qGTA2_1 were identified between the same marker interval of Sat_040-Satt233, implying that there could be some genetic factors regulating the accumulation of GC and GT. These stable QTL were responsible for large a effects ( Table 2). As suggested by Tanksley [48], QTL with higher a effects are more likely to be stable across multiple environments. Most of the QTL discussed above with higher a effects (Significant at P = 0.01) were stable across at least three environments (Tables 1 and 2). These seven novel major QTL were selected to do the GGE Bioplot analysis, and only explaineding 59% of the G and G × E variation of seed isoflavone, which was lower than other's studies [23,31]. This could be caused by few QTL involved. The contribution of G and G × E to seed isoflavone phenotypic variation could be increased to 79% after the excluding of E4 (maybe a mega environment), indicating that E4 had significant influence on isoflavone content (S1 Fig.). Moreover, in order to examine the accuracy of the results by GGE Biplot analysis, QTLNetwork 2.0 software [49] was used to analyze the interaction between QTL and environments, and the result was similar to GGE Biplot results (S1 Table), indicating that QTL detected in multiple environments were more stable. Among the newly identified QTL, the qTIF_1 shared the same marker Satt569 with qDZF_2, qGTF_1 and qTIF_2 in multi-environments, suggesting that there are some genetic elements, such as genes or factors could affect the accumulation of DZ, GT and TI (Table 1, Fig. 1). Additionally, six pairs of QTL overlapped with each other and shared the same marker interval, inferring that some genetic elements could regulate the accumulation of different isoflavone components in these intervals (Table 1). Among the 23 newly identified QTL, five QTL intervals were completely overlapped with our previously reported eQTL and a total of eleven candidate genes within the overlapped eQTL and QTL were identified [39]. For example, the newly identified QTL (qGCE_1, qGTE_1 and qTIE_1) located in the interval of Sat_124-Sat_380 shared the same marker Sat_380 with the eQTL qF3HE_1, implying the F3H gene in the phenylpropanoid pathway could affect the accumulation of GC, GT and TI. In this study, many unstable QTL were detected in different environments (Table 1, Fig. 1), which were due to the weak expression of the QTL, QTL by environment interaction in the opposite direction to a effects, and/or epistasis (Tables 2 and 3). Therefore, the information of QTL by environment interaction should be considered if MAS was applied to the manipulation of quantitative traits. Since the 194 markers were not uniformly distributed, large gaps appeared with low marker density on chromosomes Gm02, 13 and 20, implying that more markers should be developed among these gaps and the authenticity of QTL should be further clarified. The precise estimate of individual and total isoflavone content of soybean seed based on phenotype was difficult due to environment effect. Markers tightly linked to the QTL underlying isoflavone content would help to identify soybean lines containing higher isoflavone on the basis of genotype, to maximize the effectiveness of selection. Identification of stable QTL in multi-environments and fine mapping those loci could be desirable for identifying the underlying candidate genes or factors. Table. Additive × additive epistatic effect and their environmental interaction effect of QTL associated with individual and total isoflavone at RIL population using QTLNetwork 2.0 software. (DOCX)

v3-fos

2019-03-30T13:11:31.310Z

2015-07-02T00:00:00.000Z

53406314

s2

Effect of Egg Storage Temperature and Storage Period Pre-incubation on Hatchability of Eggs in Three Varieties of Japanese Quail Background: There are many factors affecting successes of quail production system , one of important factor is provide sufficient number of egg for needs of hatcheries to produce chicks . This study was conducted in poultry farm of Animal Resources – College of Agriculture – University of Diyala Iraq, to determine suitable conditions for storage of Japanese quail eggs belong to three varieties of Japanese quails( White , Black and brown plumage color ). Materials and Methods: Eggs from three varieties allocated in two groups represented two storage temperatures 7 C° and 20 C° (average room temperature ) , and each temperature group divided into four sub-groups represented storage periods length 3 , 7 , 10 and 14 days , thus the total number of egg groups were 16 groups. The experiment performed in factorial experiment 3 × 2 × 4 for three factors included variety, storage temperature and storage period , conducted in Randomized Completely Blocks Design with three replicates. The experimental flock consist of 450 birds belong to three varieties , the eggs collected daily and stored according to these various treatments before entered the incubator , and after hatching of eggs , the data recorded for hatchability and embryonic mortality percentages for treatments. Results: The results showed that the black variety quail has significant superiority in fertility (80.19 %) with compare to White and Brown varieties (69.07 and 68.03 % respectively). There were significant effect (P< 0.05) of storage period on hatchability, hence there were significantly decline in hatchability after storage period for 14 days (36.58 %), also there were significant interaction between varieties and storage periods. While there were no significant effect of storage temperature and other interactions on hatchability and embryonic mortality percentage. Introduction Japanese quail represents smallest bird used in poultry industry for egg and meat production 1 , because of his unique traits such as fast growth and high rate of egg production 2 . There were increasing interest in quail production in Iraq during the few last year, and that reflect on many studies conducted in Iraq about viability and production performance of Japanese quail in the natural environment exhibited high adaptation for Iraqi conditions (Hassan 3 and Hassan et al. 4 ). The important factor to activate the production system of quail is the production of quail chicks, involve in this process obtain a sufficient number of eggs to fill an Hatchery (Kuurmon et al. 5 ). Romao et al. 6 reported that eggs from quails usually accumulated and storage over a period from 1 day up to 3 weeks before incubation. There are many factors affecting hatchability of stored eggs before incubation , for instance storage temperature and storage length period , Romao et al. 6 also recorded that egg-type quail eggs had 85 % hatchability when storage up to 10 days at 20 C and 60 % of relative humidity . In other study Garip and Dere 7 reported that hatchability was 78.4 % for quail eggs stored for 5 days in 21 C and hatchability declined to 35.4 % when the storage period extended to 15 days in the same storage temperature. The importance of storage temperature recorded by Meijerhof 8 who reported that low temperature prevent the embryonic development before incubation , and for this purpose the eggs room temperature must be 20 -25 C for 4 -7 days . Hassan 1 reported that poultry breed classified into varieties according to plumage color or comb shape and may be depend on both. Alkan et al. 9 explained that hatchability is affected by many factors as fertility of eggs, handling of eggs and conditions during incubation. The aim of this study is to determine the effect of storage temperature and storage periods on hatchability of eggs quails belong to White, Black and Brown varieties of Japanese quail. Materials and Methods The experiment was carried out in poultry farm of Department of Animal Resources -College of Agriculture -University of Diyala / Iraq. The experimental flock consist of 450 Japanese quail birds, belong to White , Black and Brown plumage represented three varieties. Hassan 1 reported that the variety classification can be perform according to plumage color . Eggs from three varieties allocated in two groups represented two storage temperatures 7 C° and 20 C° (average room temperature ) , and each temperature group divided into four sub-groups represented storage periods length 3 , 7 , 10 and 14 days , thus the total number of egg groups were 16 groups. The experiment performed in factorial experiment 3 × 2 × 4 for three factors included variety, storage temperature and storage period , conducted in Randomized Completely Blocks Design with three replicates. The eggs collected daily and stored according to these various treatments before entered the incubator ( the number of eggs were 344 , 337 and 280 eggs in the three batches respectively ) , and after hatching of eggs , the data recorded for hatchability of total eggs, hatchability of fertile eggs and embryonic mortality percentages for treatments. The fertility of un hatching eggs determined by broken the egg and examined the embryonic development in each treatment groups . The birds fed ad libitum a diet containing 24 % protein and 2896.8 kcal / kg metabolize energy. The statistical analysis performed according factorial experiment 3 × 2× 4 in randomized complete block design with three replicates, and the significance of differences among means tested by Revised Least Significant Differences ( R.LSD) at P<0.05. Analysis of variance performed by used SPSS program . Results The statistical analysis of variance showed that there were no significant effect of varieties and storage temperature on hatchability of total eggs, hatchability of fertile eggs and embryonic mortality percentages, while there were significant effect ( P<0.05) of main effect of storage periods and also the interaction between variety and storage periods, the other interactions between factors showed no significant effect on the traits ( Table 1 ). The result reveled significant differences (P < 0.05) among varieties in fertility ( Fig. 1) hence the black variety recorded significant superiority in fertility as 80.19 %, while there were no significant differences between white and brown varieties in fertility as 69.07 % and 68.03 % respectively. Tavaniello 10 confirm that heredity has affected role in fertility of quails, so there were differences between strains in fertility of their eggs. While the results showed no significant differences among varieties in respect of hatchability of total eggs and hatchability of fertile eggs ( Table 2). The storage temperature 7 and 20 C° showed also no significant differences in hatchability of total eggs (43.27 and 35.57 % respectively) and hatchability of fertile eggs (55.50 and 45.58 %) as presented in Table 3 . Storage periods as mentioned previously showed significant effect on hatchability and embryonic mortality according to F test in the analysis of variance , and the post hoc test (Revised LSD) showed significant decline in hatchability of total eggs for 14 days storage period (24.98%) compared with other periods which have no significant differences among them (Table 4). While the result recorded no significant differences between 7 days and 14 days periods in its affect on hatchability of fertile eggs (Table 4). This results agreed with Tavaniello 10 who reported that successful hatches affected by the princubation period, and represent important factor. The interaction between varieties and storage periods appear significant differences in hatchability of fertile eggs and embryonic mortality , The highest hatchability for black variety was at 3 days storage period (73.76 %), while the highest hatchability for brown variety was at 14 days (52.09 %) , and the best hatchability for white variety appear at 10 days (63.58 %) as recorded in Table 5. These differences reflect the presence of variations in the suitable conditions needed for different genotypes as varieties and strains. In other hand, there were no significant differences recorded among hatchability result from interactions between storage temperature and varieties as showing in Table 6. The study estimates the correlation coefficient between the factors included in the study (varieties, storage temperature and storage periods) and each of fertility, hatchability of total eggs, hatchability of fertile eggs and embryonic mortality as showing in Table 7. The highly significant negative correlation between storage periods and fertility may be caused by undetected early embryonic mortality. While the storage periods have highly significant negative correlation coefficient with hatchability of total eggs (Table 7) may be reflect the un suitable conditions appear with the progress of storage as a result of loss of water from eggs and change in the PH of the egg. Discussion The results of this study showed a significant effect of varieties on fertility , the data showed superiority of black variety in this traits which may indicate for linkage or pleiotropic effect between the two traits , and significant correlation between variety and fertility may confirm this situation . The significant effect of storage periods on hatchability of total eggs and fertile eggs agreed with many previous studies (Garip and Dere 7 , Tavaniello 10 ) have the same conclusion , that the prolonged storage period caused decline hatchability because of water loss and gas which lead to change PH of the interior environment of the egg , in this direction Garip and Dere 7 reported that hatchability of quail eggs decline from 78.4 % to 35.4 % when stored in 21 C° for 5 and 15 days respectively . Also, there were highly significant negative correlation coefficient between storage periods and hatchability, and confirm the importance of the storage periods in the process of production quail chicks. Storage temperatures showed no significant differences between 7 and 20 C° included in the study which indicated that the two degrees in the suitable range of storage temperature. Conclusion According to the results of this study, in general the best storage period for Japanese quail was 7 -10 days in the room temperature (20 C°). Also, there were important interaction between variety and storage periods, that indicate to presence of genotype-environment interactions.

v3-fos

2019-04-17T15:40:18.633Z

2015-01-01T00:00:00.000Z

118361704

s2

PATH ANALYSIS OF THE MAIN ECONOMIC CHARACTERS OF THE INNER MONGOLIA WHITE CASHMERE GOATS In this paper, the correlation coefficients among the various economic characters of the Inner Mongolia white cashmere goats such as cashmere yield (Y) , bo dy weight (X1), cashmere thickness (X2), staple length (X3), cashmere fineness (X4) and cashmere stretched length(X5) are analyzed with the SAS8.1 software, and the regression equation between the cashmere yield and other economic characters is established based on the path analysis. The results shows that the main factor determining the cashmere yield of rams is the body weight and that of ewes is the cashmere thickness of the cashmere, followed by the body weight. The regression equations of rams and ewes between the cashmere yield and other economic characters were Y=7.0462X1+15.6568X2-7.2930X3+14.5744X5 and Y=176.8148+5.0321X1+45.5121X2-3.4524X3+15.6498X4+18.7189X5, respectively. It’s recommended to put emphasis on the body weight and cashmere yield and give dual attention to the cashmere thickness and cashmere fineness when breedin g the Inner Mongolia white cashmere goats, in order to obtain good breedin g effect. JunYan Bai*, Xiao Ping Jia, Yu Qin Wang and You Zhi Pang Introduction Various kinds of cashmere goats are used for various purposes. Particularly, the cashmere (referring to the downy unmyelinated fiber grown from the secondary hair follicle in the under layer of the goat hair) has the special characteristics in the international market. Being extra fine, white, soft and bright, the fiber is a traditional rare and advanced textile material. The produced fabric is thin, light, warm, smooth, comfortable, beautiful and elegant at high prices, promoting the development of the international cashmere goat industry (Li et al.,2001). Not only the traditional cashmere goat breedin g countries are actively involved in the cashmere goat breedin g and research work, but also the animal husbandry developed countries like Australia, New Zealand, the UK and the US also successively develop and establish the cashmere goat bases and control the weeds an d shrubs an d improve the grasslan d through grazing the goats (Jia et al., 1994). At the same time, they vigorously carry out scientific research to improve the production level of the cashmere goats. With the favorable advantages of the cashmere goat resources, China has made great achievements in the cashmere goat breeding and accumulated rich information, but due to the poor basis and late start of the cashmere goat genetic breeding work, the existing research level is still low an d there are a lot of work needing to be done (Jia et al., 1994;Jia et al., 1999). Formed after the long-term breeding, the Inner Mongolia white cashmere goat is an improved local variety with the dual purpose of cashmere and meat. T he Inner Mongolia white cashmere goats are found in the Inner Mongolia Autonomous Region. T hey are divided into three types, namely, Aerbasi cashmere goat, Erlang san cashmere goat and Alashan cashmere goat (Bai et al., 2006). In April 1988, they were accepted and named the Inner Mongolia white cashmere goat by the People's Government of the Autonomous Region. Since the introduction of the quantitative genetics in the 1920s, the animal breedin g method has been developing rapidly, leading to the great achievements in the breeding work and the huge progress in the production level of animal and poultry (Jia et al.,1999). Some new theories and methods of animal genetics and bree ding are widely applied in the breeding of co ws, chickens, pigs, fine-wool sheep, beef cattle, mutton sheep, milk goats and hairy goats. However, due to the late start, the cashmere goat breeding lags behind other breeds in the breedin g theory and method (Jia et al., 1999). In the cashmere goat research is currently mainly concentrated in the estimation of genetic parameters (Bai et al., 2004) and gene on production traits influence (Lan et al., 2012). T his paper conducts a regression analysis bet ween the cashmere yield of the Inner Mongolia white cashmere goat and other economic characters and discusses the relationship among the economic characters, in order to provide some references for the scientific breeding and variety breeding of the Inner Mongolia white cashmere goats. Materials and methods T he test data are from the record data of 759 rams and 3836 ewes in a cashmere goat farm in Inner Mongolia, including 6 economic characters viz cashmere yield, body weight, cashmere thickness, staple length, cashmere fineness and straight length. For the sake of convenience, these 6 indicators will be abbreviated to Y, X1, X2, X3, X4 and X5, respectively. Inner Mongolia white cashmere goat was provided by Inner Mongolia Yimeng Ar bas white cashmere goat breedin g farm, the sheep sheep herder groups according to a fixed feeding, year-round grazing in winter and spring, the quantitative feeding feed. Every spring, unified administration, immune sheep wash, insecticide, unified breeding in autumn, spring lambing. 4 to June each year between the unity of performance measurement in the same place, the cashmere yield and body weight were measured, including cashmere thickness, staple length, cashmere fineness and straight length sample parts are on the side of the body of scapula backwar d a cashmere goat cashmere, cashmere fineness and the stretched length is determined under the microscope. The correlation analysis and path analysis are conducted with MEANS, CORR and RE G of SAS8.1 software. Basic Statistics Analysis T he basic statistics of the cashmere yield, body weight and hair characters of the Inner Mongolia white cashmere ram and ewe goats have been represented in T able 1. According to T able 1, the variation coefficient was reported highest for the body weight of rams this weight was follo wed by the cashmere yield, bein g 33.10% and 30.62%, respectively. According to T able 2, the variation coefficient of the cashmere yield of ewes was the largest which followed by the body weight, being 28.54% and 22.63%, respectively, indicating that the cashmere yield and body weight of this variety change greatly and there are great potential in the cashmere yield and body weight when selecting the Inner Mongolia white cashmere goats. Correlation Analysis T he correlation coefficients among various economic characters of the Inner Mongolia white cashmere ram and ewe goats were shown in T able 2. It can be seen in T able 2 that the body weight, cashmere yield and hair character of rams are positively correlated with each other to varied extents (P<0.01), in which the coefficients correlated with the body weight were as follo w cashmere fineness > cashmere yield > cashmere thickness > staple length > length; the coefficients correlated with the cashmere yield follow: bo dy weight > cashmere fineness > length > cashmere thickness > staple length. Notes: **: that the correlation coefficient between the two traits is very significant(P<0.01), *: indicates the correlation coefficient between the two traits as a significant(P<0.05), other: that the correlation coefficient between the two traits was not significant(P>0.05). Path analysis According to the principle of the path analysis, the phenotypic correlation among the characters of rams and eves can be used to establish the path coefficient equation set 1 and 2 on the cashmere yield. For ewe s, the staple length has the largest indirect influence of 0.1463 on the cashmere yield, but its indirect influence was negative. T he influence was exerted through the cashmere thickness. T he indirect influence of other characters through the staple length was negative, either; the indirect influence of the cashmere fineness was 0.1404 and that of the cashmere thickness was 0.0999. T he cashmere stretched length has the smallest indirect influence on the cashmere yield of 0.0667. It mainly exerts the influence through the cashmere thickness and its influence through other characters was negative. T he indirect influence of the characters on the cashmere thickness through the body weight was very high and it varied from 0.0442 to 0.1031. T hus, the influence of the staple length and cashmere fineness of ewes on the cashmere yield mainly depends on the indirect effect; the body weight and cashmere fineness have the strong direct influence on the cashmere yield; the cashmere thickness not only has strong direct influence on the cashmere yield, but also assists other characters to exert great direct influence on it. Optimal regression equation is established It's found through the significance test on the intercept and 5 regression coefficients of the cashmere yield regression model that the regression coefficients of the intercept and cashmere fineness do not reach the significant level and those of other characters reach the extremely significant level. After eliminating the characters with the not significant coefficients, the regression equation is: Y=7.0462X1+15.6568X2-7.2930X3+14.5744X5. Similarly, the regression equat ion of the cashmere yield of ewes was y=-176.8148+5.0321X1+45.5121X2-3.4524X3+15.6498X4+18.7189X5. Discussions T he research of Xiaohong et al. (2004) shows that the body length (X2), chest circumference (X3), shin circumference (X4), staple length (X6) and body weight (Y1) of Liaoning white cashmere goats have the significant positive correlation with each other (P<0.01). The optimal regression equation is established as: Y1=-46.306+0.327X2+0.587X3+ 0.780X4+0.288X6. According to the equation, the body weight of the cashmere goats has the reliability of more than 95%. T hey think the chest circumference of Liaoning white cashmere goats has the biggest influence on the body weight and the body length, shine circumference and staple length indirectly affect the body weight through the chest circumference; the staple length has the biggest influence on the cashmere yield, but the optimal regression equation cannot be found through the body height and length, chest circumference, shine circumference, staple length and cashmere yield. T ong-jun et al. (2003) argue that the body size of Jianchang black goats at all ages has different degrees of correlation with the body weight. T he chest circumference plays the decisive role on the body weight when the goat is at 2 years old. It's followed by the interactive effect between the chest circumference and the back height. It's the key time for the breeding selection and mating combination. T he optimal regression model of the body size of the replacement and adult Hainan black goats on the body weight established by Xu et al. (2005) recommends to give due consideration to the chest circumference selection besides the body weight when selecting the high grow rate line of Hainan black goats. Han et al. (2006) analyzes the correlation between the body size and the body weight of Duhan F1 lambs at 6 months old and establishes the regression equitation of the body size on the body body weight. He thinks that there is a significant correlation with the body size indicators and t he body weight of Duhan F1 lambs (P<0.05); and, the body length and chest depth mainly exert the direct influence and others exert the indirect influence. T he decisive coefficient analysis shows that the chest depth and body length of Duhan F1 have a great er influence on the body weight. At preset some scholars of the cashmere goat body measurements and cashmere yield of correlation and regression analysis, for example, Zeng et al. (2014) study showe d the effect of body weight, body height of Shanbei white cashmere goat cashmere yield was significant, regression equation is Y = -574.771 + 18.067X1+19.542X2 can well reflect the linear the relationship between the indicators. Yiming et al. (2014) results showed that: the cashmere goat green Gerry weight (X1), body height (X2), body length (X3), chest circumference (X4), (X5), the fiber length (X6) and cashmere yield (Y) showed a significant positive correlation (P < 0.01). T he direct effect of body weight and chest circumference of cashmere yield and body height is large, mainly through the indirect effect of cashmere yield. T he optimal regression equation: Y= 0.267 X10.094 X30.231 X40.047 X50.089 X6. studies sho w that the farming pastoral zone feeding the measurement indexes of Liaoning cashmere goat body height, body length, chest girth, chest width, chest depth and body are important factors affecting body weight. T he results of this research are slightly different from the findings of Li et al. (2014). After the significance test on t he multiple optimal regression model of the cashmere yield of the Inner Mongolia white cashmere ram and ewe goats, the regression correlations are extremely significant (P<0.01), indicating the real multiple regression relationship between the cashmere yield and the hair characters of the Inner Mongolia white cashmere goats. T his equation is of certain reference value. It can be seen from the correlation analysis, path analysis and decisive degree analysis that the body weight determines the cashmere yield of rams. T herefore, the focus should be put on the cashmere yield with the due consideration to the body weight when selecting the Inner Mongolia white cashmere ram goats for breeding, in order to obtain good breeding effect. T he cashmere thickness determines the cashmere yield of ewes, followed by the body weight. Therefore, the focus should be

v3-fos

2016-05-04T20:20:58.661Z

2015-10-16T00:00:00.000Z

3022304

s2

Multiple antibiotic resistances among Shiga toxin producing Escherichia coli O157 in feces of dairy cattle farms in Eastern Cape of South Africa Background Shiga toxin–producing Escherichia coli (STEC) O157:H7 is a well-recognized cause of bloody diarrhea and hemolytic-uremic syndrome (HUS). The ability of STEC strains to cause human disease is due to the production of Shiga toxins. The objectives of this study were to determinate the prevalence, serotypes, antibiotic susceptibility patterns and the genetic capability for Shiga toxin production in Escherichia coli (STEC) strains isolated from dairy cattle farms in two rural communities in the Eastern Cape Province of South Africa. Methods Fecal samples were collected between March and May 2014, from individual cattle (n = 400) in two commercial dairy farms having 800 and 120 cattle each. Three hundred presumptive isolates obtained were subjected to polymerase chain reactions (PCR) for identification of O157 serogroup and Shiga toxin producing genes (stx1, stx2) on genomic DNA extracted by boiling method. Susceptibility of the isolates to 17 antibiotics was carried out in vitro by the standardized agar disc-diffusion method. Results Based on direct PCR detection, 95 (31.7 %) isolates were identified as O157 serogroup. The genetic repertoire for Shiga toxin production was present in 84 (88.42 %) isolates distributed as stx1 (37), stx2 (38) and stx1/2 (9) respectively while 11 of the isolates did not harbor Shiga toxin producing genes. Multiple antibiotic resistances were observed among the isolates and genetic profiling of resistance genes identified blaampC 90 %, blaCMY 70 %, blaCTX-M 65 %, blaTEM 27 % and tetA 70 % and strA 80 % genes among the antimicrobial resistance determinants examined. Conclusion We conclude that dairy cattle farms in the Eastern Cape Province are potential reservoirs of antibiotic resistance determinants in the province. Background Escherichia coli is an important pathogen in cattle, medicine and public health [1], and Shiga toxin-producing strain (STEC) have emerged as important food-borne pathogens, especially serotype O157:H7. Human diseases caused by this serotype that produces STEC ranges from mild diarrhoea to haemorrhagic colitis and haemolytic uraemic syndrome (HUS) and typically it affects children, the elderly, and immunocompromised patients [2]. Healthy domestic ruminants such as cattle, sheep, and goats can harbor STEC and E. coli O157:H7 in their faeces and are thus natural reservoirs of these pathogens [3][4][5]. The pathogenicity of STEC resides in a number of virulence factors, including Shiga toxins (Stx1 and Stx2), intimin, enterohaemolysin, and the STEC autoagglutinating adhesin (Saa) [6]. Shiga toxin-producing (STEC) and enteropathogenic Escherichia coli (EPEC) represent two of the six different categories of diarrheagenic E. coli that can cause disease in humans [1]. STEC, which is defined by the production of two Shiga toxins, Stx1 and/or Stx2, is a zoonotic pathogen that is a major cause of diarrhea worldwide. Stx2 is more closely related to these diseases than stx1 [7]. There are three Stx1 subtypes (Stx1a, Stx1c, and Stx1d) and seven Stx2 subtypes (Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f, and Stx2g) according to the subtyping nomenclature proposal put forth at the 7th International Symposium on Shiga Toxin (Verocytotoxin)-Producing Escherichia coli Infection, held in Buenos Aires, in 2009. The use of antimicrobial in animal feeds as growth promoters is common worldwide. Across the globe, a variety of antimicrobial agents are available for therapeutic use or as growth promotion in animals. Many studies have supported the claim that with the increased use of antimicrobial agents in animals and humans, an increased prevalence of resistant strains may be selected as a direct consequence of the antimicrobial use [8,9]. Humans, via the food chain, ingest a lot of bacteria originating from food-producing animals, which have been recognized as major reservoirs of E. coli habouring CTX-M β-lactamase an enzyme that confers resistance to β-lactam antibiotics [23]. Sasaki [10] have reported a high prevalence of CTX-M β-lactamase encoding gene among Enterobacteriaceae in stool specimens from healthy asymptomatic volunteers in a rural community in Thailand. Concerns exist about the potential spread of the β-lactam-CTX-M genes from food animal products to humans through the food chain. CTX-M βlactamase genes have been reported in E. coli from various food-producing animals worldwide raising a potential threat to public health [11][12][13][14][15][16]. Giving the frequent occurrence of O157 STEC as foodborne pathogen in North America and some parts of the western world, there is a clear need to gather data on the prevalence and distribution of STEC producing E.coli and the antibiotic resistance profiles of isolates of this organism recovered from faecal samples from commercial dairy cattle farms in the Eastern Cape of South Africa where pastoral farming is a major source of income for many families. This study therefore aimed at characterizing E.coli O157 isolates from fecal samples of two dairy cattle farms in the Eastern Cape Province of South Africa. Ability of isolates to produce Shiga toxin and their antibiotic susceptibility patterns as well as presence of some resistance determinants were screened by molecular approaches. Ethical clearance Ethical clearance was obtained from the University of Fort Hare ethics committee prior to sample collection and permission was sought from farmers from whose farms samples were collected. Study population and sampling Details on the study population and sampling procedures are as follows. Briefly, samples were collected from commercial dairy cattle farms in Nkonkobe District of Eastern Cape Province, South Africa. A total of 400 samples from two commercial dairy farms were collected for the study. Rectal fecal grab samples of approximately 10 g were collected from individual cattle using sterile gloves into appropriate capped containers. After collection, samples were shipped on ice to the University of Fort Hare Microbiology laboratory for immediate processing. Preliminary sample processing Approximately 1 g of each fecal sample was mixed in 9 ml of Trypticase soya broth (TSB) with 20 mg/L novobiocine and incubated for 6-8 h at 37°C. This was streaked out onto sorbitol MacConkey agar (SMAC) supplemented with 1 mg/L potassium tellurite and incubated for 18-24 h at 37°C. A pale colony each (sorbitol nonfermenters) was picked as presumptive E. coli O157 per sample. The pure colonies were each inoculated into separate TSB and incubated for 24 h at 37°C from which glycerol stock was made and then stored at −80°C for further analyses. DNA extraction Bacterial DNA was prepared as previously described by Bai [17]; briefly, bacterial culture from glycerol stock was resuscitated by an overnight growth in trypticase soya broth (TSB) (Oxoid Ltd, London, UK) at 37°C with slight agitation. From this culture, 2 ml was centrifuged for 5 min at 14,000 rpm and the pellet was washed with normal saline (0.85 % NaCl). After the addition of 150 μl of rapid lysis buffer (100 mM NaCl, 10 mM Tris-HCl pH8.3, 1 mM EDTA pH9.0; 1 % Triton X-100), the suspension was votexed, boiled for 15 min, centrifuged at 10,000 rpm, supernatants collected in a DNase free Eppendorf tube and stored at −20°C. These were then used as templates in all the polymerase chain reactions (PCRs) that were performed in this study. Molecular serotyping and virulence typing Molecular serotyping using the O-unit flippase gene (wzx) was performed: primers used for detection of O157 strains are shown in Table 1. E. coli O157:H7 ATCC 35150 served as the positive control. Specific primers were used to detect the presence of virulence genes encoding the Shiga-toxins (stx1 and stx2) as previously described by [17,18]. Amplification was performed using 25 μl of PCR mix containing 5 μl of bacterial DNA purified as described above, 12 μl of 2X Dream Taq Master Mix (Thermo Scientific), 10pmol of both forward and reverse primers and 6 μl of water of PCR grade. The conditions for PCR amplification performed in a thermal cycler (BioRad) were 94°C for 3 min, followed by 35 cycles of 93°C for 60 s, either 55°C for 60 s and 72°C for 60 s. The final cycle was followed by an extension step at 72°C for 7 min. The amplified products were visualized by standard gel electrophoresis using 5 μl of the PCR product on 2 % agarose gels in 0.5X TBE buffer (0.1 M Tris, 0.1 M boric acid and 0.002 M NaEDTA). Gels were stained using ethidiumbromide (1 mg/ml) and photographed under UV light in a transilluminator (ALLIANCE 4.7). PCR profiling of resistance genes Template DNA was prepared as previously stated above. The β-lactamase genes bla TEM , bla SHV, bla CMY-2 , bla-CTX-M , bla-CTX-M1 , bla-CTX-M9 and bla-ampC and two of the genes responsible for resistance to streptomycin (strA) and tetracycline (tetA) respectively were tested using specific primers in Table 2 Thong et al., 2010). PCR was performed in a 25-μl mixture of 12 μl of 2X Dream Taq Master Mix (Thermo Scientific, Pittsburgh, PA, USA), 10 pmol of each forward and reverse primer and 6 μl water of PCR grade. The PCR mixture was subjected to a 3-min denaturation step at 94°C, followed by 35 cycles of 45 s at 94°C, 45 s at 55°C/ 57°C/50°C depending on the primer set, and 60 s at 72°C , and a final elongation step of 7 min at 72°C. PCR Results Serotyping PCR-based molecular serotyping using primers designed for detection of O157 group identified 95 positive isolates as O157 serotype (gel not shown) out of the 320 presumptive isolates. PCR-based detection of virulence genes The 95 molecularly confirmed E.coli O157 isolates were analyzed by PCR for their Shiga toxin producing capabilities. Table 3 shows PCR results of the different virulence genes for 84 (88.45 %) isolates detected among the 95 confirmed O157 isolates. The remaining 11 (11.55 %) though belonging to O157 serogroup, did not harbor Shiga toxin genes and were therefore regarded as non-STEC strains. Distribution of virulence genes among the 84 STEC strains showed that 37 (44 %) isolates possessed the stx1 gene as shown in Fig. 1, 38 Table 4 below. All isolates showed reduced susceptibility to several antimicrobials agents in the panel. PCR profiling of resistance genes Prevalence of AMR genes Results of genetic profiling of the observed phenotypic resistances among the isolates showed predominance of bla ampC 90 %, bla CMY 70 %, bla CTX-M 65 %, and bla TEM 27 %, among the isolates that were resistance to ampicillin, amoxicillin/clavulanate, cephalothin, cefuroxime, ceftazidime. PCR amplification of the bla SHV , bla VEB , bla CTX-Mgroup1 and bla CTX-Mgroup9 did not yield any amplicon. The tet(A) and strA resistance genes were amplified from 70 % and 80 % respectively of the isolates that were phenotypically resistant to tetracycline, oxytetracycline and streptomycin. Altogether, AMR genes were detected in all E. coli isolates. The most frequent resistance genes were tet(A), str(A), bla-ampC, bla-CMY-I (Table 5). With a very few exceptions, susceptibility test results were consistent with genotyping results. Gel photographs of some of the amplified resistance genes are shown in Figs. 2, 3 and 4. Overall, AMR genes were identified in 92 % of the O157 isolates recovered from studied samples. Resistance genes detected from isolates as shown in Table 5. Discussions The prevalence of STEC O157 serogroup in fecal samples collected from commercial dairy cattle was investigated. Escherichia coli O157:H7 (O157) is the Shiga toxin-producing E. coli (STEC) serotype most frequently isolated and most often associated with hemolytic uremic syndrome (HUS) in the United States [2]. According to the U.S. Centers for Disease Control and Prevention, an estimated 265,000 STEC infections occur each year in the United States. STEC O157 causes about 36 % of these infections and non-O157 STEC cause the rest [2]. STEC inhabits in the guts of ruminant animals, including cattle, goats, sheep, deer, and elk [2]. The major source for human illnesses is cattle and around 5-10 % of those who are diagnosed with STEC infection develop a potentially life-threatening complication known as hemolytic uremic syndrome (HUS) with young children and the elderly more likely to develop severe illness and hemolytic uremic syndrome (HUS) than others. In this study, a total of 400 fecal samples were collected from two commercial dairy farms in the Eastern Cape Province of South Africa. These samples were analyzed for the presence of O157 E.coli strain. A total of 95 isolates confirmed by PCR targeting the O-unit flippase gene (wzx) were delineated to be O157 isolates. Results of the determination for the presence of Shiga toxin encoding gene (stx1 and stx2) among the 95 isolates showed that 35 (36.84 %) harbored the stx1 gene, 26 (27.4 %) were positive for stx2 while 9 (9.5 %) harbored both stx1 and stx2 genes. Twenty five (26.3 %) of the isolates were commensals as no Shiga toxin genes were detected in them. According to Gyles [3], ruminants especially cattle and sheep are the major reservoir of STEC and individual animal could carry more than one serogroups of STEC. During processing, meat derived from infected animals may become contaminated by STEC contained fecal materials if they are mistakenly mixed with it. Barlow and Mellor [27] had reported the presence of STEC in fecal samples of cattle from Australia where a prevalence of 10 % was observed with E.coli O157 accounting for 1.7 % of all the isolates. It is possible for fresh farm produce to be contaminated with STEC where irrigation water or soil treated with farm effluent or manure is used in growing them. A prevalence as high as 33.5 % of STEC in bulk milk has been reported internationally [28]. It is also possible for STEC to survive for a long time in soil applied with manure from cattle and sheep. The possibility of water sources being contaminated by STEC is also very high as fecal materials of animal origin could be washed through storm drains into fresh waterbodies thus posing health challenges to the people who depend on such waterbodies for several uses. WHO [29] had reported waterborne transmission of STEC in both There are increasing concerns about the use of antimicrobial products in food-producing animals and focus has been on human food safety because foods of animal origin are sometimes identified as the vehicles of food borne disease as well as resistant food borne pathogens carrying resistant genetic materials. We profiled the antimicrobial susceptibility of E.coli O157 isolates recovered from commercial dairy cattle that are constantly receiving (tylosin, advocin, ampicillin, tetracycline) antimicrobial agents. We observed a very high level of multiple antimicrobial resistances among the isolates and the most common resistance was to tetracycline. This is not surprising since tetracycline is often used as a first-line antimicrobial in disease prevention and growth promotion in food animals and its widespread use has likely contributed to high rates of resistance [30]. The frequency of tetracycline resistance among the E. coli isolates from the farms that we investigated was 96.8 %, which is within the range of values described in previous reports (68 to 93 %) [31,32]. Genetic profiling of the resistance determinants showed that the resistances were encoded by bla ampC , bla CMY , bla CTXM, bla TEM genes for the ESLBs (extended spectrum β-lactamases), while tetA and strA genes were responsible for tetracycline and streptomycin respectively. High prevalence of CTX-M βlactamase-encoding genes in Enterobacteriaceae has been reported in stool specimens from healthy asymptomatic volunteers in a rural community in Thailand [11]. The fact that bacteria which infect animals could also establish infections in humans poses concerns about the potential spread of the bla-CTX-M genes from food animal products to humans through the food chain. CTX-M β-lactamase has been increasingly reported in E. coli from various food-producing animals worldwide raising a potential threat to public health with the earliest account of CTX-M β-lactamase of food animal origin from Spain, where a CTX-M-14-producing E. coli was isolated from healthy chickens [33]. Since then, E.coli harboring CTX-M β-lactamase encoding gene has been reported from healthy cattle from Japan [34] and Hong Kong [35], and from sick or healthy cattle from France [36,37] and in the United States [38,39]. Similarly, E.coli strains isolated from pigs that have the genetic repertoire to produce CTX-M-β-lactamase have also been reported from Hong Kong [40], China [41], Spain [42], and France [36]. In this study, genetic resistance determinants were however not amplified from some of the isolates and this could be attributed to the fact that the genetic elements targeted in our PCR profiling were not responsible for the observed phenotypic resistances as there are numerous arrays of genes that encodes for resistances to the drugs. Besides, there are many resistance mechanisms like the efflux pump, intrinsic resistance, innate resistant or acquire resistance to one or few classes of antimicrobial agents. Our findings also showed that most of the isolates were susceptible to imipenem, amikacin, kanamycin, the quinolones (norfloxacin, ciprofloxacin and enrofloxxacin) and gentamicin. This finding is curious as regards the susceptibility to the quinolones because the farms that were sampled uses advocin (danofloxacin) which is a synthetic fluoroquinolone in the treatment of respiratory disease in chickens, cattle and pigs and ought to have selected for other quinolone Fig. 3 Agarose gel image of amplicons obtained from PCR with primers designed for bla-ampC resistance gene of E. coli isolates recovered from this study. Lane 1 is molecular size markers (100 bp), lane 2 is negative control (PCR mix without DNA) while lanes 3 to 18 are bla-ampC (198 bp) gene from O157 strains isolated in this study Fig. 2 Agarose gel images of amplicons obtained from PCR with primers designed for strA resistance gene of E. coli isolates recovered from this study. Lane 1 is molecular size markers (100 bp), lane 2 is negative control (PCR mix without DNA) while lanes 3 to 18 are strA (548 bp) gene from O157 strains isolated in this study resistances as they have similar structure and mode of action. To understand the bases of high tetracycline resistance among our isolates, we screened for tet determinants. The predominance of the tet(A) efflux gene observed in our study is similar to that previously documented in coliforms (73 %) of human and animal origins by Marshall [43]. In this study, we observed also a very high resistance to streptomycin among the isolates which is in partial agreement with the reported 53 % resistance of E.coli O157 isolates from feedlots by Rao [44]. Also, Sawant [45] reported a prevalence of 93 % of tetracycline resistance in E.coli of dairy cattle with tet(B) accounting for the resistance determinant in 93 % of their isolates and this is similar to our finding though mediated by different variants of the gene. The fact that bacteria from animals spread to the food products during slaughter and processing has been extensively documented [46][47][48][49]. The detection of E.coli resistant to antibiotic growth promoters (AGPs) in food products derived from animals where AGPs have been used therefore comes as no surprise. Resistant bacteria and active antibiotics, or active metabolites of antibiotics can also spread on farmland with manure. In this study, we isolated toxigenic O157 E.coli strains carrying S tx1 and stx2 genes that were also multi-resistant to many antibiotics some of which are medically important in human medicine. The possibility of these antibiotic resistant strains being shed into the environment and the eventual transmission of the resistance determinants to nonpathogenic environmental bacteria is high. Such transfer of resistance determinants could fuel the spread of antibiotic resistant bacteria (ARB) that could have grave implications on the health of humans and animals thus increasing the burden of disease in the community. There is therefore need for urgent policy formulations on the prudent use of antimicrobials in both human and veterinary medicine as failure in this regards could spell doom in the nearest future. Conclusions In conclusion, this study demonstrated that antimicrobial resistant (AMR) determinants were present in dairy cattle exposed to veterinary antimicrobials for both therapeutic and as growth promoting agents. The overall resistance rate was high and the isolates have the genetic repertoires to survive antimicrobial pressure. Though genetic capacity for Shiga toxins production have been reported among E.coli nonO157 serogroups, we did not profile for any other serogroups apart from O157 in the fecal samples analyzed. The observed phenotypic multiple resistances among the isolates were also genetically confirmed. There were high β-lactam resistances among the isolates and this poses health implications as these are the drugs of choice for the management of numerous Gram negative infections. The results of this study suggest that agricultural activities, specifically antimicrobial use may have a significant impact on AMR evolution in general. More studies with larger sample sizes and more precise AMR genes typing by DNA sequencing and molecular typing of bacterial strains are needed to further throw more light in this regard.

v3-fos

2018-04-03T05:34:18.085Z

2015-01-26T00:00:00.000Z

24731304

s2

The WRKY45-2 WRKY13 WRKY42 Transcriptional Regulatory Cascade Is Required for Rice Resistance to Fungal Pathogen1[OPEN] Three transcription factors form a sequential transcriptional regulatory cascade which is involved in rice response to the infection of Magnaporthe oryzae. Blast caused by fungal Magnaporthe oryzae is a devastating disease of rice (Oryza sativa) worldwide, and this fungus also infects barley (Hordeum vulgare). At least 11 rice WRKY transcription factors have been reported to regulate rice response to M. oryzae either positively or negatively. However, the relationships of these WRKYs in the rice defense signaling pathway against M. oryzae are unknown. Previous studies have revealed that rice WRKY13 (as a transcriptional repressor) and WRKY45-2 enhance resistance to M. oryzae. Here, we show that rice WRKY42, functioning as a transcriptional repressor, suppresses resistance to M. oryzae. WRKY42-RNA interference (RNAi) and WRKY42-overexpressing (oe) plants showed increased resistance and susceptibility to M. oryzae, accompanied by increased or reduced jasmonic acid (JA) content, respectively, compared with wild-type plants. JA pretreatment enhanced the resistance of WRKY42-oe plants to M. oryzae. WRKY13 directly suppressed WRKY42. WRKY45-2, functioning as a transcriptional activator, directly activated WRKY13. In addition, WRKY13 directly suppressed WRKY45-2 by feedback regulation. The WRKY13-RNAi WRKY45-2-oe and WRKY13-oe WRKY42-oe double transgenic lines showed increased susceptibility to M. oryzae compared with WRKY45-2-oe and WRKY13-oe plants, respectively. These results suggest that the three WRKYs form a sequential transcriptional regulatory cascade. WRKY42 may negatively regulate rice response to M. oryzae by suppressing JA signaling-related genes, and WRKY45-2 transcriptionally activates WRKY13, whose encoding protein in turn transcriptionally suppresses WRKY42 to regulate rice resistance to M. oryzae. Plants have evolved sophisticated mechanisms to respond to pathogen invasion. The plant immune system is considered to have four components: pathogen recognition, signal transduction, downstream defense-related gene activation, and cross talk among different signaling pathways (Helliwell and Yang, 2013). Many genes are involved in this defense system and can be divided into two classes, the receptor genes and the defense-responsive genes (Kou and Wang, 2010). The receptor genes include race-specific disease resistance (R) genes and host pattern recognition receptor (PRR) genes. The encoding proteins of defense-responsive genes function downstream of R or PRR proteins as either activators or repressors in defense responses. Plant-pathogen interaction usually results in transcriptional reprogramming of a large number of defense-responsive genes (Eulgem, 2005). This indicates that transcriptional regulators play crucial roles in pathogen-induced defense responses. Different types of transcription factors have been reported to be involved in plant-pathogen interactions, which include auxin/indole acetic acid, basic-domain Leu-zipper, ethylene-responsive element binding, MYB, NAC (for no apical meristem [NAM], Arabidopsis [Arabidopsis thaliana] transcription activation factor [ATAF], and cup-shaped cotyledon [CUC]), Whirly, and WRKY (Singh et al., 2002;Desveaux et al., 2005;Qu and Zhu, 2006;Eulgem and Somssich, 2007;Kazan and Manners, 2009;Puranik et al., 2012). As one of the largest transcription factor families in plants, WRKY transcription factors, which bind to W-box (W) or W-like box-type cis elements or other cis elements, such as the barley (Hordeum vulgare) sugar-responsive element (core sequence: AA/TAA) and rice (Oryza sativa) pathogen response element4 element (TACTGCGCTTAGT; Sun et al., 2003;Cai et al., 2008;Yuan and Wang, 2012), play important roles in plant-pathogen interactions. Loss-offunction and gain-of-function studies have demonstrated that WRKY transcription factors act in a complex signaling network as both positive and negative regulators of disease resistance Li et al., 2004;Zheng et al., 2006;Eulgem and Somssich, 2007;Oh et al., 2008). WRKY proteins function as either transcriptional activators or repressors (Pandey and Somssich, 2009). Interestingly, some WRKY genes carry a set of W or W-like boxes in their own promoters, suggesting the complex interaction of these transcription factors with each other (Eulgem and Somssich, 2007;Qiu et al., 2009;Tao et al., 2009). In addition, some WRKY proteins can bind to their own promoters to regulate their own gene expression (Robatzek and Somssich, 2002;Pandey and Somssich, 2009;Xiao et al., 2013). The autoregulation or cross regulation of different WRKY members may ensure quick and efficient defense signaling amplification (Eulgem and Somssich, 2007). The rice WRKY gene family consists of 98 to 102 paralogs (Ross et al., 2007). Systematic analyses of WRKY gene expression in rice-pathogen interactions have revealed that many WRKY genes are rapidly induced or repressed upon pathogen infection, suggesting that these WRKY genes may contribute to the regulation of rice response to pathogen infection (Ryu et al., 2006;Bagnaresi et al., 2012;Wei et al., 2013). In addition, 13 rice WRKY genes from 12 loci have been characterized to be involved in rice-pathogen interactions. Among the 13 genes, 11 are involved in the interaction of rice with Magnaporthe oryzae, which causes blast diseases not only in rice but also in barley . Among the 11 genes, WRKY13, WRKY22, WRKY30, WRKY45-1 (named WRKY45 in Shimono et al., 2007), WRKY45-2, WRKY47, WRKY53, WRKY55/WRKY31, and WRKY104/WRKY89 positively regulate rice resistance to M. oryzae, whereas WRKY28 and WRKY76 negatively regulate rice resistance to M. oryzae (Cheng and Wang, 2014). Rice WRKY13 is a quantitative trait locus that also confers resistance to Xanthomonas oryzae pv oryzae (Xoo), which causes bacterial blight disease (Qiu et al., 2007Hu et al., 2008). In addition, WRKY45-2 also promotes rice resistance to Xoo and X. oryzae pv oryzicola (Xoc), which causes bacterial streak disease (Tao et al., 2009). These results suggest that WRKYs play important roles in rice response to M. oryzae, and some of these WRKYs are also involved in rice response to the infection of other pathogens. However, the relationship of different WRKYs in the rice defense response to the same pathogen or the positions of different WRKYs in the rice defense signaling pathway to the same pathogen remain largely unknown. In this respect, WRKY13 is relatively more intensively studied than other characterized WRKY genes involved in rice-pathogen interactions. WRKY13 is a transcriptional repressor (Xiao et al., 2013). It can bind to the promoters of both WRKY45-1/WRKY45 and WRKY45-2, which are alleles encoding proteins with a 10-amino acid difference, in the rice-Xoo interaction, suggesting that WRKY13 may regulate the functions of the WRKY45 alleles in defense signaling (Tao et al., 2009;Xiao et al., 2013). WRKY45-1/ WRKY45 is a transcriptional activator (Shimono et al., 2007), but the transcription function of WRKY45-2 is unknown. Activation of WRKY13 transcriptionally influenced a set of WRKY genes, including WRKY42, in rice resistance to Xoo; in addition, the WRKY13 protein binds to the WRKY42 promoter in yeast (Saccharomyces cerevisiae) cells . To elucidate whether WRKY42 is a player in rice disease resistance, and to elucidate its relationship with WRKY13 and WRKY45-2 in defense signaling, we modulated WRKY42 expression and generated WRKY13 WRKY42 and WRKY13 WRKY45-2 double transgenic lines. Our results show that WRKY42 negatively regulates rice resistance to M. oryzae by functioning downstream of WRKY13 and WRKY45-2 in the defense signaling pathway. WRKY45-2 WRKY13 WRKY42 forms a transcriptional regulatory cascade to regulate rice-M. oryzae interaction. Fungal and Bacterial Pathogen Infection Influenced WRKY42 Expression To study the potential function of WRKY42 in the rice defense response to the infection of fungal pathogen M. oryzae and bacterial pathogen Xoo, we first examined the expression patterns of WRKY42 in two pairs of resistant and susceptible rice lines, with each pair having the same genetic background. The first pair of rice lines was C101A51 and CO39. C101A51 carrying the R gene Pi2 was resistant (disease index 5.6 6 3.9) to M. oryzae isolate Enshi2-2 (N2-2), whereas CO39 was highly susceptible to N2-2 (disease index 80.6 6 9.6). N2-2 infection influenced WRKY42 expression (Fig. 1A). Its expression was rapidly induced in both resistant and susceptible plants at 1 h after inoculation. However, the expression level of WRKY42 was approximately 2-fold higher in susceptible CO39 than in resistant C101A51 at 1 h after inoculation. Furthermore, WRKY42 expression showed a second induction at 8 h after inoculation in C101A51 but not in CO39, which might be due to the R gene Pi2. We further examined WRKY42 expression in susceptible (disease index 43.3 6 4.6) japonica rice var Mudanjiang 8, which was used as a recipient of transgenes in our transformation experiments. WRKY42 expression reached the highest level at 8 h after inoculation with N2-2, whereas mock inoculation (control) with water did not affect WRKY42 expression (Fig. 1A). The second pair of rice lines was Rb49 and Mudanjiang 8. Rb49 carrying the PRR-like gene Xa3/Xa26 is resistant to Xoo strain PXO61, and Mudanjiang 8 is susceptible to PXO61 (Sun et al., 2004;Cao et al., 2007). PXO61 inoculation induced WRKY42 expression in both resistant and susceptible rice lines (Fig. 1B). However, the expression level of WRKY42 was obviously higher in susceptible Mudanjiang 8 than in resistant Rb49. The rapidly induced expression of WRKY42 in rice lines suggests that this gene may be involved in rice-pathogen interaction. Modulating WRKY42 Expression Influenced Rice Resistance to the Infection of M. oryzae But Not Xoo and Xoc To explore the role of WRKY42 in rice-pathogen interactions, we modulated its expression. Twenty-seven WRKY42-overexpressing (oe; D153UM; also named WRKY42-oe in the following text) and 29 WRKY42suppressing (D153RM or RNA interference [RNAi]; also named WRKY42-RNAi in the following text) independent transformants were generated in the genetic background of rice var Mudanjiang 8. The homozygous WRKY42-oe (WRKY42-oe+) and WRKY42-RNAi (WRKY42-RNAi+) lines and wild-type siblings (WRKY42-oe2 or WRKY42-RNAi2) segregated from the same T0 plants were analyzed. The WRKY42-oe plants showed increased susceptibility (P , 0.01) to M. oryzae, with disease indexes ranging from 64.6 6 6.2 to 68.9 6 4.7 (high susceptibility) compared with 37.8 6 6.8 (moderate susceptibility) for wild-type Mudanjiang 8 ( Fig. 2A). The increased susceptibility of WRKY42-oe+ plants was associated with the overexpression of WRKY42 ( Fig. 2A). The WRKY42-oe2 plants had a level of susceptibility (disease indexes ranged from 40.4 6 5.0 to 41.1 6 2.9; moderate susceptibility) to M. oryzae similar to that of wild-type plants. In contrast, the WRKY42-RNAi+ plants showed enhanced resistance (P , 0.01) to M. oryzae, with disease indexes ranging from 27.3 6 5.0 to 28.7 6 4.0 (moderate resistance) compared with 42.6 6 6.0 (moderate susceptibility) for wild-type Mudanjiang 8 (Fig. 2B). The enhanced resistance of the WRKY42-RNAi+ plants was associated with the suppressed expression of WRKY42 (Fig. 2B). Conversely, the WRKY42-RNAi2 plants had a level of susceptibility (disease indexes ranged from 41.2 6 4.4 to 44.9 6 6.9; moderate susceptibility) to M. oryzae similar to that of wild-type plants. The fungal growth rate in WRKY42-oe+ plants was significantly higher (P , 0.01) than that in wild-type plants measured at 6 d after inoculation, whereas the fungal growth rate in WRKY42-RNAi+ plants was significantly lower (P , 0.01; Fig. 2C). The growth rate of M. oryzae in wild-type siblings showed no significant difference compared with the wild type. Although Xoo infection influenced the expression of WRKY42, the WRKY42-oe and WRKY42-RNAi plants showed no obvious difference in disease level when compared with wild-type plants after Xoo infection (Supplemental Figs. S1 and S2). These transgenic plants also showed no difference when compared with wildtype plants after Xoc infection (Supplemental Fig. S3). These results suggest that WRKY42 negatively regulates rice resistance to M. oryzae but not Xoo or Xoc. WRKY42 Affected Jasmonic Acid-Dependent Signaling in Rice after M. oryzae Infection Jasmonic acid (JA) and salicylic acid (SA) are important defense signaling-related molecules. To examine whether WRKY42-involved rice-M. oryzae interaction was related to these phytohormones, we quantified JA and SA in rice plants. The JA contents in different rice plants appeared to be negatively associated with the transcript levels of WRKY42 and with the increased susceptibility to M. oryzae infection. Modulating WRKY42 expression influenced the level of JA but not SA (Fig. 3A). M. oryzae infection reduced JA levels in both wild-type and WRKY42-transgenic plants. However, compared with wild-type plants, WRKY42-oe plants showed reduced JA levels and WRKY-RNAi plants showed increased JA levels (Fig. 3A). Consistent with the JA content, the expression of Allene Oxide Synthase2 (AOS2), encoding an allene oxide synthase that is a key enzyme in the JA biosynthetic pathway in rice (Mei et al., 2006), was slightly suppressed in WRKY42-oe plants but was increased in WRKY42-RNAi plants compared with the wild type ( Fig. 3A). The expression of JAZ8, encoding a jasmonate zinc-finger protein expressed in inflorescence meristemdomain (JAZ) protein functioning in JA-dependent signaling Yamada et al., 2012), showed a pattern similar to that of AOS2 in WRKY42-transgenic plants. These results suggested that the negative regulation of rice resistance to M. oryzae by WRKY42 may be at least partly related to the decreased JA level and JAdependent signaling. To further examine the inference that WRKY42suppressed resistance was associated with reduced JA level, we treated WRKY42-oe plants with exogenous application of JA. Studies have demonstrated that exogenous application of JA enhanced rice resistance to M. oryzae (Mei et al., 2006;Riemann et al., 2013). Consistent with previous reports, JA treatment enhanced resistance to M. oryzae not only in wild-type plants but also in WRKY42-oe plants (Fig. 3B). However, overexpressing WRKY42 compromised the effect of JA on rice resistance to M. oryzae. The JA-treated WRKY42-oe plants showed 26% reduced (P , 0.01) disease symptoms, whereas the JA-treated wild-type plants only showed 22% reduced disease symptoms. The fungal growth rates Figure 3. The negative regulation of rice resistance to M. oryzae by WRKY42 was associated with suppressed JA signaling. Plants were inoculated with M. oryzae isolate N2-2 without treatment (ck) or after being treated with 200 mM JA or the solution not containing JA (mock) at the three-to four-leaf stage. Bars represent mean (three replicates for hormone concentration and gene expression and 12-15 plants for disease index) 6 SD. The letters a and b indicate a significant difference was detected between inoculated and noninoculated ck plants or JA-and mock-treated plants at P , 0.05 and P , 0.01, respectively. Asterisks indicate a significant difference was detected between wild-type and WRKY42-transgenic plants subjected to the same treatment at **P , 0.01 and *P , 0.05. A, WRKY42-transgenic plants showed changes in levels of JA and JA synthesis-related and signaling-related genes but not in the level of SA. B, Exogenous application of JA enhanced rice resistance to M. oryzae. in JA-treated or mock-treated plants were further examined in rice leaves. The results showed that the fungal growth rate was significantly decreased (P , 0.01) in both WRKY42-oe+ and wild-type plants after JA treatment. The JA-treated WRKY42-oe plants showed 85% reduced (P , 0.05) fungal growth rate, whereas the JA-treated wild-type plants only showed 76% reduced fungal growth rate. These results suggested that overexpression of WRKY42 increased susceptibility to M. oryzae, which was associated with suppressed JA signaling. WRKY42 Had the Characteristics of a Transcription Factor The predicted encoding protein of WRKY42 consists of 253 amino acids and putatively harbors a nuclear localization signal, a WRKY domain, and zinc finger motif, which are the characteristics of a typical type II WKRY protein (Supplemental Fig. S4; Ross et al., 2007). To determine whether WRKY42 functioned as a transcription factor, we first analyzed its subcellular localization. The fusion genes of yellow fluorescent protein (YFP)-WRKY42 and cyan fluorescent protein (CFP)-Ghd7 (for Grain number, plant height, and heading date7) were cotransformed into rice protoplasts. Rice Ghd7 has been used as a marker since it was reported as a transcription factor localized in the nucleus (Xue et al., 2008). The yellow fluorescence signal produced by YFP-WRKY42 overlapped with the cyan fluorescence signal produced by CFP-Ghd7 (Supplemental Fig. S5), suggesting that WRKY42 localizes in the nucleus. In the transactivation activity assay, the complete WRKY42, the N-terminal part of WRKY42 (harboring the nuclear localization signal), and the C-terminal part of WRKY42 (harboring the WRKY domain and the zinc finger motif) did not show activity of transactivation in yeast cells as compared with rice transcription activator basic leucine zipper23 (OsbZIP23), which was the positive control (Supplemental Fig. S6; Xiang et al., 2008). Because some WRKY transcription factors, such as rice WRKY13 (Xiao et al., 2013), function as transcription repressors, we further assayed the transcriptional activity of WRKY42 using the galectin4 DNA binding domain (GAL4DB)/upstream activating sequence (UAS)/ luciferase (LUC) and GAL4DB-VP16 (Herpes simplex virus activation domain)/UAS/LUC transient expression systems in rice protoplasts. The relative activity of reporter LUC in the presence of GAL4DB-VP16 WRKY42 was significantly reduced (P , 0.05) compared with that of control (existence of GAL4DB-VP16 effector; Fig. 4). The LUC activity in the presence of the transcriptional repressor control, GAL4DB-VP16 WRKY13, was also significantly (P , 0.05) reduced compared with control. The LUC activity in the presence of GAL4DB WRKY42 and GAL4DB WRKY13 was also significantly reduced (P , 0.01), whereas the LUC activity in the presence of the positive control GAL4DB bZIP23, expressing a transcriptional activator, was significantly increased (P , 0.05; Fig. 4). These results suggest that WRKY42 is a transcriptional repressor. Considering the results presented in Figure 3A, WRKY42 may suppress rice resistance to M. oryzae by directly or indirectly suppressing JA synthesis-related and/or signaling-related genes. WRKY13 Bound to WRKY42 Promoter in Rice and Suppressed WRKY42 Expression Rice WRKY13, as a transcriptional repressor, confers resistance to both M. oryzae and Xoo (Qiu et al., 2007Xiao et al., 2013). WRKY13 interacts with the promoter of WRKY42 in yeast cells . To further characterize the relationship of WRKY13 protein and WRKY42 expression, we performed a series of analyses. First, the WRKY13 and WRKY42 promoter were transiently expressed in rice protoplasts using a chimeric reporter/effector assay. In this assay, the effector plasmid, which carried WRKY13 driven by the Cauliflower mosaic virus 35S promoter, was cotransformed with the reporter plasmid, which carried the LUC reporter gene driven by the full-length or truncated WRKY42 promoter into protoplasts (Fig. 5A). Compared with the LUC activity in protoplasts carrying control effector YFP and LUC reporter gene, the LUC activities in the protoplasts carrying WRKY13 effector and LUC reporter gene driven by the P WRKY42 , P WRKY42-530 , or P WRKY42-336 promoter were reduced (P , 0.05) by approximately 3-, 8-, and 9-fold, respectively. These results suggest that WRKY13 can suppress LUC expression by interacting with the fulllength or truncated WRKY42 promoters. . WRKY42 and WRKY45-2 functioned as a transcriptional repressor and activator, respectively. The relative LUC activities in rice protoplasts after transformation with reporter plasmid and different effector plasmids were presented. bZIP23, which is a transcriptional activator, and WRKY13, which is a transcriptional repressor, were used as the positive control. REN was used as the internal control to standardize the difference of the transformation ratio. Data represent mean (three biological repeats with each repeat having three replicates) 6 SD. The letters a and b indicate a significant difference was detected between the effector and empty vector (GAL4DB-VP16 or GAL4DB) at P , 0.05 and P , 0.01, respectively. Second, to ascertain whether stably expressed WRKY13 had an effect on the expression of WRKY42, we generated WRKY13-RNAi plants in the genetic background of Mudanjiang 8. The WRKY13-oe plants, which showed enhanced resistance to M. oryzae and Xoo, also had the Mudanjiang 8 background (Qiu et al., 2007). The WRKY13-RNAi plants of both the T0 and T1 generation showed increased susceptibility to Xoo compared with wild-type plants (Supplemental Fig. S7), which is similar to the WRKY13-RNAi plants with the genetic background of an indica var Minghui 63 . The increased susceptibility of these WRKY13-RNAi plants to Xoo was associated with reduced WRKY13 transcripts (Supplemental Fig. S7). The WRKY13-RNAi plants also showed significantly increased (P , 0.05) susceptibility to M. oryzae as compared with wild-type Mudanjiang 8, and the increased susceptibility of these plants to M. oryzae was associated with suppressed expression of WRKY13 (Supplemental Fig. S8). The WRKY13-RNAi plants showed increased expression of WRKY42, whereas the WRKY13-oe plants showed reduced expression of WRKY42 (Fig. 6A). These results suggest that WRKY13 suppresses WRKY42 in plants (Fig. 6A). Finally, to determine whether WRKY13 protein directly interacted with WRKY42 promoter in rice during ricepathogen interaction, we performed chromatin immunoprecipitation (ChIP) assay using anti-WRKY13 antibody (Supplemental Fig. S9). Three segments of the WRKY42 promoter, which harbor W and W-like boxes, were analyzed ( Fig. 6B; Supplemental Fig. S10). After immunoprecipitation (IP) with anti-WRKY13 antibody, significant enrichment of the first (N1F/N1R) and second (N2F/ N2R) DNA segments from untreated, Xoo-inoculated, and M. oryzae-inoculated samples was detected by quantitative PCR (qPCR) as compared with the corresponding controls (samples after IP without anti-WRKY13 antibody; Fig. 6B). Compared with other treated samples, there was more enrichment of the N1F/N1R than of the N2F/N2R segments in both pathogen-infected and control samples. However, the enrichment levels of the N1F/N1R in M. oryzae-infected and control samples were similar, whereas higher enrichment of the N2F/N2R was detected in the M. oryzae-infected sample compared with the control sample. No significant or obvious enrichment of the third DNA segment (N3F/N3R) was detected, although it also harbors W-like boxes ( Fig. 6B; Supplemental Fig. S10). Together, these results suggest that WRKY13 may suppress WRKY42 expression by preferentially binding to certain regions of the WRKY42 promoter even without pathogen infection, and WRKY13 may further suppress WRKY42 by binding to N2F/N2R in the rice-M. oryzae interaction. WRKY13, WRKY42, and WRKY45-2 Functioned in a Complex Regulation Loop A previous study revealed that suppressing WRKY13 induces WRKY45-2 expression, whereas transcriptionally modulating WRKY45-2 influences WRKY13 expression Tao et al., 2009). To further ascertain the transcriptional regulation relationship of WRKY13, WRKY42, and WRKY45-2, we first examined the feature of WRKY45-2 as a transcription factor using the GAL4DB/UAS/LUC transient expression system in rice protoplasts. The activity of reporter LUC in the presence of GAL4DB WRKY45-2 significantly increased (P , 0.01) compared with that of control (GAL4DB effector; Fig. 4). The LUC activity in the presence of the transcriptional activator control, GAL4DB bZIP23, also significantly increased (P , 0.05) compared with control. These results suggest that WRKY45-2 is a transcriptional activator. In the chimeric reporter/effector assay, LUC activity in rice protoplasts carrying the WRKY13 effector and Figure 5. Transient transcriptional regulatory activities of WRKY42, WRKY13, and WRKY45-2 analyzed by the chimeric reporter/effector assay in rice protoplasts. REN was used as the internal control to standardize the difference of the transformation ratio. Data represent mean (three biological repeats with each repeat having three replicates) 6 SD. The letters a and b indicate a significant difference was detected between effector WRKY13, WRKY42, or WRKY45-2 and control effector YFP at P , 0.05 and P , 0.01, respectively. A, WRKY13 directly suppressed LUC driven by full-length or truncated WRKY42 promoters. B, WRKY13 and WRKY45 regulated each other's promoter, and WRKY42 and WRKY45-2 regulated their own promoters. LUC gene driven by the WRKY45-2 promoter was significantly reduced (P , 0.05) compared with that of the control protoplasts carrying the YFP effector and LUC gene driven by the WRKY45-2 promoter (Fig. 5B). The LUC activity in the protoplasts carrying the WRKY45-2 effector and LUC gene driven by the WRKY13 promoter significantly increased (P , 0.01) compared with the control. Although WRKY13 suppressed WRKY42 (Fig. 5A), the LUC activities in the protoplasts carrying the WRKY45-2 effector and LUC gene driven by the WRKY42 promoter, carrying the WRKY42 effector and LUC gene driven by the WRKY13 promoter, or carrying the WRKY42 effector and LUC gene driven by the WRKY45-2 promoter showed no significant difference compared with corresponding controls (Fig. 5B). Together, these results suggest that WRKY45-2 and WRKY13 may regulate each other in a forward feedback loop; WRKY13 may directly suppress WRKY45-2, whereas WRKY45-2 may directly activate WRKY13. However, it appears that WRKY42 does not directly regulate the expression of WRKY13 and WRKY45-2, and WRKY45-2 does not directly regulate WRKY42 expression. In addition to transcriptional regulation of WRKY42 and WRKY45-2, WRKY13 has the ability to suppress its own gene (Cai et al., 2008;Xiao et al., 2013). In the chimeric reporter/effector transient expression system, the LUC activity in rice protoplasts carrying the WRKY45-2 effector and LUC gene driven by the WRKY45-2 promoter significantly increased compared with the control protoplasts carrying the YFP effector and LUC gene driven by the WRKY45-2 promoter (Fig. 5B). The LUC activity in rice protoplasts carrying the WRKY42 effector and LUC gene driven by the WRKY42 promoter was significantly suppressed compared with control. These results suggest that WRKY45-2 and WRKY42 can also regulate their own gene expression, which may be important for the balance of defense signaling transduction. To analyze the relationship of WRKY13, WRKY42, and WRKY45-2 in the defense signaling pathway against M. oryzae, we generated double transgenic lines by crossing the transgenic lines of the three WRKY genes. The WRKY13-oe WRKY42-oe double transgenic line (disease index 64.8 6 6.4) was highly susceptible to M. oryzae compared with WRKY13-oe plants (disease index 19.4 6 4.4) but showed a similar level of susceptibility to M. oryzae as the WRKY42-oe plants (disease index 67.6 6 9.1; Fig. 7A). The WRKY13-RNAi WRKY45-2-oe double transgenic line (disease index 29.2 6 8.9) showed slightly increased susceptibility to M. oryzae compared with the WRKY45-2-oe plants (disease index 24.4 6 9.8) but showed reduced susceptibility to M. oryzae compared with the WRKY13-RNAi plants (disease index 47.2 6 9.5; Fig. 7B). Furthermore, transcriptional modulating of WRKY42 did not influence WRKY13 expression (Supplemental Fig. S11). Together, these results suggest that WRKY42, WRKY13, and WRKY45-2 may function in the same defense transduction pathway in which WRKY45-2 functions in the upstream, followed by WRKY13 and WRKY42, leading to resistance to M. oryzae. DISCUSSION A large number of WRKYs have been reported to regulate rice response to M. oryzae (Cheng and Wang, Figure 6. The interaction of WRKY13 and WRKY42. Bars represent mean (three replicates) 6 SD. A, Transcriptionally modulating WRKY13 influenced WRKY42 expression. The letters a and b indicate that a significant difference between WRKY13-transgenic and wild-type plants was detected at P , 0.05 and P , 0.01, respectively. B, WRKY13 protein bound to the promoter regions of WRKY42 analyzed by ChIP assay. Samples were from rice var Mudanjiang 8 that was untreated (ck), at 1 d after inoculation with Xoo, or at 1 d after inoculation with M. oryzae. The qPCR was conducted before immunoprecipitation (input), after IP without anti-WRKY13 antibody (-), or after IP with anti-WRKY13 antibody (+). The presented percentage of PCR product from IP is relative (IP/input %) to that from input. The letters a and b indicate that a significant difference was detected between the PCR products from IP with and without anti-WRKY13 antibody at P , 0.05 and P , 0.01, respectively. ATG, Start codon. 2014). However, the relationship of these WRKYs in rice resistance to the same disease is largely unknown. The present results add another gene, WRKY42, to the list of WRKY defense regulators in rice-M. oryzae interaction. Furthermore, the present results suggest that the WRKY42 gene with the previously characterized WRKY13 and WRKY45-2 form a WRKY transcriptional regulatory cascade in rice response to M. oryzae invasion. WRKY42, as a Transcriptional Repressor, Negatively Regulates Rice Disease Resistance by Suppressing JA-Dependent Signaling WRKY proteins can function as either transcriptional activators or transcriptional repressors (Pandey and Somssich, 2009). Among the 13 characterized rice WRKY proteins involved in rice-pathogen interactions, only six have been examined for their transcriptional activity in rice cells (Cheng and Wang, 2014). WRKY45-1/WRKY45 and WRKY53 show transactivation activity (Chujo et al., 2007;Shimono et al., 2007), whereas WRKY13, WRKY28, WRKY71, and WRKY76 are transcriptional repressors (Chujo et al., 2008Xiao et al., 2013;Yokotani et al., 2013). Interestingly, the four repressors belong to group II WRKYs, which harbor one WRKY motif and one C2H2-type zinc-finger motif, and WRKY53 and WRKY45-1/WRKY45 belong to group I WRKYs, which harbor two WRKY motifs and two C2H2-type zinc-finger motifs, and group III WRKYs, which harbor one WRKY motif and one C2HC-type zinc-finger motif, respectively, based on the classification of rice WRKY proteins (Wu et al., 2005). WRKY42 also belongs to group II. The present results suggest that WRKY42 appears to be a transcriptional repressor. This inference is proposed based on the evidence that WRKY42 did not display transactivation activity in yeast cells and rice protoplasts but expressed transcriptional repressor activity in rice protoplasts ( Fig. 4; Supplemental Fig. S6). Thus, future studies are needed to determine whether all rice group II WRKYs involved in rice-pathogen interactions function as transcriptional repressors. Rice resistance to M. oryzae is dependent on JA or SA. Increasing the expression of AOS2, which is required for JA synthesis, enhanced the rice resistance to M. oryzae (Mei et al., 2006). Mutation of Allene Oxide Cyclase, which is also a JA synthesis-related gene, increased rice susceptibility to M. oryzae (Riemann et al., 2013). Benzothiadiazole, the analog of SA, serves as a defense activator and enhances rice resistance to M. oryzae (Shimono et al., 2007). JA-and SA-dependent defense signaling frequently interact with each other either synergistically or antagonistically (Durrant and Dong, 2004). However, the present results showed that WRKY42-regulated rice-M. oryzae interaction was associated with reduced JA levels but not with SA levels (Fig. 3A). The reduced JA level was related to the suppressed expression of JA synthesis-related and signaling-related genes. Exogenous application of JA had a larger effect on WRKY42-oe plants than on the wild-type plants in reducing the susceptibility to M. oryzae (Fig. 3B). These results suggest that WRKY42 may negatively regulate rice resistance to M. oryzae by direct or indirect suppression of the JA synthesis-related genes, which in turn suppresses JAdependent defense signaling. WRKY42 Functions in a WRKY Transcriptional Regulatory Cascade in Rice Resistance to M. oryzae A set of WRKY proteins is frequently involved in plant resistance to a pathogen in a given species (Pandey and Somssich, 2009). Modulating the expression of a WRKY gene frequently influences the expression of other WRKY genes. For example, both AtWRKY11 and AtWRKY17 negatively regulate basal resistance in Arabidopsis (Arabidopsis thaliana), and the two WRKYs also influence the expression of each other (Journot-Catalino et al., 2006). Rice WRKY13 positively regulates a broadspectrum resistance to both bacterial and fungal pathogens (Qiu et al., 2007). Activation of WRKY13 influenced the expression of at least nine WRKY genes in rice response to Xoo infection . However, the relationship of different WRKYs in the plant defense response to the same pathogen remains largely unknown. Only a few studies revealed that a WRKY gene directly regulates the expression of another WRKY gene in plant-pathogen interactions. Arabidopsis AtWRKY46 is involved in disease resistance, and two defense-related WRKYs, AtWRKY8 an AtWRKY48, directly activate AtWRKY46 (Hu et al., 2012;Gao et al., 2013). Rice WRKY13 directly suppresses WRKY45-1/WRKY45, which negatively regulates rice resistance to Xoo, in the rice-Xoo interaction (Tao et al., 2009;Xiao et al., 2013). The present results suggest that WRKY13 directly suppresses WRKY42, which functions downstream of WRKY13 in the rice defense signaling pathway to M. oryzae. This inference is supported by the following evidence. First, WRKY13 suppressed the function of WRKY42 promoter in rice protoplasts (Fig. 5A). Second, transcriptional activation of WRKY13 suppressed the WRKY42 expression, and suppressing WRKY13 increased WRKY42 expression, whereas transcriptional modulation of WRKY42 did not influence WRKY13 expression ( Fig. 6A; Supplemental Fig. S11). Third, WRKY13 selectively bound to two regions that harbor W and W-like boxes known for WRKY protein binding in the WRKY42 promoter in rice (Fig. 6B). Finally, the WRKY13 WRKY42 double transgenic line showed the phenotype of the WRKY42 transgenic line in the rice response to M. oryzae (Fig. 7A). This inference is consistent with the fact that WRKY42 negatively regulates rice resistance only to M. oryzae, but WRKY13 positively regulates rice resistance to both M. oryzae and Xoo (Qiu et al., 2007). However, WRKY13 functions in association with activation of SAdependent signaling and suppression of JA-dependent signaling (Qiu et al., 2007, whereas WRKY42 only suppresses JA signaling. This inconsistency may be due to the fact that both SA-and JA-dependent signaling are involved in rice resistance to M. oryzae (Mei et al., 2006;Shimono et al., 2007;Riemann et al., 2013). The SA-and JA-dependent defense pathways may function antagonistically; however, the two pathways may have crisscross roles at different processes in rice resistance to M. oryzae just like in rice resistance to Xoo . A previous study has shown that WRKY13 binds to the promoter of WRKY45-2, which confers broad-spectrum resistance to both bacterial and fungal pathogens (Tao et al., 2009). The present results suggest that WRKY45-2 can also bind to the promoter of WRKY13 (Supplemental Fig. S12). WRKY45-2 is a transcriptional activator that can directly promote the expression of the reporter gene driven by the WRKY13 promoter (Figs. 4 and 5B). In addition, WRKY13 can directly suppress the expression of the reporter gene driven by the WRKY45-2 promoter (Fig. 5B). These results suggest that WRKY45-2 and WRKY13 mutually regulate the expression of each other's gene. The WRKY13-RNAi WRKY45-2-oe double transgenic line showed increased susceptibility to M. oryzae compared with WRKY45-2-oe plants, but showed enhanced resistance to M. oryzae compared with WRKY13-RNAi plants (Fig. 7B). WRKY45-2 confers resistance to three pathogen species, M. oryzae, Xoc, and Xoo, and WRKY13 only confers resistance to M. oryzae and Xoo (Qiu et al., 2007;Tao et al., 2009). Furthermore, the present results have revealed that WRKY45-2 is a transcriptional activator, but WRKY13 is a transcriptional repressor (Xiao et al., 2013). These results suggest that WRKY45-2 may function upstream of WRKY13 in rice resistance to M. oryzae. WRKY45-2 functions in association with activation of JA-but not SA-dependent signaling (Tao et al., 2009). The previous studies and present results further suggest that the SA-and JA-dependent signaling may have crisscross roles in rice resistance to M. oryzae. However, WRKY45-2-mediated resistance to M. oryzae may also require a subpathway that is WRKY13 independent. This hypothesis is supported by evidence that the WRKY13-RNAi WRKY45-2-oe double transgenic line showed a disease index between those of WRKY13-RNAi and WRKY42-2-oe plants (Fig. 7B). Furthermore, WRKY13 may regulate WRKY45-2 by negative feedback regulation. CONCLUSION The present results suggest that WRKY45-2, WRKY13, and WRKY42 form a sequential transcriptional regulatory cascade, which is required in rice resistance to M. oryzae (Fig. 8). The function of this cascade may be balanced via the negative feedback regulation of WRKY45-2 by WRKY13, and it also may be balanced by the autoregulation of the three WRKY proteins on their own genes. Because WRKY45-2 and WRKY13 are negatively involved in the regulation of rice response to abiotic stresses Tao et al., 2011), this cascade may provide rapid amplification of pathogen-induced defense signaling and balancing adaptation to different environmental stimuli by reprogramming the transcriptome. Rice Materials and Treatment Indica rice (Oryza sativa ssp. indica) C101A51 and CO39 are near-isogenic lines with the genetic background of CO39 (Jiang and Wang, 2002). Transgenic line Rb49 carries the PRR-like gene Xa3/Xa26 driven by its native promoter and has the genetic background of japonica rice ssp. japonica var Mudanjiang 8 (Sun et al., 2004;Xiang et al., 2006). Transgenic lines WRKY13-oe (D11UM7-2) and WRKY45-2-oe (D114UM4) were also used in this study. WRKY13 and WRKY45-2 genes from indica rice var Minghui 63 driven by the maize (Zea mays) ubiquitin promoter were separately transformed into Mudanjiang 8 to generate WRKY13-oe and WRKY45-2-oe plants (Qiu et al., 2007;Tao et al., 2009). Plant treatment with JA was performed as reported previously (Ke et al., 2014). Rice plants at the three-to four-leaf stage were sprayed with 200 mM JA in 0.1% (v/v) methanol and 0.015% (v/v) Tween 20 or 0.1% (v/v) methanol and 0.015% (v/v) Tween 20 (mock treatment). The sprayed plants were kept in sealed plastic shade for 2 d before further treatment. Vector Construction and Rice Transformation To make an overexpressing construct of rice WRKY42, the genomic DNA fragment harboring the complete coding region (1,187 nucleotides) of this gene was obtained from rice var Minghui 63 by PCR amplification using primers 153U3F and 153U3R (Supplemental Table S1), and the PCR product was digested with KpnI and inserted into vector pU1301, which contained a maize ubiquitin gene promoter in the multiple cloning site . To construct the RNAi vectors for WRKY42 and WRKY13, the 313-nucleotide fragment of WRKY42 and the 366-nucleotide fragment of WRKY13 amplified from the complementary DNA (cDNA) of rice var Mudanjiang 8 using primer pairs 153R4F/153R4R and WRKY1322F/WRKY1322R (Supplemental Table S1), respectively, were separately inserted into the pDS1301 vector (Yuan et al., 2007). These constructs were transferred into Agrobacterium tumefaciens strain EHA105. A. tumefaciens-mediated transformation was performed using the callus derived from mature embryos of rice var Mudanjiang 8 (Lin and Zhang, 2005). Pathogen Inoculation To evaluate fungal blast disease, rice plants were inoculated with isolate N2-2 of Magnaporthe oryzae at the three-to four-leaf stage by the spraying method (Chen et al., 2003). The inoculated plants were first treated in the growth chamber at 28°C in darkness for 36 h, and then under alternating light and darkness every 12 h with 95% humidity. Disease was scored for individual plants using a scale rating system (scale of 0-9) at 7 d after inoculation (Tao et al., 2009). Disease index was calculated with the individual leaf ratings using the following formula: disease index = [sum of numerical ratings from all leaves/(number of leaves assessed 3 maximum lesion rating)] 3 100. The average disease index of all plants was presented. A disease index of $0 and #5 indicates high resistance, .5 and #15 indicates resistance, .15 and #30 indicates moderate resistance, .30 and #45 indicates moderate susceptibility, .45 and #60 indicates susceptibility, and .60 indicates high susceptibility. The fungal growth rate in rice leaves was determined by detecting the ratio of the Pot gene of M. oryzae and the ubiquitin gene of rice via qPCR using primer pairs PotF and PotR (Supplemental Table S1; Duan et al., 2014). The PCR product level of the rice ubiquitin gene amplified using primers UbiqF and UbiqR (Supplemental Table S1) was used to standardize the DNA sample for analyzing fungal growth rate. To examine bacterial blight disease, rice plants were inoculated with Xoo strain PXO61 by the leaf-clipping method . Disease was scored by measuring the percentage of the lesion area (lesion length/leaf length) at 2 weeks after inoculation. To examine bacterial streak disease, rice plants were inoculated with Xoc strain RH3 by using a penetration method (Ke et al., 2014). Disease was scored by measuring the lesion length at 2 weeks after inoculation. Gene Expression For quantitative reverse-transcriptase PCR, total RNA was isolated from rice leaves. To examine the influence of M. oryzae infection on gene expression, whole leaves were used for RNA isolation. To examine the influence of bacterial blight infection on gene expression, 3-cm leaf fragments next to bacterial infection sites were used for RNA isolation. The qPCR was performed as described previously using primers listed in Supplemental Table S2 (Qiu et al., 2007). The expression level of the rice actin gene was used to standardize the RNA sample for each quantitative reverse-transcriptase PCR. Because the gene primers used in this study have different amplification efficiency, the expression level of each gene in transgenic or treated plants was calculated relative to that in wild-type or untreated plants. Database Search The known motif and domain of the WRKY42 protein were determined by motif scanning (http://hits.isb-sib.ch). Hormone Quantification Whole leaves were used for phytohormone quantification. Samples were prepared, and JA and SA were quantified using an ultrafast liquid chromatography/ electrospray ionization/tandem mass spectrometry system as described previously . Protein Subcellular Localization To determine the subcellular localization of WRKY42, the full coding region of WRKY42 was amplified from the cDNA of rice var Mudanjiang 8 using primers 1536F and 1536R (Supplemental Table S1) and fused to the coding region of YFP driven by the 35S promoter in the pM999 vector (kindly provided by Jian Xu, Huazhong Agricultural University, Wuhan, China). Rice Ghd7 has been used as a marker since it was reported as a transcription factor localized in the nucleus (Xue et al., 2008). 35S:YFP-WRKY42 and 35S:CFP-Ghd7, kindly provided by Lei Wang of Huazhong Agricultural University, were cotransformed and transiently expressed in rice protoplasts prepared from an etiolated shoot of japonica rice var Zhonghua 11 by polyethylene glycol treatment (Ning et al., 2010). After 24 h of transformation, the fluorescence signal was observed using a confocal microscope. Transactivation and Transsuppression Activity Assays The transactivation activity of WRKY42 was analyzed in both yeast (Saccharomyces cerevisiae) cells and rice protoplasts using the known rice transcription activator OsbZIP23 as the positive control. The yeast assay was performed as described previously (Deng et al., 2012). In brief, the complete coding region, the N-terminal region, and the C-terminal region of WRKY42 were amplified using primer pairs 1532F/R, 153Y1F/R, and 153Y2F/R, respectively (Supplemental Table S1). The PCR products were respectively ligated into the pGAL4-BD vector. The pGAL4-BD-OsbZIP23 vector was kindly provided by Xiang et al. (2008). These recombinant plasmids and empty vector (negative control) were respectively transformed into yeast strain Y187. Yeast transformants were screened by culture in synthetic dropout medium-Trp-Leu-Ade medium. For study of the transactivation or transsuppression activity of WRKY42 and WRKY45-2, the GAL4DB/UAS/LUC or GAL4DB-VP16/UAS/LUC transient assays were performed in rice protoplasts according to the process reported previously (Jing et al., 2013;Weng et al., 2014). This system contains three vectors: the effector, reporter, and control. The complete coding region of WRKR42 or WRKY45-2 was fused with GAL4DB or GAL4DB-VP16 driven by the 35S promoter as the effector. The LUC linked to four tandem repeats of UAS, which harbors DNA sequence for GAL4DB binding (43UAS-LUC), was the reporter. The Renilla LUC (REN) driven by the 35S promoter was the control for standardizing the difference of the transformation ratio. The three vectors were cotransferred into rice protoplasts. The LUC activity was detected with a luminescence kit using the LUC assay substrate (Promega). The expression level of reporter gene LUC was determined by counting the ratio of LUC to REN. The transcriptional activity of the target transcription factor was calculated relative to that of the empty vector. To study the role of a transcription factor in the target gene, the chimeric reporter/effector transient expression assay was performed in rice protoplasts according to the process reported previously (Gao et al., 2013). In this assay, LUC was driven by the target promoter as the reporter, the target transcription factor gene driven by the 35S promoter was used as the effector, and the YFP driven by the 35S promoter was used as the effector control. The reporter and effector vectors were cotransferred into rice protoplasts with the control REN vector. Protein-DNA Interaction ChIP assay was performed as described previously (Tao et al., 2009). Samples were collected from the shoot of rice var Mudanjiang 8 at the three-to four-leaf stage. Sonicated chromatin fragments were immunoprecipitated with WRKY13-specific antibody (Tao et al., 2009), which was custom synthesized by NewEast Biosciences. The IP chromatin was analyzed by qPCR using primer pairs N1F/N1R, N2F/N2R, and N3F/N3R (Supplemental Table S1). The non-IP and sonicated chromatin was used as the total input DNA control. The percentage of immunoprecipitation (IP%) was used to compare different samples by calculating the ratio of the amount of target PCR product from IP relative to that from the input. The IP% was calculated by using the following formula: IP% = 2 2DCt (normalized ChIP) , in which DCycle threshold (DCt) = Ct [ChIP] 2 (Ct [input] 2 log 2 input dilution factor) and input dilution factor = (fraction of the input chromatin saved) 21 in the qPCR, according to a previous report (Haring et al., 2007). The interaction of the WRKY protein with the DNA regulatory element was determined by a yeast one-hybrid assay as described previously (Chen et al., 2014). In brief, the full-length cDNA of WRKY protein obtained by PCR amplification was ligated into the pB42AD vector (Clontech). The target cis-acting DNA fragment obtained by PCR amplification of the promoter region of the target gene was ligated into the p8op-lacZ vector containing the b-galactosidase reporter. The two recombinant vectors were cotransformed into yeast strain EGY48. Transformants were grown on synthetic dropout medium/-Uracil-Trp plates containing 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside for blue color development. Statistical Analysis The correlation analysis between disease area and gene expression level was performed using the CORREL analysis in Excel (Microsoft). The significant differences between control and treatment of the samples were analyzed by the pairwise t test in the Excel program. Supplemental Data The following supplemental materials are available. Supplemental Figure S4. The schematic diagram of rice WRKY42 protein structure. Supplemental Figure S5. WRKY42 colocalized with transcription factor Ghd7 in the nucleus of rice protoplast. Supplemental Figure S6. WRKY42 displayed no transactivation activity in yeast cells. Supplemental Figure S7. Increased susceptibility to Xoo is associated with suppressed expression of WRKY13. Supplemental Figure S8. Modulating WRKY13 expression influenced rice response to M. oryzae infection. Supplemental Figure S9. The specificity of anti-WRKY13 antibody was examined. Supplemental Figure S10. The W displayed in the promoter region of WRKY42. Supplemental Figure S12. Interaction of the three WRKY genes detected by yeast one-hybrid. Supplemental Table S1. PCR primers used for construction of vectors, gene structure analysis, and ChIP analysis. Supplemental Table S2. Primers used for qPCR in gene expression analysis.

v3-fos

2016-03-22T00:56:01.885Z

2015-11-01T00:00:00.000Z

14238284

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Polyphenolic Compositions and Chromatic Characteristics of Bog Bilberry Syrup Wines Phenolic compounds determine the color quality of fruit wines. In this study, the phenolic compound content and composition, color characteristics and changes during 6 months of bottle aging were studied in wines fermented with bog bilberry syrup under three different pHs. The total anthocyanins and total phenols were around 15.12–16.23 mg/L and 475.82 to 486.50 mg GAE/L in fresh wines and declined 22%–31% and about 11% in bottle aged wines, respectively. In fresh wines, eight anthocyanins, six phenolic aids and 14 flavonols, but no flavon-3-ols were identified; Malvidin-3-O-glucoside, petunidin-3-O-glucoside and delphinium-3-O-glucoside were the predominant pigments; Chlorogentic acid was the most abundant phenolic acid, and quercetin-3-O-galactoside and myricetin-3-O-galactoside accounted for nearly 90% of the total flavonols. During 6 months of bottle storage, the amounts of all the monomeric anthocyanins and phenolic acids were reduced dramatically, while the glycosidyl flavonols remained constant or were less reduced and their corresponding aglycones increased a lot. The effects of aging on blueberry wine color were described as the loss of color intensity with a dramatic change in color hue, from initial red-purple up to final red-brick nuances, while the pH of the fermentation matrix was negatively related to the color stability of aged wine. Introduction Vaccinium uliginosum, known as bog bilberry, belongs to the Ericaceae family of the Vaccinium genus [1]. It is one of the most abundant wild blueberries in the Greater Khingan Range, Northeast China. V. uliginosum berries are not only rich in monosaccharides, amino acids, dietary fiber, vitamins and trace elements such as potassium, iron, zinc and manganese [1], but also contain a variety and high contents of bioactive substances, including anthocyanins and flavonols [2][3][4][5][6]. During past years, it was well documented that V. uliginosum berries have several kinds of high nutritional values and medicinal effects, such as preventing cranial nerve aging, strengthening cardiac functions, combating cancers, softening blood vessels and enhancing immunity [7][8][9][10]. Due to their low sugars and high organic acids contents, V. uliginosum berries are not consumed fresh but rather usually steeped in a solution with high sugar content for hours and then dried into Molecules 2015, 20,[19865][19866][19867][19868][19869][19870][19871][19872][19873][19874][19875][19876][19877]; doi:10.3390/molecules201119662 www.mdpi.com/journal/molecules fruit snacks for local markets. During the soaking process, some berries are unavoidably crushed due to their quite thin skins. The sugar solutions are then turned into blueberry syrup as by-products after the snack batch production cycles. The related syrup, which not only contain a high content of sugars, but also have considerable amounts of organic acids and polyphenols, are an ideal raw material for fruit wine production [11,12]. Our research groups have developed a process to convert these syrup into fermented red wines by supplementation of an appropriate nitrogen source such as dibasic ammonium phosphate (DAP) [13]. The colors matter a lot for the sensory perception of red wines when the consumers make choices. Anthocyanins, which give young red wines their typical purple red color and decide their color intensity, are the most important phenolic substances, but they are also unstable during the enology process. Previous studies on grape wines found a progressive loss of anthocyanins during the winemaking and aging process, which can be caused by different mechanisms including co-pigmentation, cycloaddition, polymerization or oxidation [14]. It is well known that these reactions are relevant to both the molecular structure of anthocyanins and the matrix of a specific wine. Most of the previous research about the relationships between chromatic characteristics and chemical composition were focused on red grape wines [15][16][17][18]. The anthocyanin, flavonol and organic acid composition of bog bilberries is well documented and distinct from that of wine grapes [2][3][4][5][6]. However, currently, there is a lack of studies regarding the color stability and phenolic compound changes of wines fermented with non-grape fruits, including blueberries. This research aims to figure out: (1) the content, composition and changes of anthocyanins and other polyphenols in wines fermented from bog bilberries syrup; (2) the influences of fermentation pH and bottle aging on their color stability. Conventional Analysis of Blueberry Wines Fermented with Bog Bilberry Syrup under Different pHs As described previously, the wild blueberry syrup, which are the byproducts of dried berry snack processing and contain plenty of sugars, organic acids and phenolic compounds, were used as raw materials to produce fruit wines by dilution and pH adjustment. Finally, three kinds of wines fermented under three different pH conditions (3.1, 3.3 and 3.5) were obtained and named Wine A, Wine B and Wine C, respectively. As shown in Table 1, pH had no obvious effect on the alcoholic strength of the final blueberry wines, while Wine B had the lowest total sugar and reducing sugar content, indicating that a pH 3.3 environment was more conducive for yeast to use the sugars in blueberry syrup. The pH of all the resulting wines decreased during the fermentation process, but still showed a certain difference between the different treatments. Note: * from the start of fermentation to when fermentation naturally terminated; when the relative density dropped to 0.990 and did not change for three consecutive days it was considered the end of fermentation; Different letters in each row indicate the significant differences in the mean at p < 0.05. Changes of Total Anthocyanin and Total Phenol Content in Bog Bilberry Syrup Wines with Different Treatments during the Aging Process Anthocyanins are mostly responsible for the characteristic red-purplish colors presented by young wines [19], while other polyphenols can stabilize the wine color through co-pigmentation or protecting the anthocyanins by competitively reacting with O 2 [20,21]. There are a number of studies on the changes of anthocyanins and other polyphenols during the grape wine aging process [16,22,23], but nearly none focused on wines made from other fruits with distinct phenolic compositions. We compared both the total anthocyanin (TA) and total phenol (TP) content in young (after fermentation) and aged (after 6 months bottle storage) blueberries wines. As shown in Figure 1, after fermentation, the contents of TA in Wine A-0M, Wine B-0M and Wine C-0M samples were 15.12, 16.32 and 16.23mg/L, respectively; a little higher than rose grape wines (about 11 mg/L) [24], but much lower than common red grape wines (ranging from 127 to 358 mg/L) [22,23]. As usually reported, the anthocyanins originating from similar berries (such as strawberries, elderberry, and also grape berries) were quite unstable and their degradation rates depended on the pH, temperature and O2 concentration of the processing and storage conditions [21,25,26]. TA content decreased considerably during bottle storage, independently of wine type [20,27]. After 6 months of bottle storage, the TA contents in Wine A-6M, B-6M and C-6M declined 21.96%, 30.02% and 30.76%, respectively, which were comparable to rose grape wines (34.5%, from 11.00 mg/L to 7.21 mg/L) [24], but lower than sweet red grape wines (79.75%, from 186 mg/L to 37.7 mg/L) [22] and dry red grape wines (50.15%, from 358.26 mg/L to 178.61 mg/L) [23]. Anthocyanins might undergo complex reactions during aging, among which the oxidative degradation [28] and the formation of insoluble polymeric pigments are the most relevant to the loss of TA and color strength in red wines [15,29]. The total phenols (TP) in our fresh wines ranged from 475.82 to 486.50 mg GAE/L, similar to white grape wines (240 to 500 mg /L), but less than in red grape wines 700 and 4095 mg/L [30]. Over 6 months of aging, the TP in all the samples decreased around 11%, slower than in the white grape wines (about 17%) [31]. In red grape wines, a 15%-22% drop was found during 3 months of bottle aging [16]. It has been proved that the predominant reasons for the decline in TA and TP during wine aging are mainly: (1) oxidative degradation; (2) the polymerization of polyphenolic compounds themselves or reactions with other small compounds such as acetaldehyde and pyruvic acid [32][33][34]. The polymerization reactions result in a more stable color and a better taste and organoleptic quality [33,34], while the irreversible decrease in some bigger polymer pigments, together with the oxidation of monomeric pigments, would make the color intensity decrease [26,35]. The phenolic composition differences between bog As usually reported, the anthocyanins originating from similar berries (such as strawberries, elderberry, and also grape berries) were quite unstable and their degradation rates depended on the pH, temperature and O 2 concentration of the processing and storage conditions [21,25,26]. TA content decreased considerably during bottle storage, independently of wine type [20,27]. After 6 months of bottle storage, the TA contents in Wine A-6M, B-6M and C-6M declined 21.96%, 30.02% and 30.76%, respectively, which were comparable to rose grape wines (34.5%, from 11.00 mg/L to 7.21 mg/L) [24], but lower than sweet red grape wines (79.75%, from 186 mg/L to 37.7 mg/L) [22] and dry red grape wines (50.15%, from 358.26 mg/L to 178.61 mg/L) [23]. Anthocyanins might undergo complex reactions during aging, among which the oxidative degradation [28] and the formation of insoluble polymeric pigments are the most relevant to the loss of TA and color strength in red wines [15,29]. The total phenols (TP) in our fresh wines ranged from 475.82 to 486.50 mg GAE/L, similar to white grape wines (240 to 500 mg /L), but less than in red grape wines 700 and 4095 mg/L [30]. Over 6 months of aging, the TP in all the samples decreased around 11%, slower than in the white grape wines (about 17%) [31]. In red grape wines, a 15%-22% drop was found during 3 months of bottle aging [16]. It has been proved that the predominant reasons for the decline in TA and TP during wine aging are mainly: (1) oxidative degradation; (2) the polymerization of polyphenolic compounds themselves or reactions with other small compounds such as acetaldehyde and pyruvic acid [32][33][34]. The polymerization reactions result in a more stable color and a better taste and organoleptic quality [33,34], while the irreversible decrease in some bigger polymer pigments, together with the oxidation of monomeric pigments, would make the color intensity decrease [26,35]. The phenolic composition differences between bog bilberry syrup wines and grape wines, which are associated with these chemical reactions occurring along with aging, is valuable to expound. Comparison of Phenolic Compositions in the Young and Aged Bog Bilberries Syrup Wines HPLC-ESI-Trap-MS n coupled with a DAD detector was applied to elaborate the anthocyanin and non-anthocyanin phenolic composition of bog bilberry syrup wines and the results are shown in Table 2. Note: Nd, "Not-Detected"; Tr, "Trace"; Different letters in each row indicate the significant differences in the mean at p < 0.05. were detected in all the wine samples, but in trace amounts. The type of anthocyanins, especially the pyranoanthocyanins and acylated anthocyanins, was far less than in red grape wines [36], and even less than in rose grape wines, which had comparable TA contents to the samples studied here [24]. In all the samples, malvidin-3-O-glucoside was the predominant pigment, accounting for about 40% of the total anthocyanin content, followed by petunidin-3-O-glucoside (about 18%) and delphinium-3-O-glucoside (about 16%). This was similar to the previously found monomeric anthocyanin composition of bog bilberries [2,3]. The proportions of malvidin-3-O-glucoside (with maximum numbers of methoxy groups on the B rings) were less than in grape wines (about 60%) [16,20], while the proportions of delphinidin-3-O-glucoside and petunidin-3-O-glucoside, which have more hydroxyl groups and are less stable compared to malvidin-3-O-glucoside, were more in our wines than in grape wines (which ranged from 2.11% to 3.41% and 2.64% to 6.34%, respectively) [17,20,36,37]. The numbers of hydroxyl groups on the B ring of a specific anthocyanin is related with its color hue and stability [38]. These differences in anthocyanin composition would make the blueberry wines display a more blue color hue, but less color stability. During 6 months of bottle storage, all six of the monomeric anthocyanins detected decreased significantly, and it was speculated that they degraded into small phenolic acids and anthocyanone A through oxidation and this finally reduced the related wine color intensity [28], or they changed into more stable oligomers and polymers of pigments through direct and acetaldehyde-mediated anthocyanin-flavanol condensation reactions or other anthocyanin condensation reactions leading to pyranoanthocyanins [18,36,39,40], which could lead to the wine color hue changing from bright-red to brick-red [33]. Although the polymerization products of anthocyanins are quite commonly identified in red grape wines [35,36], they were not detected in our aged blueberiy wines, which might related to their low contents of monomeric anthocyanins. Phenolic Acids A total of six phenolic acids were found in all wine samples ( Table 2). Among them, chlorogentic acid was the most abundant phenolic acid, and followed by protocatechuic acid, caffeic acid and salicylic acid (also called 2-hydroxybenzoic acid). Both 4-hydroxycinniamic acid and gallic acid were identified in all our wine samples, but under their limit of quantitation (LOQ). It was also worth noting that some other phenolic acids and their esters, including fertaric acid, coutaric acid and caftaric acid which are quite common in grape wines, were not detected in the wine samples studied here. These results demonstrated that the phenolic acid compositions of blueberry wines were quite distinct from those of grape wines which have been studied in detail previously [16,19,24,41,42]. Chlorogentic acid, the ester of caffeic acid and quinic acid, was previously found with a content of 20 mg/L in bog bilberry juice [6]. Its concentration was around 7.15-9.02 mg/L and with no significant differences in our original wines. Chlorogentic acid was also identified in wines made from other fruits including cherries (1.1-21.3 mg/L) [43][44][45][46], peaches (3.59 mg/L) [47] and apples (8.27 mg/L) [48] and found to be a major contributor to the characteristic taste of cherry wine [43]. To our best knowledge, this compound has not been reported in grape wines yet, although a similar compound, caftaric acid (an ester of caffeic acid and tartaric acid), is quite common in fresh grape wines. Plenty of studies have shown that caftaric acid was unstable during bottle aging, independently of the wine type [20,27]. Similarly, we observed a 17%-42% decrease of chlorogentic acid during the bottle aging periods and the proportion of the decline was related to the pH values of the original wines. This might be the first report on the fate of chlorogentic acid in fruit wines during aging. The concentrations of protocatechuic acid and salicylic acid in all our fresh wines, were around 3 mg/L and 0.2 mg/L respectively, without any significant differences. Protocatechuic acid was found at the range of 1.3-2.4 mg/L in sweet grape wines [41,49] and 6-7 mg/L in young red wines [16]. The caffeic acid contents in our fresh wines were as follows: Wine A-0M (0.13 mg/L) < Wine B-0M (0.29 mg/L) < Wine C-0M (0.66 mg/L), far less than in red grape wines (4.7-18 mg/L) [16,30], and even less than in rose grape wine (around 1 mg/L) [24]. After 6 months of bottle aging, the levels of all these quantitative phenolic acids were reduced dramatically, which might result from oxidation to the corresponding quinones or polymerization with monomeric anthocyanins to form complex pigment polymers [50,51]. In grape wines, protocatechuic acid and salicylic acid levels were also found to be reduced with bottle aging; while contrarily, caffeic acid levels increase over this period because of the hydrolysis of caftaric acid [23,24,27,52]. Many studies have proved that phenolic acids could serve as cofactors of co-pigmentation to stabilize the flavylium cation chromophore of monomeric anthocyanins and thus enhance the red color intensity and bluish hues of young red wines [16,20,38,53]. Flavonols Previously studies showed that flavonol compounds could also be involved in the co-pigmentation reactions with anthocyanins and might play an important role in stabilizing wine color [20,23,54]. As shown in Table 2, a total of 14 kinds of flavonol compounds were identified in the samples studied here, including 11 flavonol glycosides (five glucosides, four galactosides, one glucuronide and one rhamnoside) and three free flavonols (quercetin, syringetin and myricetin). In the fresh wines, only two free flavonols were detected, but their contents were at trace levels. Quercetin-3-O-galactoside and myricetin-3-O-galactoside were the most abundant glycosides-bound flavonols (accounting for around 51% and 38% of the total flavonols, respectively), followed by syringetin-3-O-galactoside, quercetin-3-O-glucuronide, syringetin-3-glucoside, quercetin-3-O-glucoside and myricetin-3-O-glucoside. It was noteworthy that the galactoside-type compound contents were much higher than those of the glucoside-type, in line with the flavonol composition of bog bilberries found previously [3,55], but contrary to the grape and grape wines in which the glucosides were dominant [20,54]. After 6 months of storage, the contents of free flavonols (myricetin, quercetinand syringetin) increased significantly; meanwhile, inversely, the concentrations of most of the flavonol glycosides (except for quercetin-3-O-glucuronide) decreased. This resulted from the hydrolysis of the glycosylated derivatives to aglycones during the wine storage time, as previously observed during bottle aging of grape wines [20,22,23]. The changing proportions of flavonol glycosides were quite lower than those of other types of phenolic compounds (anthocyanadins and phenolic acids), which indicate the former are the most stable phenolic components during wine aging. Interestingly, bog bilberries were found to have the capacity to accumulate amounts of flavon-3-ols [56] during berry development; but unexpectedly, none of the monomeric flavon-3-ols (catechin, epicatechin, gallocatechin and epigallocatechin gallate) were detected in both the young and aged blueberry syrup wines. The method used in this study was originally developed for grape wines and has been applied to the analysis of hundreds of samples and showed good sensitives for these compounds [36], so we can be sure that the lack of flavon-3-ol compounds (not due to any limitation of the instrumental method but to the samples themselves) was another critical distinction between wines fermented with the bog bilberry syrup and wine grapes. It has been well documented that flavon-3-ol compounds not only contribute to the astringent mouth feel and serve as antioxidants during storage, but also could form unstable non-covalent co-pigments with monomeric anthocyanidins in young red grape wines, or finally changed into stable covalently connected complexes in aged wines, both of which are beneficial to the color stability of aging wine [35,[38][39][40]. Without flavon-3-ol compounds, the monomeric anthocyanidins was deduced to more easily degrade into small and colorless compounds through oxidation [28], which further might make the blueberry syrup wines more sensitive to free O 2 during storage. Chromatic Characteristics Wine chromatic characteristics, lightness (L*), red/green component (a*), yellow/blue component (b*), chroma (C*), hue angle (h*) and chromatic differences (∆E*), were evaluated using the CIELab method and the results are shown in Table 3. Note: Different letters in each row indicate the significant differences in the mean at p < 0.05. The CIELab parameter L* (which ranges from 0 for black to 100 for white) is inversely related to the intensity of color. The L* values of the three fresh blueberry syrup wines were compared to the red grape wines, but lower than rose grape wines [24]. Wine A-0M had a higher L* value than B-0M and C-0M. After 6 months of aging, the L* value of wine A did not change significantly, but in contrast, Wine B and Wine C increased from 56.85 to 64.43 and from 58.30 to 71.02, respectively, which means they lost color intensity. The a* values of our fresh were around 30, lower than young red grape wines (about 45~55) found previously [57,58]; this may be associated with their different anthocyanin compositions, as described in Section 2.3.1. It was also worth noting that the a* value of Wine A was higher than those of Wine B and Wine C. Anthocyanins exist in two structures that can be mutually interconverted when the pH of wine is below 3.2: the flavylium cation (red) and the quinoidal base (blue); With the rise of pH value, the flavylium cations lose protons forming the quinoidal base, at the same time, the flavylium cation is hydrolyzed into the hemiketal or carbinol pseudo-base (colorless), and the hemiketal or carbinol pseudo-base slowly ring opens into a chalcone (colorless) [38]. After 6 months of storage, the a* values of all the studied wines decreased [A (11.19%), B (13.74%), C (16.07%)], which might mainly be due to the precipitation of insoluble polymeric anthocyanin-derived pigments and/or the oxidative degradation of free anthocyanins [21,22,26,35]. With regard to the yellow-blue color component (b*: positive values, yellow component; negative values, blue component) and h*(hue angle): Wine A-0M had lower b* and h* values than the other two wines, which might result from that the lowest pH of A-0M among the three fresh wines, as described in Section 2.1. The values of b* and h* increased during the 6 months of bottle aging. The evolution of b* and h* values with aging time were associated with the formation of anthocyanin-derived yellow-orange pigments like pyranoanthocyanins, and also due to the oxidation of red wine pigments [18,59]. The chromatic differences (∆E*) is the distance between two points in a three-dimensional space. Our eyes can discriminate samples if their ∆E* more than 3.0 [60,61]. The ∆E* values of Wine A, B and C between 0 month and 6 months of bottle aging were 6.53, 9.59 and 13.78, respectively, which were all higher than 3.0 and easily recognized by human eyes. It was noteworthy that the ∆E* of Wine C was about twice that of Wine A, showing that the pH of the fermentation matrix was indeed negatively related to the color stability of aged wine. The Fermentation of Bog Bilberry Syrup Wines The bog bilberry syrups (78.1˝Brix, pH 2.88), byproducts of dried berry snack production and provided by Xinganlieshen Original Products Ltd., (Hulunbeier City, Inner Mongolia, China), were diluted with volumes of potable water and used as raw materials for wine fermentation. Next, 300 mg/L of dibasic ammonium phosphate (DAP) were added as nitrogen source and various amounts of sodium hydrogen carbonate were added to adjust the pH to 3.1, 3.3 and 3.5, respectively. After that, a commercial dry yeast (red fruit , Enartis Ltd., Novara, Italy) was activated and inoculated and all fermentations were conducted under controlled temperature (20-22˝C). When the relative density did not drop in three consecutive days, 100 mg/L of K 2 S 2 O 7 were added and the temperature were reduced to 4˝C to stop the fermentation and three kinds of wines were obtained and stored in 100 L sealed stainless steel tanks for further research. For the aging tests, some of the fresh wines were filtered with a membrane (0.45 µm average pore size) and packaged into 750 mL glass bottles (dark green color, Bordeaux type, Changyu Ltd., Yantai, China), sealed with natural corks (45 mm in length) and stored in a dark cellar with a constant temperature of 15˘1˝C and a humidity of 80%˘5% for a period of 6 months. Three bottles of wine for each treatment were used for further analysis. Standard Chemical Analysis of Wines The amount of total sugar, reducing sugars, total acidity, pH and alcoholic content (% v/v) were analyzed according to the methods proposed by the National Standard of the People's Republic of China (GB/ T15038-2006, 2006) [62]. Total Anthocyanin Content Measurement Total anthocyanin content was determined by using a pH differential method according to the AOAC Official Method as described previously [63], but with some modifications. Two dilutions of the extracts were prepared, one for pH 1.0 using KCl buffer and the other for pH 4.5 using sodium acetate buffer, samples were diluted to a factor of 1:3 using the different buffers. Sample spectral absorbance measurements were read at 521 and 700 nm on a spectrophotometer. The total anthocyanins were expressed as malvidin-3-O-glucoside equivalents in milligram per liter using the following equation: where A = (A 521´A700 ) pH1.0´( A 521´A700 ) pH4.5 ; MW is 493.2, the molecular weight of malvidin-3-O-glucoside (g/mol); Df is the dilution factor; ε denotes the extinction coefficient of malvidin-3-O-glucoside [28,000 Lˆmol´1ˆcm´1]; L is the constant at path-length 1 cm; 1000 is the factor to convert g to mg. Total Phenol Content Measurement The total phenol contents were determined using the Folin-Ciocalteu method [42], adapted to a microscale experiment. A total of 790 µL of distilled water, 10 µL of sample dissolved in methanol, and 50 µL of Folin-Ciocalteu reagent were added in an Eppendorf tube and vortexed. After 1 min, 150 µL of sodium carbonate solution (20%) was added and vortexed again and stand at room temperature in the obscurity for 2 h. The absorbance was read at 750 nm, and the total phenol concentration was calculated from calibration curve, using gallic acid as the standard. The results were expressed as mg¨L´1 gallic acid (GAE). Determination of Phenolic Compounds An Agilent-1200 HPLC system equipped with a UV detector and an LC-MSD Trap VL ion-trap mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) via an ESI source, were used to analysis the phenolic composition of the wine samples according to the method published by Gao et al.,previously [36] with some modifications. For anthocyanins-50 µL of filtered samples (cellulose acetate and nitrocellulose, CAN, 0.45 µm) was injected to the system for quantitative and qualitative analyses. A reversed-phase column (Kromasil C18, 250ˆ4.6 mm, 5 µm) was used and the mobile phases were as follows: Solvent A (water:formic:acetonitrile = 92:2:6 [v/v/v]) and solvent B (water: formic: acetonitrile = 44:2: The flow rate was set at 1 mL/min and the gradient was from 0%-10% B for 1 min, from 10%-25% B for 17 min, 25% B for 2 min, 25%-40% B for 10 min, from 40%-70% B for 5 min, 70%-100% B for 5 min. MS conditions were the same with [36]. The anthocyanins were identified by their order of elution time with respect to malvidin-3-O-glucoside and the weights of the molecular ion and the fragment ions compared with standards and references [2,36,37]. For quantitative analyses, the detection wavelength used by the diode array detector (DAD) was 525 nm. The concentration of all anthocyanins was expressed as malvidin-3-O-glucoside. For non-anthocyanin phenolic compounds-100 mL of each wine sample was diluted with an equal volume of pure water and extracted three times with ethyl acetate. The organic phase was collected and evaporated to dryness by a rotary evaporator at 30˝C and re-dissolved in 5 mL methanol (chromatographic grade). 2 µL of filtered methanol solution were injected to the HPLC-MS system and a reversed Zorbax SB-C18 column (3ˆ50 mm, 1.8 µm) was used for separation. A gradient consisting of solvent A (pure water with 1% acetic acid) and solvent B (acetonitrile1% acetic acid), was applied at the flow rate of 1.0 mL/min as follows: 0%-5% B for 5 min, 5%-8% B for 5 min, 8%-12% B for 5 min, 12%-18% B for 5 min, 18%-22% B for 2 min, 22%-35% B for 2 min, and 35%-100% B for 4 min. The column temperature was 25˝C. MS conditions: negative ion mode; nebulizer pressure, 35 psi.; dry gas flow, 10 mL/min; dry gas temperature, 325˝C; scans at 100-1500 m/z. For quantification, the detection wavelength was set at 280 nm and an external standard method were used: flavanols using catechin, flavonol using quercetin, hydroxybenzoic acids using gallic acid; hydroxycinnamic acids using caffeic acid and chlorogentic acid using itself, respectively. All of the standards were dissolved with ethanol (HPLC quality) as stock solution, and then this mixed standard solution was diluted into seven levels in succession with the synthetic model wine solution. Mixed standards of each level were analyzed under the same condition as the samples and calibration curves were obtained with their regression coefficients all above 95%. Data Analysis Calculation of average and standard deviation were performed using Microsoft Excel 2007 software. IBM SPSS Statistics 20 (IBM, New York, NY, USA) for Windows was used for statistical calculations. The differences were considered to be statistically significant when p < 0.05. Conclusions In summary, we obtained three kinds of blueberry wines fermented with wild bog bilberry syrup under different pHs. Polyphenolic content, composition and the chromatic characteristics of these wines, both at the end of fermentation and after 6 months of bottle aging, were studied. The TAs contents in our blueberry wines were similar to those of rose grape wines, and the TPs were comparable to white wines and rose wines. Distinct compositions of both anthocynidins and non-anthocynidins phenolic compounds in our young wines were found compared to grape wines, especially characterized by the lack of flavan-3-ol compounds. During bottle aging, the polyphenolic contents decreased mainly through oxidative degradation, which induced color fading. The effects of aging on blueberry wine color were described as the loss of color intensity with a change in color hue, from initial red-purple to final red-brick nuances. pH was negatively related to the color stability of related wines. These results might help us understand the color stability mechanism in non-grape red wines and improve the organoleptic quality of related wine products further.

v3-fos

2020-07-02T10:32:19.293Z

2015-04-01T00:00:00.000Z

221691944

s2

A NOVEL VISION USING IMAGING ANALYSIS FOR TOMATO QUALITY DETECTION The quality of products is very important for the human health. Sorting tons of fruits and vegetables manually is a slow, costly, and an inaccurate process. The objective of this study was to develop a computer vision and image analysis program to serve as a simple and suitable technique for external fruit inspection and for predicting orange fruits maturity through the image analysis technique. The MATLAB software package was used image processing tools to analysis image of tomato. The study also investigated the effectiveness of some color bands, average intensity of RGB bands and HSI. The results revealed that the computer vision and image analysis program could be used to differentiate tomato maturity stages. The results also showed that there is a strong response between both RGB band and HSI of tomato fruits and maturity stage also storage period during 21 days. Automatic sorting of food products is an important process to get high quality food. Vision based sorting system is an accurate and fast process compared to manual sorting. The accuracy of this system can be improved by increasing the dataset of images. INTRODUCTION he quality of products is very important for the human health. Sorting of agricultural products is accomplished based on appearance (color and absence defects), texture, shape and sizes. Manual sorting is based on traditional visual quality inspection performed by human operators, which is tedious, time-consuming, slow and nonconsistent. *Assistant Prof of Agric. Eng. Dep., Fac. of Agric., Minoufiya Univ., **Post graduate student The basic principle of computer vision is described in Figure (1). Image processing and image analysis are the core of computer vision with numerous algorithms and methods available to achieve the required classification and measurements (Krutz et al., 2000) Fig (1). Principles of computer vision system. Helmy et al. (2003) used a digital camera to take images for fresh tomato fruits sample; each fruit had two images, from up and down. The intensity of used light was 530 lux. And classified fresh tomatoes into fifteen maturity grades namely green, spring green, light green, breaker, sand, light orange, peach, turning, color, faded pink, soft pink, pink, tropical pink, light red, neon red and red. They gave average, variance, and standard deviation of reflectance and RGB values of the classified tomatoes. Sorting is a separation based on a single measurable property of raw material units, while grading is the assessment of the overall quality of a food using a number of attributes. Grading of fresh product may also be defined as sorting according to quality, as sorting usually upgrades the product (Brennan, 2006). Walailak and Tech (2006) mentioned that, color grading is an important process for the agriculture industry especially in food processing, fruit and vegetable grading. The color of products is often used to determine quality and price. Consumers have developed distinct correlations between color and the overall quality of a specific product. In the agriculture industry, color-grading applications are implemented by using color image processing. The advancement of color grading is based on the development of color charge coupled device (CCD) camera. In recent ten years, operations in grading systems for fruits and vegetables became highly automated with mechatronics, and robotics technologies. Machine vision systems and near infrared inspection, systems have been introduced to many grading facilities with mechanisms for inspecting all sides of fruits and vegetables (kondo, 2009). It has become increasingly difficult to hire personnel who are adequately trained and willing to undertake the tedious task of inspection (Amer Eissa and Khalik, 2012). Computer vision systems have been used increasingly in the food and agricultural industry for inspection and evaluation purposes as they provide suitably rapid, economic, consistent and objective assessment. They have proved to be successful for the objective measurement and assessment of several agricultural products. Over the past decade, advances in hardware and software for digital image processing have motivated several studies on the development of these systems to evaluate the quality of diverse and processed foods. Computer vision has long been recognized as a potential technique for the guidance or control of agricultural and food processes. Fouda and Salah (2014) tested a computer vision and image analysis program as a suitable technique for external orange fruit inspection. The results showed the relationships between hue and saturation and total soluble solid (Tss), ph, acidity and percentage of liquid. The results demonstrated that hue and saturation indices gives understanding about between total soluble solid (tss), ph, acidity and percentage of liquid. Tomatoes (lycopersicon esculentum), is a major vegetables crop in Egypt which is cultivated in about 216385 thousand faddans to produce 8.5 million tons/year. Egypt is ranked fifth in the world in the production of tomatoes (FAO of agricultural statistics, 2014). Quality and productivity control is a big issue in tomato crop due existence of large number of defects. Based on these considerations, the proposal of this research work was conceived as to adapt the MATLAB code used Image processing toolbox to open software to enable the classification system in recognizing color and possibly bruises at a unique glance, driving at to develop low cost and reliable techniques applicable to fruit sorting. MATERIALS AND METHODS The present work investigated the potential of image analysis techniques to detect the response of tomato maturity. The experimental work was undertaken at the Department of Agricultural Engineering -College Agriculture -Minoufiya University. The tomato fruit and varieties The samples were hand harvest and selected randomized. All samples were individually numbered, four image for each sample occurred .The tomato fruit and varieties under study were (Commercial items),Super Strain (fresh) and Super Mar mend (storage) were stored at refrigerator temperature a range of (10-15º), without exposed to light for 21 days and pictures of tomato samples were taking every three days as shown in Figure (2). Computer visioning system The system consists of an imaging box with non-reflective black color cloth connected to a digital camera of 14 Mega pixels. The camera was mounted at 15 cm from the surface of tomato fruit. Samples were illuminated using two parallel lamps with two fluorescents tubes in each lamp. Both lamps (60 cm long) were situated 35 cm above the sample and at an angle of 45• to the sample. Following capturing images were stored on a personal computer for the analysis. Image Analysis system: tomato samples were captured by the camera, transferred to Color evaluation: Using the most popular color model RGB color space and HSI. The color was presented with R, G and B, the amount of information is tripled. The RGB system is sensitive to lighting and other surrounding conditions. To evaluate the color of captured images of fruits, the acquired RGB color as shown in Figure (3) information was transformed by MATLAB 9 (image processing tools). The following expressions show the relationship between HSI values and RGB values: RESULTS AND DISCUSSION The results of this work are discussed under two heading: 1-Physical characteristics of tomato fruit. 2-Image processing of tomato fruit. Experimental results obtained for both an unripe and a ripe fruit. The mean value of RGB color and intensity in different maturity stage of fresh tomato fruit are discussed. The relationship between maturity stage of tomato fruit and both of Seasons and RGB: Maturity of tomato fruit, (M) with red color, green color and blue color content is displayed in Fig (6) and (7) and Table (1) and (2) in summer and winter season. (R) and (G) increased then decreased with maturity stage, but was increased with maturity stage .Maturity of tomato fruit, (M) with red color, green color and blue color (G) and (R) decreased with increased maturity stage, but (B) was increased with increased maturity stage. The relationship between maturity stage of tomato fruit and intensity in summer. Maturity stage of tomato fruit, with intensity content is displayed in Fig. (8). Intensity increased from 0.21 to 0.23 with increased Storage time (T) then decreased to 0.13. Fig (8): The relationship between maturity stage of tomato fruit and intensity in summer. The relationship between maturity stage of tomato fruit and intensity in winter. Maturity stage of tomato fruit, with intensity content is displayed in Fig (9). Intensity decreased from 0.24 to 0.17 with increased maturity stage. Fig (9): The relationship between maturity stage of tomato fruit and intensity winter. Storage tomato fruit Ripe tomatoes fruit were stored at refrigerator temperature arrange of (10-15º), without exposed to light for 21 days and was taking pictures of tomato samples every three days. Maturity intensity The relationship between storage day of tomato fruit and RGB. Storage time (T) of tomato fruit, with red color (R), green color (G) and blue (B) color content is displayed in Fig. (10) and Table (3). (G) Color decreased with increased Storage time (T), but (R) and (B) color was increased with Storage time (T). The relationship between storage day of tomato fruit and intensity. Storage time (T) of tomato fruit, with intensity content is displayed in Fig. (11). Intensity increased with increased Storage time (T) then decreased. Relationships between Blue color (B) and both of Green color (G) and Red color(R) of tomato fruit (fresh). The obtained results were presented in Table (4) showed that, the correlation between Blue color and both of Green color and Red color of tomato fruit., were significant, where, they were 0.648 and -0.565 at P<0.01, respectively. The curve represents all the values of the red, green and blue color of the different of maturity stage. Pairs of values Blue color and Green color specific contour line dark red on the horizontal plane shows the highest values for Red color as shown in Fig. (12) (>500). Relationships between Green Color (G) and both of Blue Color (B) and Red Color (R) of storage tomato fruit). The obtained results were presented in Table ( Relationships between Intensity (I) and both of Saturation (S) and Hue (H) of storage tomato fruit. The obtained results were presented in Table ( CONCLUSION An image analysis technique was found to serve as a suitable and accurate method for external tomato fruit inspection. Relationships were determined between average of RGB bands and HSI. Multiple regression analysis and correlation coefficient tested the association between RGB and HSI identify the optimum index sensitive to maturity. Possible discrimination by analyzing the colors between the different stages of maturity Tomato Accordingly machine vision can be used successfully in sorting tomatoes based on color. It is recommended using a sophisticated system by connecting the processor directly online by using camera and is connected to a computer program to give signals to the gates of each degree of sorting by color on a continuous basis. The quantitative color appearance evaluation is an adequate system for access to a system that works directly on the sorting and grading machines.

v3-fos

2019-04-25T13:03:14.716Z

2015-01-01T00:00:00.000Z

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Agronomic characterization variety Quebranta in the Ica region, Peru The study was to identify the best strains of the Quebranta variety cultivated in Peruvian region of Ica during campaigns 2011 to 2014. The evaluations were conducted in fourteen vineyards and the criteria to evaluate each one of them was that the same owner vineyard, would identify the best strain Quebranta for their good performance and sanitary quality. Productive parameters as grape weight and number of bunches per vine, average cluster weight, length and width of cluster and Berry weight were evaluated. Within the parameters of vegetative growth was assessed Ravaz index and as parameter for the composition of the grape was evaluated the concentration soluble solids (°Brix), total acidity and pH. Four phenological stages were recorded and defined from observed events in the branch of the year: Phase I comprising bud of winter to sprouting; Phase II of sprouting to full bloom; Phase III of full bloom to veraison and Phase IV of veraison to maturity. At the time of fruit setting were taken leaf samples to assess the State of health of each strain to Grapevine fanleaf virus, Grapevine fleck virus, Grapevine leafroll virus 1, Grapevine leafroll virus 3 and Tomato ringspot virus. The variables average bunch weight (22%), berry weight (20%), bunch length (13%) and bunch width (11%) presented the lowest values coefficient of variation. The variables of weight of grape per vine (52%), number of bunch (42%) and index of Ravaz (60%) has the highest values of coefficient of variation. Four variables were used that showed lower values (25%) coefficient of variation for the weighted average. The variables that presented perfect correlation were berry weight and width of bunch, berry weight and Ravaz index, length of bunch and Ravaz index. The analysis of conglomerate allowed to group the strains in study in two groups which showed a significant difference between them (p < 0.0001). The principal component analysis identified that the variable of weight per bunch, index Ravaz and berry weight distinguished the 77 strains studied. Soluble solids, acidity and pH of the grapes from the fourteen vineyards present significant difference. The phenology of the 77 study strains does not present significant difference. Samples of tender leaves were used for virus scanning. Only the variables of length and width of bunch showed a coefficient of variation acceptable accuracy. The weighted average analysis allowed choosing the best strains which are: QT1; QT5; QCH2; QCH3; QYJ1; QT2; QT3; QYJ4; QT4; QCH1 which will serve as base material for future certifications and improvement of this variety. The variables such as the number of bunch and bunch width feature the highest perfect correlation (0.89%). The grape presented in average 24.67 °Brix, acidity 4.14 g/L tartaric acid and 3.92 of pH. The phenological period total average of the 77 strains was 202 days and shows no significant difference. The results of the analysis of virus were negative for the 77 strains and for the five viruses that were analyzed. Introduction There are 6154 varieties of vine, in 35 states, between members and non-members of the International Organization of Vine and Wine [1]. In spite of this enormous diversity, only a small number of these have commercial importance. Our country was the first in South America in cultivating vineyards and also to produce wines [2]. In the Peru between 1532 and 1580 were founded more than 700 population centers. On the coast, characterized by it is aridity was adapted in a surprising way [3]. The Inca Garcilaso de la Vega, in their Real-world feedback, is the only one that speaks clearly about the introduction of the grapevine. The grape was introduced the Prieta, variety of red ink, black and clear quality of a very particular. It was the only one that was until the early eighteenth century and this it was derived numerous clones as the Quebranta grape. The origin of the varieties of wider dissemination in the country that could be considered as native or creole by its antiquity would correspond to subsequent imports; this would be the case of the Muscatel inks, Black (Tintilla) and Mollar, or the white as the Albilla and Italy [2]. At present there are eight varieties called pisco grapes (Quebranta, Uvina, Negra criolla, Mollar, Albilla, Italy, Muscatel and Torontel), of which the Pisco is elaborated, a grape brandy, colorless considered as the drink flag of Peru and its Appellation of Origin includes the departments of Lima, Ica, Arequipa, Moquegua and Tacna [4]. History tells us that the epicenter and where it is established widely the viticulture is the Valley of Ica, where he founded the city of Valverde in 1563 [5] and continues to enjoy this feature to occupy 55% of national production [6]. The traditional variety and more important in the province of Ica is the Quebranta, object of study in the present work. Is red and black, pleasant-tasting, considered non aromatic, tolerant to phylloxera. It is the result of natural hybridization of Negra criolla with the Mollar [7]. It is possible that although some vineyards are separated a few kilometers, different areas have their dominant a Corresponding author: [email protected] cultivars, fruit of their differences of soil and microclimate [8]. There are few studies of agronomic characterization of the vine in Peru that relates their results with the plant material to spread and future breeding programs. In Ica, the references are based on improving the production and morphological characterization. As a result, there are no commercial nurseries in the material to meet the demand of the new plantations and replants that need certified plants free of viruses and other diseases. The improvement of the propagation material generates: increased vegetative growth of the grapevines, greater longevity of the plants, greater uniformity in the vineyards, greater performance and productive potential in time, better quality of the grapes, greater content of sugar, uniform maturity, best curdle, and uniformity of the caliber of the berries [9]. In spite of the importance of the Quebranta and its derivative the Pisco, there are no studies that treat on the characterization of the plant material. In this sense, the present study aims to obtain propagation material for being able to choose the best strains of each plot of Quebranta representative in the study area, covering a need for future plantations with healthy plant material and agronomic qualities of good. Study area Grape vines (Vitis vinífera) variety Quebranta (Fig. 1), were selected and coded 2011 in eight districts (fourteen vineyards) in the province of Ica (Pueblo Nuevo, Cercado de Ica, San Juan Bautista, Subtanjalla, Pachacutec; Salas Guadalupe, Santiago) ( Table 1). Ica is located in the central coast of Peru. Its climate is typically arid. The daily solar radiation has an average value of 7 hours; the average temperature is 21°C with an average maximum of 29°C and an average minimum of 14°C. As the annual rainfall is nearly zero, the vineyards are irrigated in its majority by flooding during rainy periods (January-March) and in the dry season (April-November) with groundwater. Plant material This work has been started in the year 2011 and its application arrived until 2014. The criterion that was used to mark was that the owners themselves identify as the variety mentioned, that this good performance and the absence of external symptoms of pests and diseases. The conduction system was generally in "Galera from Ica" or modifications of this by each owner of the vineyard. The pruning system that used consisted of leave thumbs of the replacement of two to three buds. The plants should have more than 15 years of sown. Agronomic characterization To determine the agronomic performance of each of the strains of Quebranta, three parameters were measured. [8]. 4. Parameters for the composition of the grape: when the plants reached the of optimum ripeness, the grapes were harvested; these are squeezed and obtained which juice be assessed °Brix, total acidity and pH [8]. 5. The phonological period four phonological stages was evaluated and defined from events observed in the branch of the year: Phase I is from pruning to sprouting; phase II is sprouting to full flowering; phase III is in full flowering to veraison and phase IV is veraison to harvest or ripening. 6. It was also evaluated the health connection to the virus, in the Virus Laboratory at the National Service of Agrarian Health of Peru, located in the city of Lima, the test was conducted DAS-ELISA (Double Antibody Sandwich-Enzyme Linked Immuno sorbent Assay) to assess the health status of the strains regarding on the following viruses: Grapevine fanleaf virus, Grapevine fleck virus, Grapevine leafroll virus 1, Grapevine leafroll virus 3 and Tomato ringspot virus. The samples analyzed were a mix of tender and almost mature leaves collected in time flowering. Statistical analysis The data from the agronomic characterization were analyzed by means of INFOSTAD/Professional version 2013 program. The mean, standard deviation, variance, standard error, the coefficient of variation, minimum and maximum value were used to have an overview on the variability of the quantitative characteristics of the strains under study. Performed was a weighted average to determine the best allocation of the 77 strains, considering the variables with less variability. An analysis of variance was performed multivariate analyzes to determine the significant difference in the composition of the 77 strains under study. We performed a multivariate analysis using the hierarchical cluster analysis using the algorithm of Ward and the Euclidean distance squared, to group the 77 strains of the variety Quebranta in groups with agronomic characteristics of maximum similarity with respect to the variables studied. The principal components analysis was used to identify the quantitative variables that have more weight to differentiate the 77 strains of Quebranta. With a graphic Biplot was observed the relationship between variables and accessions. The canonical discriminate analysis was used to determine the quantitative features that discriminate against varieties defined a priori. Results The descriptive statistics relevant to the studied variables are described in Table 2. To evaluate the weighted average is took four variables and allocated the weight according to importance in the performance of the crop. It was not considered the weight of grapes per plant, number of clusters per plant and Ravaz index, due to the high variability that presented. From the results shown in Table 4 we can see that there is significant difference in the composition of the grapes from some vineyards. The average total phenological period of the 77 strains was 202 days and shows no significant difference (p > 0.05). The result of the analysis of virus was negative for the 77 strains and for the five viruses that were analyzed (Table 5). To find the conglomerate analysis was used all the quantitative agronomic characteristics (total weight and number of bunch per vine, bunch weight, berry weight, length and width of bunch and Ravaz index). The structure obtained by the method of hierarchical clustering of Ward and the Euclidean distance squared, is represented by means of a dendogram in Fig. 2. Two groups were identified at a distance of 135.85. In the Table 6 shows the multivariate analysis of variance to the two groups and sub groups generated from group 2, where it was noted that there was a significant difference for all. In Figure 3 shows that the variable weight of grapes per vine has a greater association with the grapes from Yajasi LC, the variable weight of berry has a greater association with the grapes from Macacona. The grape coming from Yanquiza and The Pobres was not significant in the production in relation to the agronomic characteristics. The canonical discriminate analysis allows corroborating the analysis of principal components above, and determining the characteristics discriminates. The variable number of clusters per vine is the most important to discriminate the 77 strains of vine Quebranta, with the highest positive value (0.63) on the shaft. However the axis 2 shows two important variables of bunch length and weight of berry with a value of (0.58) indicating that they are the most important variables to discriminate among the 77 strains of Quebranta grapevine. Discussion The five best strains are QT1, QT5, QCH3, QCH2 and QYJ1, are found in the vineyards located on Tres Esquinas, The conglomerate analysis and multivariate variance it allowed observe that strains are grouped into two groups. The second group has three subgroups in turn. The grouping of vineyards are not given exactly for the location the vineyard respect to the location in the province of Ica, but strains reached the best weighted average. Much so that the vineyards grouped in the first group (17 strains), are characterized by being located in up town and low prevalent strains with the best weighted average QT, QYJ and There aren't others studies where have been characterized the variety Quebranta, therefore we compared with a table grape variety well known Red Globe, that on average bunch weight 559 g and 9.42 g berry [10]. The high variability observed for grape weight per strain, number bunches and Ravax index, this is due to the 01023-p.7 high modification of the conduction system "Galera from Ica" in each of the vineyards evaluated with many arms and pythons. The Ravaz index must be between 4 and 9. Above these values is considered that there may be a risk from overwork harvest [8]. In CITEagroindustrial, to carry out the fermentation process of the musts for making Pisco, this must be within the range of °Brix 22-26, acidity 3.5-7.0 g/L tartaric acid and pH between 3.2 and 3.7. The 77 strains are within the ranges for °Brix and total acidity but they exceed the threshold of pH in the majority of strains. Bear in mind that when you exceed the 26 °Brix, it is lost grape aromas and starts a process of dehydration with less acidity as notes in the grape evaluated in San Antonio (4.55 g/L tartaric acid). Quebranta is called a non-aromatic variety [11] in comparison with Muscat of Alexandria (called in Peru, "Italy") [1]. The phenological period of this variety can be compared with other that are cultivated in Ica. Table Grapes Red Globe (total 190 days) and Thompson Seedless (total 176 days) and Syrah wine varieties (total 193 days) and Sauvignon blanc (total 168 days). The variety Quebranta has the longest phenological cycle because it is considered maturation when it fluctuates between 24 -26 °Brix unlike other varieties of table and wine are harvested at 16 and 22 °Brix, respectively (unpublished data). About healing, it isn't excluded that strains susceptible to infection for other asymptomatic virus affecting the grapevine and present in Ica (results not shown). The strains with less weighted average are found in soils with high salinity. In this regard the grapevine is little tolerance to salinity (EC < 2-3 dS/m) as to overcome the high osmotic potential that produce salts in the soil, the plant should increase their respiratory activity getting the energy needed; causing a decrease in force, adversely affecting the biomass of stem, and leaves thumbs and fertility of the buds, accompanied by a reduced harvest. And also the effects of high pH cause low availability (shortages) of the elements phosphorus, manganese, boron, copper and zinc. Increases availability (toxicity) of the elements molybdenum, sulfur and calcium. Modifying the structural stability of the soil by flocculation of clays [12]. The strains chosen are an alternative for the production of grapevine Quebranta in the agroclimatic conditions of Ica in virtue of the performance advantages and agronomic characteristics, therefore it is suggested your validation agronomic. The parameter of grape composition is within the thresholds with which the CITEagroindustrial (Quilloay, YajasiMR y huaranga), therefore it is suggested your validation agronomic of this strains. The vineyards study are located in the zone high, medium, and low in the province of Ica, each one of them was independently managed, it is known that is carried out by each producer, do not receive direct advice of specialized professionals, only by the knowledge acquired in training specific. It performs a basic NPK fertilization and guano. Pests are treated with sulfur until that the bunch is in pea size, and then in some cases apply synthetic fungicides. The time of pruning is important and is performed in the majority of vineyards when the moon is in the full moon phase. Usually the "galleries" are located around the lots, where there is an irrigation ditch and such have good moisture. These plants have more than 15 years of installed and in only a vineyard are appreciation fungi of wood where it was observed that the weighted average was one of the lowest QY5. Other tasks are very similar in all the vineyards. Entirely strains are free; you are expected to know the agronomic performance when they are grafted a pattern that to be drought tolerant or phylloxera and strains are grown in a same microclimate and are brought to the same cultural and agronomic management. Conclusion The best five strains are QT1, QT5, QCH3, QCH2 and QYJ1, are found in vineyards Tres Esquinas, Cachiche and YajasiLC. This strains will serve to start plans mass propagation and for further research.

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2016-05-18T17:47:16.466Z

2015-08-21T00:00:00.000Z

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Effect of dietary formic acid and astaxanthin on the survival and growth of Pacific white shrimp (Litopenaeus vannamei) and their resistance to Vibrio parahaemolyticus A 90-day feeding trial was conducted to evaluate the effects of formic acid (FA) and astaxanthin (AX) on growth, survival, immune parameters, and tolerance to Vibrio infection in Pacific white shrimp. The study was divided into two experiments. In experiment 1, postlarvae-12 were randomly distributed into six groups and then fed four times daily with six experimental diets contained 0.3 % FA, 0.6 % FA, 50 ppm AX, 0.3 % FA + 50 ppm AX, 0.6 % FA + 50 ppm AX, or none of these supplements (control diet). After 60 days of the feeding trials, the body weight of all treatment groups was not significantly different from the control group, although shrimp fed formic acid had significantly lower body weight than shrimp fed 50 ppm AX. However, the 0.6 % FA + 50 ppm AX group had a significantly higher survival rate (82.33 ± 8.32 %) than the control group (64.33 ± 10.12 %). In experiment 2, Vibrio parahaemolyticus was added to each tank to obtain a final concentration of 104 colony-forming units/mL. Each treatment group received the aforementioned diets for another 30 days. At the end of this experiment, there was no difference in the weight gain among all experimental groups. However, the survival rate of shrimps whose diet included FA, AX, and their combination (in the range of 45.83–67.50 %) was significantly higher than the control group (20.00 ± 17.32 %). FA-fed shrimps also had significantly lower total intestinal bacteria and Vibrio spp. counts, while immune parameters [total hemocyte count (THC), phagocytosis activity, phenoloxidase (PO) activity, and superoxide dismutase (SOD) activity] of AX-fed groups were significantly improved compared with the other groups. In conclusion, FA, AX, and their combination are useful in shrimp aquaculture. Electronic supplementary material The online version of this article (doi:10.1186/s40064-015-1234-x) contains supplementary material, which is available to authorized users. coloration owing to pigment loss, as well as an atrophied hepatopancreas. These signs may become apparent as early as 4 days after stocking (Munkongwongsiri et al. 2013). Vibrio parahaemolyticus is the suspected agent that causes mass mortality as it induced 100 % mortality with typical EMS pathology to experimental shrimp (Tran et al. 2013). Because the usage of antibiotics in shrimp aquaculture is discouraged, it is necessary to find an alternative solution to prevent bacterial infection. Organic acids are among the most promising substances as they have been reported to possess anti-Vibrio spp. activities (Mine and Boopathy 2011;Adams and Boopathy 2013;da Silva et al. 2013), and increased survival rate of shrimps (Walla et al. 2012;Su et al. 2014;Romano et al. 2015;Ng et al. 2015). Astaxanthin, a type of carotenoid, can also improve shrimp survival rate and enhance resistance to several stress conditions, such as low dissolved oxygen, low salinity, low temperature, and ammonia stress (Chien et al. 2003;Pan et al. 2003;Chien and Shiau 2005;Flores et al. 2007;Niu et al. 2009). Therefore, both organic acids and astaxanthin have the potential to be used in shrimp farming as feed additives. The objectives of this study were to evaluate the effect of dietary supplementation of formic acid and astaxanthin on growth, survival and tolerance to V. parahaemolyticus infection in Pacific white shrimp under laboratory conditions. The effects of these substances on total intestinal bacterial counts, intestinal Vibrio spp. counts, and some immune parameters of shrimp were also examined. Experiment 1 The effects of formic acid and astaxanthin on growth and survival of Pacific white shrimp postlarvae. Shrimps and experimental protocol The experiments were carried out at the Aquaculture Business Research Center Laboratory, Faculty of Fisheries, Kasetsart University, Thailand. Postlarvae-9 (PL-9) of Pacific white shrimp were obtained from a hatchery in Chachoengsao Province, Thailand. After 3 days of acclimation, shrimps (PL-12) were randomly distributed into 24 × 500-L fiberglass tanks (four replicate tanks per treatment). Each tank was stocked with 75 shrimp. Each treatment group was fed with one of the six diets four times daily to satiation for 60 days. Salinity throughout the experiment was maintained at 25 ppt, dissolved oxygen above 4 ppm, and water temperature at 29 ± 1 °C. Leftover feed and feces were siphoned daily, and 10 % of the water was exchanged every 3 days. The average body weight and survival rate of shrimp were recorded after a 60-day experimental period. Ten shrimps from each tank were randomized and weighted individually by two-decimal point balance. Experiment 2 The effects of formic acid and astaxanthin on growth, survival, intestinal bacteria, and immune responses of Pacific white shrimps challenged with Vibrio parahaemolyticus. Shrimps and experimental protocol Shrimps from each tank in experiment 1 were randomly distributed into new 24 × 500-L fiberglass tanks (four replicate tanks per treatment). The stocking density was 30 shrimps per tank. At the beginning of this experiment (0 day), Vibrio parahaemolyticus was added into each tank to obtain final concentration of 10 4 colony-forming units (CFU)/mL, which is the normal concentration of Vibrio in the water of shrimp farm as described by Sung et al. (2001) and Lavilla-Pitogo et al. (1998). V. parahaemolyticus used for immersion challenge test in this study was collected from the EMS farm in Thailand using method described by Joshi et al. (2014). Each treatment group received the same diet as in experiment 1 four times daily for another 30 days. Salinity, dissolved oxygen, and water temperature were maintained as in the experiment 1. Leftover feed and feces were siphoned every 2 days. Growth and survival study The weight of shrimp from each treatment was measured and their survival rate was recorded on the 30th day after being challenged with V. parahaemolyticus at 10 4 CFU/ mL. Intestinal bacterial study Five shrimp from each group were randomized and their intestines collected on the 10th, 20th, and 30th day. The intestine of each shrimp was homogenized and spread on TCBS (selective media for Vibrio spp. culture) or NA (general media for most bacterial cultures) by the spread plate technique, then incubated at 37 °C for 24 h. Finally, all colonies of bacteria were counted and calculated as CFU/g unit. Immune parameters study The immune parameters were measured at the end of the feeding trial. Ten shrimp per treatment were used for immunological tests. A hemolymph sample of 250 µL from each shrimp was withdrawn from the base of the 3rd walking leg using a syringe containing 750 µL of precooled (4 °C) anticoagulant (0.114 M trisodium citrate, 450 mM NaCl, 10 mM KCl, 10 mM HEPES at pH 7.4) (Nonwachai et al. 2010). The hemolymph-anticoagulant mixture was used to measure total hemocyte count (THC), phagocytosis activity, phenoloxidase (PO) activity, superoxide dismutase (SOD) activity, and bactericidal activity. Phagocytosis activity Phagocytotic activity was determined according to Itami et al. (1994). Collected shrimp hemocytes were rinsed with shrimp saline (a solution of NaCl 28.4 g, MgCl 2 ·6H 2O 1.0 g, MgSO4·7H 2O 2.0 g, CaCl 2 ·2H 2O 2.25 g, KCl 0.7 g, glucose 1.0 g, and HEPES 2.38 g/L) and the viable cell number adjusted to 1 × 10 6 cells/mL. The cell suspension (200 µL) was inoculated onto a cover slip. After 20 min, the cell suspension was removed and rinsed with shrimp saline three times. Heat-killed yeast preparation (2 mL) was added and incubated for 2 h. Next, the heat-killed yeast preparation was removed and the cell suspension rinsed with shrimp saline five times to reach a concentration of 5 × 10 8 cells/mL, and fixed with 100 % methanol. Then, the cover slip was stained with Giemsa stain and mounted with Permount slide mounting fluid. Two hundred hemocytes were counted for each sample. Phagocytic activity, defined as percentage phagocytosis was expressed as: 3. Phenoloxidase activity Phenoloxidase activity was measured spectrophotometrically by recording the formation of dopachrome produced from l-dihydroxyphenylalanine, following a modification of a published protocol (Supamattaya et al. 2000). The hemolymph-anticoagulant mixture was washed three times with shrimp saline and centrifuged at 1000 rpm and 4 °C for 10 min. Hemocyte lysate was prepared from hemocytes in cacodylate buffer (pH 7.4; 0.01 M sodium cacodylate, 0.45 M sodium chloride, 0.01 M calcium chloride, and 0.26 M magnesium chloride; pH 7.0) by using a sonicator at 30 amplitude for 5 s, and the suspension was then centrifuged at 10,000 rpm at 4 °C for 20 min and the supernatant collected. Then 200 µL of 0.25 % trypsin in cacodylate buffer was mixed into the 200 µL of hemocyte lysate followed by 200 µL of l-dihydroxyphenylalanine at 4 mg/mL as substrate. Enzyme activity was measured as the absorbance of dopachrome at 490 nm wavelength. The protein content in hemocyte lysate was measured following a published protocol (Lowry et al. 1951). The phenoloxidase activity was calculated as the increase in optimum density per minute per milligram of protein. 4. Superoxide dismutase activity SOD activity was measured by its ability to inhibit superoxide radical-dependent reactions using a Ransod Kit (Randox, Crumlin, UK). This method is based on the formation of red formazan during a reaction of 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride (INT) and superoxide radical, which is assayed in a spectrophotometer at 505 nm. The reaction mixture (1.7 mL) contains 0.05 mM xanthine and 0.025 mM INT dissolved in 50 mM CAPS (pH 10.2) and 0.94 mM EDTA. In the presence of xanthine oxidase, superoxide and uric acid are produced from the xanthine. The superoxide radicals then react with INT to produce a red formazan dye. The hemolymph-anticoagulant mixture was centrifuged at 3000 rpm and 4 °C for 10 min. Plasma was removed, and the pellet was resuspended with 3 mL of 0.9 % NaCl and centrifuged again. The supernatant was discarded, and the pellet was resuspended with 2 mL of triple distilled water at 4 °C. A 50 µL aliquot of resuspended hemocytes was placed in each well of a 96-well plate that contained 200 Percentage phagocytosis = (phagocytic hemocytes/total hemocytes) × 100 µL of reaction mixture. Fifty microliters of xanthine oxidase solution was added to each well, and the absorbance measured at 505 nm and 37 °C. The rate of reaction was estimated from the absorbance readings of 0.5 and 3 min after adding xanthine oxidase. A reference standard of SOD was supplied with the Ransod Kit. One unit of SOD was defined as the amount required to inhibit the rate of xanthine reduction by 50 %. The specific activity was expressed as SOD units/mL. 5. Bactericidal activity Bactericidal activity was measured as described by Supamattaya et al. (2000). Serum was separated from the hemocytes of each shrimp sample before diluting in 2.6 % NaCl at the following ratios: 1:2, 1:4, 1:8, 1:16, and 1:32. Then 0.5 mL of each serum dilution was used for the assay. For the negative control, 0.1 mL of NaCl was used in the assay. One tenth of a milliliter of Vibrio harveyi suspension (8.2 × 10 6 CFU/ mL) was added to each serum dilution and the control. The treatments were incubated at room temperature for 3 h before enumerating the bacteria. The results were recorded from a dilution that could decrease 50 % of V. harveyi compared with the control. Statistical analysis Results are presented as the mean ± standard deviation. One way ANOVA and Duncan's New Multiple Range test were used to compare data among treatments. Differences were considered significant if p < 0.05. Experiment 1 The effects of formic acid and astaxanthin on growth and survival of Pacific white shrimp postlarvae After 60 days of dietary administration, shrimp fed with 50 ppm AX had the highest average body weight (4.45 ± 0.45 g), followed by shrimp fed with 0.3 % FA + 50 ppm AX (4.38 ± 0.37 g), 0.6 % FA + 50 ppm AX (4.05 ± 0.21 g) and the control group (4.18 ± 0.05 g). However, the body weight of all FA and AX-fed shrimps were not significantly different from the control group. The average survival rate of shrimp fed with 0.6 % FA + 50 ppm AX was 82.33 ± 8.32 % which was highest among all the other groups and significantly higher than the control group (64.33 ± 10.12 %) (Additional file 1: Table S1). Experiment 2 The effects of formic acid and astaxanthin on growth, survival, intestinal bacteria, and immune responses of Pacific white shrimps challenged with Vibrio parahaemolyticus. At the end of the feeding trial, the average weight gain of 0.3 % FA + 50 ppm AX-fed shrimp was highest, being 2.97 ± 0.83 g. Nevertheless, no significant differences among the six experimental groups were observed. The average survival rate of all FA and AXfed shrimps were significantly higher than the control group (20.00 ± 17.32 %) and the best result was obtained in the 0.6 % FA + 50 ppm AX-fed group (67.50 ± 3.33 %) (Additional file 1: Table S2). For the intestinal bacterial study, both Vibrio spp. count and total bacterial count of all four FA-fed groups (namely, 0.3 % FA, 0.6 % FA, 50 ppm AX + 0.3 % FA, and 50 ppm AX + 0.6 % FA) were significantly lower than the control and the 50 ppm AX-fed groups throughout the feeding trial. The lowest intestinal bacterial counts were observed in shrimps with a diet containing the high dose of FA (i.e. 0.6 % FA). On the 30th day of the experiment, the two lowest intestinal Vibrio spp. counts were observed in 50 ppm AX + 0.6 % FA and 0.6 % FA groups (1.30 ± 0.58 and 1.60 ± 0.70 × 10 6 CFU/g, respectively), whereas the highest count was in the control group (47.20 ± 25.40 × 10 6 CFU/g). Similarly, the two lowest total bacterial counts were in the 0.6 % FA and 50 ppm AX + 0.6 % FA groups (2.80 ± 1.30 and 3.10 ± 0.70 × 10 6 CFU/g, respectively), while the highest count was from the control group (45.00 ± 27.40 × 10 6 CFU/g) (Figs. 1, 2). The immune parameters of shrimp were significantly influenced by AX in the shrimp feed. Shrimps fed with diets containing AX (namely, 50 ppm AX + 0.3 % FA, 50 ppm AX + 0.6 % FA, and 50 ppm AX) had a total hemocyte count (THC) (Fig. 3), phagocytosis activity (Fig. 4), and phenoloxidase (PO) activity (Fig. 5) significantly higher than the control and the FA groups. Superoxide dismutase (SOD) activity (Fig. 6) of 50 ppm AX-fed shrimps but not the 50 ppm AX + 0.3 % FA and 50 ppm AX + 0.6 % FA groups showed a significant increase compared with shrimps that not fed AX. However, the bactericidal activity of the shrimp's hemolymph from all groups was at the same serum dilution, being 1:4 (Additional file 1: Table S3). Discussion Organic acids are widely used as animal food additives and preservatives for preventing food deterioration. As a group these compounds primarily include the saturated straightchain monocarboxylic acids and their derivatives (Ricke 2003). Many of them are available as sodium, potassium, or calcium salts because they are generally odorless, easier to handle, less corrosive, and may have higher solubility than free acids (Papatsiros and Billinis 2012). Organic acids possess antimicrobial activity against several pathogenic Fig. 1 The total number of Vibrio spp. (10 6 CFU/g) in the intestine of Pacific white shrimp (n = 5) after being challenged with V. parahaemolyticus at 10 4 CFU/ml. The data are presented as the mean ± standard deviation. Different letters above the bars indicate whether means are significantly different from each other (p < 0.05) bacteria such as Escherichia coli, Salmonella spp., and Vibrio spp. (Ricke 2003;Papatsiros and Billinis 2012;da Silva et al. 2013). Undissociated forms of organic acids can easily penetrate bacterial cell membranes, and dissociate into anions and H + within the cytoplasm (Ricke 2003;Beales 2004;Lückstädt and Mellor 2011). Once inside the bacterial cells, they reduce intracellular pH and disrupt the cytoplasmic membrane, protein synthesis system, genetic materials, and metabolic enzymes. In addition, because the bacterial cell uses ATP to pump the excess H + out of cells, organic acids also deplete ATP levels and affect the cell's ability to maintain pH homeostasis (Ricke 2003;Beales 2004;Lückstädt and Mellor 2011). However, not all organic acids have effects on bacteria. In Fig. 2 The total number of bacteria (10 6 CFU/g) in the intestine of Pacific white shrimp (n = 5) after being challenged with Vibrio parahaemolyticus at 10 4 CFU/mL. The data are presented as the mean ± standard deviation. Different letters above the bars indicate whether means are significantly different from each other (p < 0.05) Fig. 3 The total hemocyte count (10 5 cells/ml) of Pacific white shrimp (n = 10) after being challenged with Vibrio parahaemolyticus at 10 4 CFU/mL. The data are presented as the mean ± standard deviation. Different letters above the bars indicate whether means are significantly different from each other (p < 0.05) fact, organic acids associated with specific antimicrobial activity are short-chain acids (C1-C7) and are either simple monocarboxylic acids such as formic, acetic, propionic, and butyric acid, or are carboxylic acid bearing a hydroxyl group such as lactic, malic, tartaric, and citric acids (Dibner and Buttin 2002; Papatsiros and Billinis 2012). Organic acids are mainly used as feed additives for improving growth performance of pigs and poultry (Dibner and Buttin 2002;Franco et al. 2005;Lückstädt and Mellor 2011;Papatsiros and Billinis 2012); there are also reports on the benefit of organic acids in aquatic animals, including red hybrid tilapia (Ng et al. 2009;Koh et al. 2014), yellowtail (Sarker et al. 2012), sturgeon (Khajepour andHosseini 2012), rohu (Baruah et al. 2007), black tiger shrimp , and Pacific white shrimp (Walla et al. 2012;da Silva et al. 2013;Su et al. 2014;Romano et al. 2015). Nevertheless, the result Fig. 4 The phagocytosis activity (%) of Pacific white shrimp (n = 10) after being challenged with Vibrio parahaemolyticus at 10 4 CFU/mL. The data are presented as the mean ± standard deviation. Different letters above the bars indicate whether means are significantly different from each other (p < 0.05) Fig. 5 Phenoloxidase activity (units/min/mg protein) of Pacific white shrimp (n = 10) after being challenged with Vibrio parahaemolyticus at 10 4 CFU/ml. The data are presented as the mean ± standard deviation. Different letters above the bars indicate whether means are significantly different from each other (p < 0.05) from experiment 1 showed that shrimp fed FA had significantly lower body weight than shrimp fed 50 ppm AX and the growth was slightly less than the control group. This indicated that formic acid did not promote the growth of the shrimp and might have some negative effect on shrimp growth. Other short chain fatty acids may enhance the growth, for example, 2 % organic acids blend (consisted of a blend of formic, lactic, malic and citric acids) or 2 g/kg citric acid (Su et al. 2014). Despite no clear improvement of growth and survival of uninfected shrimp postlarvae in our study, the use of formic acid did increase the survival rate of V. parahaemolyticusinfected juvenile shrimps significantly compared with the control group. This result was consistent with the intestinal bacterial study, i.e. shrimp fed formic acid had significantly lower Vibrio spp. and total bacterial counts compared with those fed no formic acid. The similarity between Vibrio spp. counts and total bacterial counts suggested that Vibrio spp. are significant component of shrimp's intestinal microflora (Moss et al. 2000;Oxley et al. 2002;Liu et al. 2011). The antimicrobial effect of formic acid against Vibrio spp. was reported in vitro as well (Mine and Boopathy 2011;Adams and Boopathy 2013;da Silva et al. 2013). Considering all of these aspects, the antibacterial property of formic acid may reduce Vibrio infection to Pacific white shrimp (Papatsiros and Billinis 2012;Adams and Boopathy 2013;da Silva et al. 2013) by penetrating the cell wall of bacteria in the undissociated form, then releasing H + and destabilizing the intracellular pH of the bacterial cytoplasm, leading to death (da Silva et al. 2013). Astaxanthin is a pigment that belongs to the xanthophyll class (the oxygenated derivatives of carotenoids) and widely used in salmon and crustacean aquaculture to provide a desirable reddish-orange color. Astaxanthin possesses a potent antioxidant property and has an important role in larval growth and reproductive success of crustaceans. It occurs naturally in green microalgae Haematococcus pluvialis and red yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma). However, since farmed crustaceans often do not have the opportunity to access a natural source of astaxanthin, and they cannot synthesize Superoxide dismutase activity (SOD units/mL) of Pacific white shrimp (n = 10) after being challenged with Vibrio parahaemolyticus at 10 4 CFU/ml. The data are presented as the mean ± standard deviation. Different letters above the bars indicate whether means are significantly different from each other (p < 0.05) carotenoids de novo, total astaxanthin must be obtained from their feed (Higuera- Ciapara et al. 2006;Seabra and Pedrosa 2010). The growth of AX-fed shrimp in the experiment 1 was significantly better than the FAfed shrimp, but was not significantly different from the control group. The survival rate of shrimp fed with AX was not significantly different from the control group. However, the survival rate of V. parahaemolyticus-infected shrimp in the experiment 2 was improved significantly. Nevertheless, unlike formic acid, astaxanthin did not suppress intestinal bacterial populations, suggesting that other mechanisms may be responsible for the increased survival rate. In fact, many immune parameters of astaxanthin-fed shrimps were improved, including total hemocyte count (THC), phagocytosis activity, phenoloxidase (PO) activity, and superoxide dismutase (SOD) activity. These outcomes suggest that astaxanthin had an immunostimulatory property preventing V. parahaemolyticus infection in Pacific white shrimp. Antioxidant activity of carotenoids may be involved in the immunomodulatory effect; by quenching singlet oxygen and free radicals, carotenoids can protect white blood cells from oxidative damage (Bendich 1989). Superoxide dismutase (SOD) is an antioxidant enzyme that protect cells against oxidative stress by scavenges superoxide anion (O 2 − ) and it is used as an indicator of immune responses (Campa-Córdova et al. 2002a, 2002b. Given that astaxanthin also possesses an antioxidant property, this is suggests that such mechanism must be take part in immunomodulation. Furthermore, the effects of carotenoids on enhancing cell-mediated and humoral immune responses of vertebrates are also documented (Bendich 1989;Chew and Park 2004). Several studies have reported that dietary carotenoids can increase the immune parameters, enhance the survival rate, or act as a prophylactics to pathogens for many aquatic animals such as common carp Even if formic acid and astaxanthin have different modes of action to shrimp, both had positive effects on their resistance to bacterial challenge. Meanwhile, our results showed that a combination of formic acid and astaxanthin was no better than using singly or in combination. The only exception was that uninfected shrimp fed 0.6 % FA + 50 ppm AX had a significantly higher survival rate compared with the control group. In general, formic acid (FA 0.3 and 0.6 %) and astaxanthin (50 ppm AX) were equally effective in preventing V. parahaemolyticus infection of Pacific white shrimp. Given the fact that formic acid is less expensive than astaxanthin, using formic acid as feed additive in shrimp farming may be considered more economically worthwhile. Conclusion Astaxanthin (50 ppm AX) can be used as growth promoter in uninfected Pacific white shrimp, while formic acid (0.3 and 0.6 % FA) and AX can enhance the survival rate of Vibrio parahaemolyticus-infected shrimp in laboratory conditions. In addition, FA-fed shrimps had lower intestinal Vibrio spp. and total bacterial counts, whereas AX-fed shrimps showed improvement in many immune parameters. The results of our study suggest that using FA, AX, and their combination as a feed additive can prevent V. parahaemolyticus infection in shrimps.

v3-fos

2015-09-18T23:22:04.000Z

2015-08-01T00:00:00.000Z

14749754

s2

The Efficacy and Underlying Mechanism of Sulfone Derivatives Containing 1,3,4-oxadiazole on Citrus Canker The objectives of the current study were to isolate and identify the pathogen responsible for citrus canker and investigate the efficacy of sulfone derivatives containing 1,3,4-oxadiazole moiety on controlling citrus canker caused by Xanthomonas citri subsp. citri (Xcc) under in vitro and field conditions. In an in vitro study, we tested eight sulfone derivatives against Xcc and the results demonstrated that compound 3 exhibited the best antibacterial activity against Xcc, with a half-maximal effective concentration (EC50) value of 1.23 μg/mL, which was even better than those of commercial bactericides Kocide 3000 (58.21 μg/mL) and Thiodiazole copper (77.04 μg/mL), respectively. Meanwhile, under field experiments, compound 3 treatments demonstrated the highest ability to reduce the disease of citrus canker in leaves and fruits in two different places relative to an untreated control as well as the commercial bactericides Kocide 3000 and Thiodiazole copper. Meanwhile, compound 3 could stimulate the increase in peroxidase (POD), polyphenol oxidase (PPO), and phenylalanine ammonia lyase (PAL) activities in the navel orange leaves, causing marked enhancement of plant resistance against citrus canker. Moreover, compound 3 could damage the cell membranes, destruct the biofilm formation, inhibit the production of extracellular polysaccharide (EPS), and affect the cell membrane permeability to restrain the growth of the bacteria. Introduction Citrus canker, a serious disease of most commercial citrus cultivars in subtropical citrus-producing areas of the world, has a significant impact on national and international citrus markets and trade [1][2][3]. Citrus canker is a disease that is characterized by the formation of necrotic raised lesions on leaves, stems, and fruit of citrus trees, including limes, oranges, and grapefruit. Once infected with the disease, citrus canker can cause defoliation, twig dieback, general tree decline, blemished fruit, and premature fruit drop [4]. Citrus canker is caused by the bacterial pathogen Xanthomonas citri subsp. citri (Xcc) [5][6][7]. This bacterium is dispersed in rain splash often associated with wind [8][9][10][11][12] and enters the plant directly through stomata or through wounds, and then it grows in the intercellular spaces of the spongy mesophyll [1]. At present, copper-containing bactericides are the primary control measure for citrus canker. However, long-term use of copper bactericides not only led to resistance to copper in xanthomonad populations but also affected the environment and plant health [13]. Therefore, searching for new antibacterial agents for controlling the disease remains a daunting task in pesticide science. Over the past few years, we have attracted considerable attention on the studies of the synthesis and bioactivity of sulfone derivatives containing 1,3,4-oxadiazole moiety and demonstrated that sulfone derivatives containing 1,3,4-oxadiazole moiety ( Figure 1) display potent antibacterial activities against rice bacterial leaf blight and leaf streak. Specifically, compound 2-(methyl sulfonyl)-5-(4-fluorobenzyl)-1,3,4-oxadiazole (CAS Registry Number: 1596304-56-1) showed the best antibacterial activity against rice bacterial leaf blight and leaf streak caused by Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), with the half-maximal effective concentration (EC50) values of 1.07 and 7.14 μg/mL, respectively [14]. However, in our previous work, we only reported and discussed the compound activities in the control of rice bacterial leaf blight and leaf streak. The biological effects and the underlying mechanism of these sulfone derivatives containing 1,3,4-oxadiazole moiety on citrus canker were not reported. The objectives of the current study were to isolate and identify the pathogen responsible for citrus canker and investigate the efficacy and the underlying mechanism of sulfone derivatives containing 1,3,4-oxadiazole moiety on controlling citrus canker caused by Xcc under in vitro and field conditions. To the best of our knowledge, this is the first report on the bioactivity evaluation and the underlying mechanism of sulfone derivatives containing 1,3,4-oxadiazole moiety on citrus canker. Then, the genomic DNA was conducted to PCR amplification using the bacterial universal primer pair 27F/1492R. After PCR analysis, the whole PCR reaction volume was electrophoresed for 25 min onto 1.5% agarose gel in Tris-acetate-EDTA (TAE) buffer with 5 μL 4S green nucleic acid stain. As shown in Figure 2, the PCR amplicon with a molecular weight of about 1500 bp was obtained. Then, the whole 16S rDNA sequence of the sample, sequenced at Sangon Corporation (Shanghai, China), showed that the sequence identity between the sample and Xanthomonas citri subsp. citri (accession number: CP008989) was 99%. Thus, it appears to be likely that the strain is Xanthomonas citri subsp. citri. In Vitro Antibacterial Bioassay In this study, the inhibitory effect of the target compounds 1-8 were evaluated for their antibacterial activities in vitro against Xcc via the turbidimeter test. For comparison, the activity of the commercial bactericides Kocide 3000 and Thiodiazole copper, two positive controls, were evaluated in the same conditions. The results of the preliminary bioassays, as listed in Field Trials against Citrus Canker Field trials of compound 3 against citrus canker were conducted in two different places, Congjiang and Luodian, Guizhou Province, in May 2014. Results were summarized in Table 2. Table 2 indicated that, 14 days after the third spraying in Congjiang, Guizhou Province, the control efficiencies in leaves and fruits of compound 3 against citrus canker were 66.31% and 69.03%, respectively, which were even better than those of Kocide 3000 (55.13% and 53.76%, respectively) and Thiodiazole copper (61.47% and 57.73%, respectively). Meanwhile, the result, shown in Table 2, indicated that, 14 days after the third spraying in Luodian, Guizhou Province, the control efficiencies in leaves and fruits of compound 3 against citrus canker, with the values of 60.43% and 64.51%, respectively, were also superior to those of Kocide 3000 (50.93% and 52.77%, respectively) and Thiodiazole copper (55.72% and 56.52%, respectively). Determination of Peroxidase (POD), Polyphenol oxidase (PPO), and Phenylalanine ammonia lyase (PAL) Activities As shown in Figure 3, seven days after the third spraying, the POD, PPO, and PAL activities of the navel orange leaves, treated with compound 3, were 61.66, 83.80, and 29.73 U/mg·min, respectively. Meanwhile, the POD, PPO, and PAL activities of the untreated blank control were 15.36, 19.30, and 6.86 U/mg·min, respectively. Obviously, Figure 3 showed that, compared with the untreated blank control, the POD, PPO, and PAL activities in the treatment group increased by approximately 4.01, 4.34, and 4.33 times, respectively. In conclusion, the enzyme activities changes of POD, PPO, and PAL preliminarily demonstrated that compound 3 can improve the disease resistance of plants that rely on inducible defense responses in the form of enzymes that are activated for controlling citrus canker. Effect on the Integrity of Bacterial Cell Membranes The release of intracellular components that absorb at 260 nm is an indication of membrane damage. As shown in Figure 4, when bacterial suspensions were treated with different concentrations of compound 3, the A260 increased rapidly at first, then slowed its rate up to 120 min. A260 values were greater in suspensions treated with 20 μg/mL than with 10 μg/mL of compound 3. Thus, the damage of cell membranes by compound 3 is concentration-dependent, which agrees with the findings for bactericidal activity. Effect on the Biofilm Formation To study the effect of compound 3 on biofilm formation, compound 3 was hypothesized to be involved in biofilm formation. As shown in Figure 5, compound 3 could significantly affect the biofilm formation with the reduction percentages of 4.67%, 13.65%, 26.54%, and 43.32% at the concentration of 2.5, 5, 10, and 20 μg/mL, respectively. The results revealed that the destruction of biofilm formation may play an important role in the antibacterial activity of compound 3 against Xcc. Effect on Cell Membrane Permeability of Xcc We determined the electric conductivity of the cell suspensions for Xcc treated with compound 3 at the ultimate concentrations of 10 and 20 μg/mL, respectively. As shown in Figure 6, the electric conductivity showed a time-dependent increasing manner. It was found that the electric conductivity of suspensions for Xcc began to increase after being treated with compound 3, with the concentration of 10 and 20 μg/mL, respectively, and when the incubation time was 120 min, the relative conductivity increased by 50.28% and 60.69% for Xcc compared with the untreated blank control. Figure 6. Electric conductivity (mean value ± SD) of cell suspensions for Xcc treated with compound 3. T1: 10 μg/mL, T2: 20 μg/mL. Determination of Exopolysaccharide (EPS) Content The EPS content was determined by comparison of absorbance at 490 nm of inoculated Xcc either treated or untreated with compound 3. EPS content was calculated using the standard curves ( Figure 7a). As shown in Figure 7b, compound 3, at the concentrations of 2.5, 5, 10, and 20 μg/mL, could obviously inhibit the EPS production of Xcc, with inhibition rates of 21.69%, 51.60%, 75.95%, and 94.34%, respectively. These results demonstrated that compound 3 could reduce EPS production to lower the pathogenic ability of Xcc. Bacteria Isolation and Purification Bacteria were isolated from infected leaves and fruits of navel oranges collected from Congjiang, Guizhou Province of China using the previously described method [15] with some modifications. Small portions of infected fruits and leaves were disinfected using 75% ethanol, and then washed three times with sterile distilled water. These tissue portions were transferred to Nutrient Agar (NA) plates and incubated at 28-30 °C for approximately 48 h. Bacteria colonies growing around the tissue mass were aseptically moved using an inoculation loop and transferred to flesh NA plates and incubated at 28-30 °C for approximately 48 h. Discrete bacterial colonies were selected and re-streaked on flesh NA plates. Individual colonies were isolated, sub-cultured twice to ensure purity, and the single-spore isolates were stored in sterile distilled water at 4 °C for later use. DNA Extraction, PCR Amplification, and Sequencing of Species Prior to DNA extraction, each isolate was sub-cultured on Nutrient Broth (NB) medium at 28-30 °C for 48 h. Approximately 25 mg of bacteria were collected for genomic DNA extraction using the TIANamp bacteria DNA distilling kit (Tiangen-Biotech Corporation LTD, Beijing, China) and DNA concentration and quality were estimated using an ASP-3700 spectrophotometer (ACTGene, Piscataway, NJ, USA). The sequence of the 16S rDNA of the sample was obtained from the total of the bacteria by PCR amplification with the bacterial universal primer pair 27F/1492R [16], which consisted of a forward primer 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and a reverse primer 1492R (5′-TACGGCTACC TTGTTACGACTT-3′). Reactions were conducted in a final volume of 20 μL, which contained 10 μL of Premix Taq Ver., 2.0 μL of plus dye (Takara, Dalian, China), 7.2 μL of sterile distilled water, 0.4 μL of each primer, and 2 μL of genomic DNA. The PCR amplification conditions in the thermocycler were set as follows: 5 min at 95 °C followed by 30 cycles at 95 °C for 30 s, 1 min at 55 °C and 90 s at 72 °C with a final extension of 5 min at 72 °C. After PCR analysis, the whole PCR reaction product was electrophoresed for 25 min onto 1.5% agarose gel with 5 μL 4S green nucleic acid stain in Tris-acetate-EDTA (TAE) buffer. Then, the amplicons were sequenced at Sangon Corporation (Shanghai, China). The DNA sequences of the isolates were used to search for sequence similarity against the National Center for Biotechnology Information (NCBI) database using the Standard Nucleotide BLAST program. In Vitro Antibacterial Bioassay In this study, eight of the title compounds were evaluated for their antibacterial activities against Xcc, isolated from the infected fruits of navel oranges, by the turbidimeter test [17] in vitro. Dimethylsulfoxide in sterile distilled water served as a blank control and Kocide 3000 and Thiodiazole copper, commonly used as the principal tools for controlling citrus canker in China at present, served as two positive controls. Approximately 40 μL of Nutrient Broth (NB) medium containing Xcc, incubated on the phase of logarithmic growth, was added to 5 mL of NB medium containing the test compounds or the commercial bactericides Kocide 3000 and Thiodiazole copper. The inoculated test tubes were incubated at 28-30 °C and continuously shaken at 180 rpm for 24-48 h until the bacteria were incubated on the phase of logarithmic growth. The growth of the cultures was monitored on a Model 680 microplate reader (BIO-RAD, Hercules, CA, USA) by measuring the optical density at 595 nm (OD595) and then the inhibition rate I was calculated by the following formula: where C is the corrected turbidity value of bacterial growth on untreated NB (blank control), and T is the corrected turbidity value of bacterial growth on treated NB, and I represents the inhibition rate. On the basis of previous bioassays, the results of antibacterial activities (expressed by EC50) of the title compounds against Xcc were also evaluated and calculated with SPSS 17.0 software. The experiment was repeated three times. Field Trial against Citrus Canker In order to further determine the activities of the title compounds, which showed better antibacterial activities against Xcc in vitro, field trials of compound 3 against citrus canker were conducted in Congjiang and Luodian, Guizhou Province, respectively, in 2014. The citrus variety is navel orange and the effect of the natural infection of Xcc was studied in a field having suffered citrus canker for several years. Sterile distilled water served as a blank control, whereas the commercial bactericides Kocide 3000 and Thiodiazole copper served two positive controls. The solutions of compound 3 (20% suspension concentrate (SC), 500 fold dilution, 300 g ai/ha) and the commercial bactericides Kocide 3000 (46% water dispersible granule (WDG), 1000 fold dilution, 300 g ai/ha) and Thiodiazole copper (20% SC, 500 fold dilution, 300 g ai/ha) were sprayed three times on the foliage once every seven days. Approximately 1.5 L per tree of spray, depending on the size of the testing trees, was applied with a backpack sprayer (Model 3WBS-16C, Shun Industrial Co., Ltd, Taizhou, China). The experimental design area of the plot was about 20 m 2 with five trees, three replicates were conducted. The disease incidence was investigated on the 14th day after the third spraying and the control efficiencies in leaves and fruits were calculated by the following formula: where CK represents the disease incidence of the untreated plot, PT represents the disease incidence of the treatment plot, and I represents the control efficiency. Determination of POD, PPO, and PAL Activities Disease resistance in plants is associated with the activation of a wide array of defense responses that slow down or halt infection at certain stages of the host-pathogen interaction. The defense mechanisms include preexisting physical and chemical barriers that interfere with pathogen establishment. Other methods of protection rely on inducible defense responses in the form of enzymes that are activated upon infection [18] or plant activators [19][20][21][22][23]. The interaction between the pathogen or plant activators and the host plant induces some changes primarily in the activity of enzymes, particularly PAL, POD, PPO, etc. [24][25][26]. PAL is the primary enzyme in the phenylpropanoid pathway, which leads to the conversion of L-phenylalanine to trans-cinnamic acid with the elimination of ammonia, and it is the key enzyme in the synthesis of several defense-related secondary compounds such as phenols and lignin [27]. Meanwhile, PPO is a nuclear-encoded enzyme that catalyzes the oxygen-dependent oxidation of phenols to quinones, and PPO levels in a plant increase when a plant is wounded or infected [18]. Moreover, POD constitutes a class of enzymes extensively distributed in plants and it has been shown that POD plays an active role in metabolism and has been suggested as a defense response of plants to stress [28]. In view of the above findings and as an extension of our studies on the further research of whether the testing compound can improve the disease resistance of plants that rely on inducible defense responses in the form of enzymes that are activated, the enzymatic activities of PPO, POD, and PAL were determined. Seven days after the third spraying of compound 3, the leaves were collected and powdered by liquid nitrogen and the leaves that were untreated were used as a blank control. Powdered samples of leaves (0.5 g) were homogenized with cold extraction buffer containing 20 mL of 0.01 M sodium phosphate buffer (PBS, pH 5.9) for the assay of the enzymatic activities of PPO and POD. PAL activity was measured in powders extracted with 0.01 M PBS (pH 8.8) containing 5 mM β-mercaptoethanol and 5% polyvinylpyrrolidone (PVP). The extracts were filtered through two layers of miracloth and the filtrates were centrifuged at 12,000 rpm at 4 °C for 15 min. PPO and POD activities were determined according to the reported methods [29,30]. For PPO analysis, 1 mL of supernatant was mixed with 3 mL of PBS (0.01 M, pH 5.9) and 1 mL pyrocatechol (0.2 M). A control was similarly prepared by adding 1 mL of PBS instead of 1 mL of protein extraction. Change in absorbance at 410 nm was measured by spectrophotometer. One unit of PPO activity was defined as a change of one in absorbance per minute [31]. POD activity was measured in a reaction mixture consisting of 0.1 mL of supernatant, 0.4 mL of 0.05 M guaiacol, and 3.5 mL of 0.01 M PBS (pH 5.9). The increase in absorbance at 470 nm was measured by spectrophotometer after 1 mL H2O2 was added. A control was similarly prepared by adding 1 mL of PBS instead of 1 mL of H2O2. One unit of enzyme activity was defined as a change of one in absorbance per minute. POD activity was calculated as follows: PAL activity was assayed according to Assis et al. [32] with slight modifications. First, 1 mL of supernatant was mixed with 2 mL of 50 mM sodium borate buffer (BBS, pH 8.8) and 1 mL of 20 mM L-phenylalanine and incubated in a water bath at 40 °C for 30 min. Then the reaction was stopped by adding 1 mL of 1 M hydrochloric acid (HCl). A control was similarly prepared by adding 1 mL of BBS (pH 8.8) instead of 1 mL of protein extract. PAL activity was assayed by spectrophotometer at 290 nm. One unit of enzyme activity was defined as the increase of one in absorbance per hour. PAL activity was calculated as follows: where W (mg) is the total protein content of the leaves, t (min) is the reaction time, VT (mL) is the total volume of protein extract, and Vs (mL) is the amount of protein extract used for detection. Effect on the Cell Membrane Integrity Bacterial cell membrane integrity was examined by determination of the release of material absorbing at 260 nm following the reported method [33] with slight modifications. Bacterial cultures, in the mid-exponential growth phase, were harvested, washed, and re-suspended in 0.75% NaCl solution. The final cell suspension was adjusted to an absorbance at 595 nm (OD595) of 0.6. A 0.25 mL portion of compound 3 was mixed with 0.25 mL of bacterial cell suspension to give the final concentration of 10 and 20 μg/mL, respectively, and the release over time of materials absorbing at 260 nm was monitored with a Perkin-Elmer model 554 UV-Vis recording spectrophotometer. Effect on the Biofilm Formation It is reported that biofilm formation plays an important role in early infection of Xcc on host leaves [13]. In this study, the effect on the biofilm formation was studied in 96-well plates based on the method described previously [34,35] with some modifications. Compound 3, at the final concentration of 2.5, 5, 10, and 20 μg/mL, respectively, was added into the mid-exponential growth phase bacterial suspension, while the same volume of sterile distilled water was added to the untreated blank controls. The mixture was incubated at 28 °C for 36 h. Following that, 200 μL of the suspension was added to individual wells of 96-well plates and incubated at 28 °C for 12 h without shaking. Each treatment consists of three wells. Then the wells were washed three times with sterile distilled water to remove non-adhered bacteria and the remaining attached bacteria were dried and stained with 200 μL of 0.1% (w/v) crystal violet for 15 min. The wells were washed to remove non-adsorbed crystal violet solution and immediately solubilized with 200 μL of 33% acetic acid. The solution was monitored on a Model 680 microplate reader (BIO-RAD) by measuring the optical density at 595 nm (OD595). Effect on the Cell Membrane Permeability To determine the effect of compound 3 on cell membrane permeability of Xcc, an isolated single colony of Xcc was sub-cultured in 250 mL flasks containing 100 mL of NB medium. The flasks were placed on a rotary shaker at 180 rpm at 28 °C. After 36 h, partial flasks were amended with compound 3 at the ultimate concentration of 10 and 20 μg/mL. The flasks were shaken for an additional 36 h, the cells were collected by centrifugation at 12,000 rpm for 10 min and washed twice with 0.75% NaCl solution. After centrifugation for 10 min, approximately 25 mg of bacteria was suspended in 1 mL of 0.75% NaCl solution. After 0, 30, 60, 90, and 120 min, the electrical conductivity of the 0.75% NaCl solution was measured with a conductivity meter (CON510 Eutech Ltd., Oaklon, Singapore) to assess the extent of leaching of cell contents through cell membranes. After 180 min, the bacteria were boiled for 5 min, and final conductivity was measured. Each experiment was repeated three times. The relative conductivity of cells was calculated as: Relative conductivity (%) = × 100 EPS Content The quantity of EPS produced by Xcc was determined by the phenol-sulfuric acid method [36][37][38] with some modifications. For preparation of an EPS standard curve, 2 mL of a glucose solution (0, 20, 40, 60, 80, 100, 120, 140, 160, 180, and 200 μg of glucose/mL of double-distilled water) and 1 mL of a 5% phenol solution were added to the test tube, respectively, and mixed with a vortex mixer. Then 5 mL of concentrated H2SO4 was added slowly to the test tubes. The test tubes were then closed with rubber plugs, mixed with a vortex mixer for 10 s, and then incubated for 30 min at 25 °C. The solution absorbance was measured using a Model 680 microplate reader (BIO-RAD) by measuring the optical density at 490 nm (OD490). In this case, the greater the absorbance, the higher the glucose concentration. A standard curve was generated by plotting absorbance against glucose concentration. For the determination of EPS content, an isolated single colony of Xcc was sub-cultured in 250 mL flasks containing 100 mL of NB medium at 28 °C with continuous shaking at 180 rpm for 36 h. Then, the partial flasks were supplemented with compound 3 at the ultimate concentration of 2.5, 5, 10, and 20 μg/mL, respectively. The flasks were shaken continuously for an additional 36 h. Then, the flask contents were centrifuged at 12,000 rpm for 10 min, and the supernatants were collected. EPS was precipitated from 1 mL of each supernatant with three volumes of absolute ethanol, collected via centrifugation and dried. Finally, the EPS were dissolved in 10 mL of distilled water, the optical density was measured at 490 nm (OD490) using a Model 680 microplate reader (BIO-RAD), and the standard curve was quantified. Sterile-distilled water was used as an untreated blank control. There were three replications for each treatment. Conclusions In this study, the pathogenic bacterium Xcc, the cause of citrus canker, was isolated from an infected corm of navel orange fruit and the species was identified via PCR analysis and the amplicons were sequenced. We investigated the efficacy of eight sulfone derivatives containing 1,3,4-oxadiazole moiety on controlling citrus canker caused by Xcc under in vitro and field conditions. Antibacterial bioassay results indicated that compound 3 demonstrated appreciable control efficiencies against citrus canker under in vitro and field conditions, which were even better than those of Kocide 3000 and Thiodiazole copper. Meanwhile, the changes of the enzyme activity of POD, PPO, and PAL in navel orange leaves demonstrated that compound 3 could improve the disease resistance of plants that rely on inducible defense responses in the form of enzymes that are activated for controlling citrus canker. Moreover, compound 3 could damage the cell membranes, destruct the biofilm formation, inhibit the production of EPS, and affect the cell membrane permeability to restrain the growth of the bacteria. This work demonstrated that sulfone derivatives containing 1,3,4-oxadiazole moiety can be used to develop potential bactericides for controlling citrus canker.

v3-fos

2018-04-03T03:54:01.301Z

2015-07-31T00:00:00.000Z

12286304

s2

Short- and long-term dynamics in the intestinal microbiota following ingestion of Bifidobacterium animalis subsp. lactis GCL2505 Bifidobacterium animalis subsp. lactis GCL2505 (B. lactis GCL2505) is able to survive passage through the intestines and proliferate. The daily dynamics of the intestinal bifidobacteria following ingestion of probiotics are not yet clear. Moreover, the effects of long-term ingestion of probiotics on the intestinal microbiota have not been well studied. Two experiments were performed in the present study. In Experiment 1, 53 healthy female volunteers received B. lactis GCL2505; B. bifidum GCL2080, which can survive but not proliferate in the intestine; or yogurt fermented with Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus for 2 weeks, and the daily dynamics of intestinal bifidobacteria were investigated. The number of fecal bifidobacteria significantly increased on day 1, and this was maintained until day 14 in the B. lactis GCL2505 ingestion group. However, no significant change in the number of fecal bifidobacteria was observed in the other groups throughout the ingestion period. In Experiment 2, 38 constipated volunteers received either B. lactis GCL2505 or a placebo for 8 weeks. Both the number of fecal bifidobacteria and the frequency of defecation significantly increased throughout the ingestion period in the B. lactis GCL2505 ingestion group. These results suggested that the proliferation of ingested bifidobacteria within the intestine contributed to a rapid increase in the amount of intestinal bifidobacteria and subsequent maintenance of these levels. Moreover, B. lactis GCL2505 improved the intestinal microbiota more effectively than non-proliferating bifidobacteria and lactic acid bacteria. INTRODUCTION The human intestinal tract is normally inhabited by 400-500 types of bacteria, and it harbors a large, active, and complex community of microbes [1]. The intestinal microbiota play several significant roles in the digestion of food, the metabolism of endogenous and exogenous compounds, immunomodulation, and the inhibition of colonization by pathogenic bacteria, thus making them important for maintaining human health [2,3]. Members of genus Bifidobacterium are among the most predominant organisms in the human intestine and are important for general health, which means that their diversity and number provides a marker for measuring the stability of human intestinal microbiota, as well as the intestinal environment [4,5]. Probiotics are defined as "live microorganisms which when administered in adequate amounts confer a health benefit on the host" (Food and Agriculture Organization of the United Nations/World Health Organization 2002). Many studies have investigated the effects of probiotic consumption on intestinal microbial imbalance, on suppression of pathogens and prevention and treatment of intestinal and other disorders, and on inflammatory bowel disease, diarrhea, infection, colon cancer, constipation, and atopic diseases [6][7][8][9][10][11]. In particular, numerous attempts have been made to increase the number of intestinal bifidobacteria and improve intestinal disorders such as constipation and diarrhea through use of probiotics [12][13][14][15]. In most of these studies, however, the numbers of intestinal bifidobacteria were investigated using cultivation-based techniques [12][13][14][15], which are widely known to be labor-intensive and timeconsuming. In addition, classification and identification based on phenotypical traits do not always provide clear-cut results and are sometimes unreliable because the recovery of bifidobacteria from feces depends on the composition of the medium and the culture conditions [16][17][18]. Currently, 16S rRNA-targeted oligonucleotide probes are used with fluorescent in-situ hybridization and genus-and species-specific PCR as a culture-independent method [19][20][21][22][23]. These methods enable rapid and specific detection of a wide range of bacterial species. Genusspecific primers or probes are expected to provide a good overall picture of the fecal bifidobacterial population, although there are few reports describing the effect of probiotic administration on bifidobacterial composition, especially those that focus on the daily dynamics of both endogenous and exogenous (ingested) bifidobacteria. Bifidobacterium animalis subsp. lactis (B. lactis) GCL2505 is a probiotic that originates from healthy human intestines and is used in fermented milk products in the Japanese market. We previously showed that B. lactis GCL2505 reached the intestine in a viable form and was subsequently able to proliferate after a single ingestion, which led to an increase in the amount of intestinal bifidobacteria and more frequent defecation after 2 weeks of ingestion [24]. However, the daily dynamics of intestinal bifidobacteria at the species level following ingestion of B. lactis GCL2505 are not yet clear. Moreover, the effects of long-term ingestion of B. lactis GCL2505 on the composition of intestinal bifidobacteria and the changes in the frequency of defecation lasting over 2 weeks have not been well studied. In this study, we compared the dynamics of intestinal bifidobacteria after ingestion of B. lactis GCL2505 and other bifidobacteria that can survive but not proliferate in the intestine, as well as those of lactic acid bacteria used in yogurt fermentation. Quantitative real-time PCR using Bifidobacterium species-and subspecies-specific primers were used to elucidate the daily dynamics of endogenous and ingested strains at the species level. Moreover, we investigated the change in the intestinal microbiota and the frequency of defecation following long-term ingestion. Test beverages The test beverages included a milk-like drink, a yogurt drink, or a placebo drink (100 g of each). B. lactis GCL2505 or B. bifidum GCL2080 was added to the milklike drink. The viable cell count of B. lactis GCL2505 or B. bifidum GCL2080 in a test beverage was 1.5 × 10 10 cfu or 2.6 × 10 10 cfu, respectively. The yogurt drink was fermented with L. delbrueckii subsp. bulgaricus GCL1031 and S. thermophilus GCL1122, both of which are commonly used in the production of conventional yogurt. The viable cell count of lactic acid bacteria (L. bulgaricus and S. thermophilus) in a test beverage was 3.0 × 10 10 cfu. The placebo drink was prepared with the same ingredients as the milk-like drink and had a similar flavor but it did not contain bacteria. Subjects and study design of a short-term trial (Experiment 1) Sixty-four healthy female subjects were recruited (age range: 18-25 years). These subjects claimed that they had a frequency of bowel movements of ≥5.0 times/ week in a questionnaire performed in advance. The aim of Experiment 1 was to elucidate the daily dynamics of intestinal microbiota; thus, subjects were required to provide daily fecal samples. We therefore selected subjects who claimed in a preliminary questionnaire that they had a frequency of bowel movements of ≥ 5.0 times/week. The study was designed as a double-blind, parallelgroup comparison and consisted of two consecutive 2-week periods: a non-ingestion period and an ingestion period. Subjects were randomized and assigned into one of three groups that received a test beverage containing either B. lactis GCL2505 (BL group), B. bifidum GCL2080 (BB group), or L. bulgaricus GCL1031 and S. thermophilus GCL1122 (LBST group). During the ingestion period, the subjects consumed one test beverage daily. Each subject provided fecal samples for microbial analysis at the end of the non-ingestion period and on days 1-4, 7, and 14 of the ingestion period ( Fig. 1). Subjects were instructed to avoid the intake of fermented milks, lactic acid bacteria beverages, probiotic and prebiotic products, and fermented foods such as natto (soybean fermented with B. subtilis) for the duration of the study. Subjects and study design of a long-term trial (Experiment 2) Forty-two mildly constipated female subjects were selected (age range: 25-59 years). These subjects claimed that they had a frequency of bowel movements of ≤5.0 times/week in a questionnaire completed in advance. The study took the form of a double-blind, parallelgroup comparison and consisted of a 2-week non-ingestion period in which initial parameters were obtained for baseline measurements, followed by an 8-week ingestion period. During the ingestion period, subjects consumed 1 test beverage daily. Subjects were randomized and assigned to two groups (active or placebo group). The active group consumed a test beverage containing B. lactis GCL2505, and the placebo group consumed a test beverage without B. lactis GCL2505. Each subject provided fecal samples for microbial analysis at the end of the non-ingestion period and in weeks 2, 4, and 8 of the ingestion period and recorded their daily number of defecations ( Fig. 2). As in Experiment 1, subjects were instructed to avoid the intake of other fermented milks, lactic acid bacteria beverages, probiotic and prebiotic products, and fermented foods such as natto (soybean fermented with B. subtilis) for the duration of the study. Determination of fecal microbiota Fecal samples were delivered in a refrigerated, anaerobic state using an AnaeroPack Kenki (Mitsubishi GAS Chemical Co., Inc., Tokyo, Japan), diluted 10fold with phosphate-buffered saline (pH 7.4) and homogenized using a Stomacher. Suspensions were kept at −80°C until assayed. Bacterial DNA was extracted from the 10-fold dilutions of fecal samples according to the procedure described by Matsuki et al. [25]. The number of intestinal bifidobacteria was quantified by real-time PCR using Bifidobacterium species-and subspecies-specific primers, according to the procedure described by Ishizuka et al. [24]. Briefly, PCR amplification and detection procedures were performed using a CFX-96 Real-Time PCR Detection System (Bio-Rad Laboratories Inc., Hercules, CA, USA). Each reaction mixture (10 µl) was composed of 5 µl of SYBR Premix Ex Taq I or II, 1 µl of each primer ( Ethics These experiments were performed with the approval of the ethical committees of Fuji Women's University (Experiment 1), and Kenshokai Medical Co., (Osaka, Japan) (Experiment 2). The contents and methods were explained in full to all prospective subjects, and written informed consent was obtained according to the principals of the Declaration of Helsinki (adopted in 1964; revised in 1975, 1983, 1989, 1996, and 2000). Statistical analysis IBM SPSS Statistics for Windows Version 22.0 J (IBM Corp., Armonk, NY, USA) was used for statistical analyses. Within-group comparisons between the baseline and each subsequent time point were conducted using repeated-measures ANOVA followed by Dunnett's multiple comparisons. Between-group comparisons of the amount of change from baseline to each subsequent time point were conducted by one-way ANOVA followed by unpaired student's t tests. p<0.05 was considered statistically significant, and 0.05 ≤ p<0.10 was considered marginally significant. Short-term changes in fecal bifidobacteria (Experiment 1) Three subjects failed to complete the trial due to personal reasons. Eight subjects were excluded from analysis because of noncompliance with the study requirements, so 53 subjects were analyzed. Background characteristics for the subjects in Experiment 1 are shown in Table 2. There were no significant differences in any of the characteristics or parameters among the three groups. Figure 3 shows the changes in the numbers of fecal bifidobacteria. In the BL group, the total number of bifidobacteria significantly increased on day 1 compared with before ingestion. The percentage of B. lactis reached nearly 50% of the total amount of bifidobacteria on day 2, and this level was maintained until day 14. On the other hand, no significant change was observed in the composition and number of endogenous bifidobacteria in the BL group at the species level throughout the ingestion period. No significant changes were observed in either the total number of bifidobacteria or the composition of fecal bifidobacteria in the BB and LBST groups, throughout the ingestion period. Values are expressed as the mean ± SD. Long-term changes in the fecal bifidobacteria (Experiment 2) Four subjects were excluded from analysis because of noncompliance with the study requirements. Two subjects were excluded because they had a frequency of defecation of >5.0 times a week during the non-ingestion period, so 38 subjects were analyzed. Background characteristics for the subjects in Experiment 2 are shown in Table 3. There were no significant differences in any of the characteristics or parameters between the active and placebo groups. Figure 4 shows the changes in the number of fecal bifidobacteria. Before ingestion, there was no difference in the total amount of fecal bifidobacteria between the two groups. At 2 and 4 weeks after ingestion, the total number of bifidobacteria significantly increased in the active group compared with the placebo group. Moreover, the amounts of fecal bifidobacteria in the active group tended to increase 8 weeks after the ingestion period began compared with the placebo group. However, the total number and the number of each endogenous Bifidobacterium species did not significantly differ between the active and placebo groups. The changes in the frequency of defecation significantly increased in weeks 6 and 8 in the active group compared with the placebo group (Table 4). DISCUSSION There are many reports on the effects of probiotics on intestinal bifidobacteria [12-15, 26, 27]; however, the effects of consumption of probiotics on the daily dynamics of intestinal bifidobacteria or ingested probiotics are unclear. Values are expressed as the mean ± SD. In Experiment 1, we elucidated the daily dynamics of intestinal bifidobacteria and ingested probiotics. Ingestion of B. lactis GCL2505 increased the total number of intestinal bifidobacteria on day 1 compared with before ingestion, and this level was maintained throughout the ingestion period. According to speciesspecific real-time PCR, the fecal cell count for B. lactis reached 7.9 × 10 9 cells/g feces and represented half the population of intestinal bifidobacteria from day 2 onward. On the other hand, ingestion of B. bifidum GCL2080, found to be viable on reaching the intestine in our preliminary investigation (data not shown), did not lead to significant changes in the total number of intestinal bifidobacteria, either ingested or endogenous. B. bifidum (including ingested B. bifidum GCL2080 and endogenous B. bifidum) reached a total of 2.8 × 10 8 cells/g feces and represented a small proportion of the intestinal bifidobacterial microflora (approximately 2%) following ingestion of B. bifidum GCL2080. In addition, no significant changes were observed in either the composition or numbers of endogenous bifidobacteria at the species level following ingestion of L. bulgaricus GCL1131 and S. thermophilus GCL1122. Nevertheless, lactic acid bacteria from yogurt cultures themselves are also considered probiotics and are believed to increase intestinal bifidobacterial levels [28][29][30][31]. These results indicated that the effects of probiotics on the total amount of intestinal bifidobacteria were strain specific and exerted within a few days of consuming fermented milk produced by B. lactis GCL2505. To the best of our knowledge, this is the first report describing the daily dynamics of intestinal bifidobacteria, including endogenous and ingested strains, at the species level during the short-term ingestion of probiotics. In Experiment 1, no significant changes were observed in either the composition or numbers of endogenous bifidobacteria at the species level in any of the three groups. These results suggested that the probiotics had no effect on the number of endogenous bifidobacteria during short-term ingestion in the healthy human volunteers. In other words, the increase in the total amount of bifidobacteria was largely attributable to ingested (exogenous) probiotics. Therefore, the proliferation of ingested probiotics such as B. lactis GCL2505 was an important factor leading to the increase in the total amount of bifidobacteria in the intestine. In most previous studies, the number of intestinal bifidobacteria has been investigated only at the genus level using cultivation-based techniques [12][13][14][15]. In contrast, we measured the presence of each bifidobacterium species by quantitative PCR in this study to determine accurate numbers. Moreover, we used species-or subspecies-specific primers to determine the total number of intestinal bifidobacteria as a sum of the counts of ten species because multiple copy numbers of rRNA operons and genes in different bacterial chromosomes may affect the apparent relative abundance of bacteria in the sample. For example, it is reported that B. adolescentis carries 5 copies of the 16S rRNA gene [32], whereas B. longum and B. lactis carry 4 and 2 copies of the 16S rRNA gene, respectively [23,[33][34][35]. Therefore, our method of quantifying the total amount of bifidobacteria gave a more accurate representation than previous culture-based quantification techniques or genus-specific quantitative PCR. However, the numbers determined by quantitative PCR include both viable and dead cells. Previously, we revealed that B. lactis GCL2505 was detected at the same level in feces on the day after ingestion by culture-based quantification techniques, followed by PCR identification and species-specific quantitative PCR [24]. Therefore, it was considered that most of the B. lactis GCL2505 detected in the present study was in a viable form and proliferated in the intestine. We revealed in our previous study that ingestion of B. lactis GCL2505 over 2 weeks significantly increases the amount of intestinal bifidobacteria and improves the frequency of defecation [24]. However, the effects of long-term ingestion for more than 2 weeks have not yet been clarified. Thus, in this study, we investigated the effects of long-term ingestion of B. lactis GCL2505, using the amounts of fecal bifidobacteria and the frequency of defecation as indices of improvement in the intestinal environment, in addition to the effects resulting from short-term ingestion of probiotics. During the 8 weeks of B. lactis GCL2505 ingestion, the level of intestinal bifidobacteria and the frequency of defecation significantly increased compared with those in the placebo group. These results indicated that the effects of B. lactis GCL2505 ingestion on the intestinal microbiota were sustained for at least 8 weeks. On the other hand, the amounts of endogenous bifidobacteria were not significantly greater after 8 weeks of B. lactis GCL2505 ingestion, which showed that even longterm ingestion of probiotics had no effect on either the number or composition of endogenous bifidobacteria. In our previous study, ingestion of B. lactis GCL2505 over 2 weeks was found to significantly improve the frequency of defecation [24]. However, in the present study, improvements compared with the placebo group were observed only after 6 weeks of intervention. This is one reason why the present study was designed as a parallel-group trial, in contrast to the cross-over trial design used in the previous study. A larger sample size was required to reliably detect significant differences. It was estimated, based on the present data, that a sample size of 118 would be necessary for detecting significant differences between the active and placebo groups at 2 weeks with 80% power. Overall, however, ingestion of B. lactis GCL2505 increased the frequency of defecation in the active compared with placebo groups in both the previous and present studies. Therefore, it appears that ingestion of B. lactis GCL2505 is effective against constipation. Our results indicated that the proliferation of B. lactis GCL2505 in the intestine, which may cause production of short-chain fatty acids such as acetate and stimulate smooth muscle contractions and transepithelial chloride secretion [36][37][38][39][40][41][42][43], improved the function of the large bowel throughout the long-term ingestion period. In conclusion, we found that B. lactis GCL2505 increased the total number of intestinal bifidobacteria after a few days of ingestion and that long-term ingestion improved the frequency of defecation. Moreover, we showed that the proliferation of B. lactis GCL2505 in the intestine contributed significantly to the increase in the total number of intestinal bifidobacteria, although there was no significant change in the amounts of endogenous bifidobacteria. Based on these results, we propose that probiotics that are able to proliferate in the intestine, such as B. lactis GCL2505, appear to improve the intestinal microbiota more effectively than non-proliferating probiotics.

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Morphological, physical and chemical properties of soils associated in toposequence forestablishing taxonomy classes in Pratapgarh District of Rajasthan, India The present study was conducted to study the variability in soil properties in relation to landforms, in the present investigation, two transects that is, Aravalli mountain ranges and Malwa plateau, were selected in the Pratapgarh district having eight landforms namely hill, pediments, valley, and plain in the Aravalli Mountain ranges and Malwa plateau, respectively. Total eight pedons were examined in the field and investigated in the laboratory using standard laboratory procedures. The soils on hill top and pediment were shallow, gravely sandy loam to clay loam single grain in texture with medium coarse weak sub angular blocky structure and exhibited dark yellowish brown to dark reddish brown colour. The soils of valley were deep, sandy loam to loam and silty clay loam to clay loam in texture with medium coarse weak sub angular blocky to medium fine moderate sub angular blocky structure and exhibited dark yellowish brown to dark reddish brown colour. The soils of plain were found deep, silty clay in texture with medium moderate to strong angular structure (angular and sub angular) and exhibited dark brown to very dark grayish brown colour. The available water capacity were recorded higher in the plain soils as compared to soils of other landforms as well as in Aravali mountain ranges and Malwa plateau. The pH was relatively higher in the soils of Aravali mountain ranges than Malwa plateau but EC was relatively lower in the soil of Aravali mountain ranges then Malwa plateau. Distribution of organic carbon was low in soils of all pedons but comparatively higher in soils of Malwa plateau. Base saturation was comparatively lower in the soils of lower topographic position. Cation exchange capacity was found positively correlated with clay and increases as clay increased down the slope as well as with depth. Concentration of exchangeable bases was in order of Ca 2+ >Mg 2+ >K + >Na + in all the pedons soils. INTRODUCTION Pratapgarh is newest constituted district of Rajasthan state, which is a tribal dominant with an area of 411736 ha. Pratapgarh is situated in the southern part of Rajasthan. It is situated on the junction of Aravali mountain ranges and the Malwa Plateau; hence characteristics of both are prominent in the area. Pratapgarh is located at 29.03° North and 74.78° East. It has an average elevation of 491 m (1610 feet). The western, southern and northern parts of the district are somewhat plains. North and southern part of the district having black cotton soil in abundance. The major irrigation project of the district is the Jakham Dam. The north-west part of this region had dense forests. In the traditional method of soil map compilation, much emphasis is not being given on study of variability in pedogenic factors and quantification of soil properties used for soil classification. As such the information, dealing with variability in genetic related soil properties and their relationship with properties, which have key role in natural resource management, is generally scanty in India and so particularly in context to Rajasthan. Besides, sound and reliable database, dealing with information is needed to prepare base line of indicators to maintain the sustainability of system. So, detailed studies on morphological, physical and chemical properties are required to comprehend the extent of soil variability and to optimizing land use in Pratapgarh district. The present investigation is taken up to study the pathways of soil formation in relation to topography. Particle size description International pipet method as described by Black (1980) was followed for estimation of various soil separates. Texture of the soil sample was determined from the composition of separates using triangular chart. S/No. Soil properties Methods References (A) Physical properties 1. Calcium carbonate Acid neutralization method Allison and Modi (1965) Morphologial features Soil morphology or pedomorphic features has been studied mainly under field conditions. The morphology of a soil can be best evaluated from the in-situ examination of the soil profiles. The pedomorphic features of the soil profiles are the mirror image of the processes as responsible for the formation of a particular type of soil. Soil morphology is the stepping stone to the thorough appreciation of the physical, chemical and biochemical properties of the soil (Tables 1 and 2). Soil colour The colour of the soils ranged between very dark gray brown (10YR 3/1) to dark reddish brown (5YR 2.5/2) In Aravali mountain ranges, the colour variation was observed from brown (10YR 4/3) to dark brown (7.5YR 3/2). In case of soils of Malwa plateau colour was found varies from very dark grayish brown (10YR 3/1) to dark reddish brown (5YR 2.5/2). The colour of Aravali mountain range soils was brown to dark brown in almost all samples. And in case of soils of Malwa plateau the colour of soils of higher topographic positions (hill top, pediment and valley) varied from dark reddish brown (5YR 2.5/2) to yellowish brown (7.5YR 4/4), while plain soils were ranged in colour from very dark gray (10YR 3/1) to very dark grayish brown (10YR 3/2). After thorough examination of data (Table 2) it was observed that the soils of hill top in Aravali mountain ranges showed a shade of dark yellowish brown and as topography gets gentler the colour of soil becomes brown (10YR 4/3) or dark brown (7.5YR 3/2) in plain. It was also found that the colour of surface horizon was brighter then lower horizons. While in case of soils of Malwa plateau it was observed that hill top soil and valley soils showed dark reddish brown in all upper horizon and radish brown in lower most horizon of both transect and in pediments it found dark brown (7.5YR/3/2) while in plain it found very dark gray (10YR 3/1) in upper and (10YR 3/2) very dark grayish brown in lower horizons. Maximum samples in both transect were found under the category of dark yellowish brown followed by dark brown colour. Similar pattern of soil colour variation were also reported by Rajkumar et al. (2005) and Sarkar et al. (2001). Depth of soil solum Solum thickness is a combined expression of pedogenetic horizons and varied from 50 to 75 cm and 40 to 80 cm in Aravali mountain ranges (P 1 -P 4 ) and Malwa plateau (P 5 -P 8 ), respectively ( Table 2). Soils of Aravali mountain ranges were very shallow on pediments (P 2 ) and shallow on hill top (P 1 ) while remaining soils of Aravali mountain ranges on valley (P 3 ), plain (P 4 ) were deep. Whereas soils of Malwa plateau were very shallow on hill top (P 5 ), shallow on pediments (P 6 ), while the remaining soils of Malwa plateau on valley (P 7 ), plain (P 8 ) were moderately deep. Gentle to moderate slope, rapid runoff and severe erosion account for very shallow to shallow soils on the elevated segment of transect. These altogether cast rapid removal of weathering product from the site of formation, resulting in shallower depth of soils. Sidhu et al. (2000), Rathore (2003) and Singh (2004) also reported shallow soils at higher elevation. However, shallow depth in very gently sloping alluvial plain must be due to resistance of parent material to weathering, which prevents soil development and its accumulation. Therefore, the depth of soils was found to be a function of the topography, type of basement rock and configuration of the landscape. Texture The variation in soil texture of different landforms is shown in Table 2. The soils of Aravali mountain ranges were gravel sandy loam on hill top (P 1 ), Loam at the surface and gravel clay loam in sub surface of pediment (P 2 ), all horizons having different texture (sandy loam to loam) in valley (P 3 ) while clayey in charpotiya (P 4 ). Malwa plateau had sandy loam to gravel sandy loam on hill top soil (P 5 ), and loam to clay loam in pediment (P 6 ), silty clay loam at the upper surface and clay loam in sub surface of valley (P 7 ) had sandy clay loam texture. In case of pedon of plain (P 8 ) the texture was found silty clay. The variation in the intensity of erosion and deposition explains the variation in soil texture topographically. Thus the variation in type of parent rock, the portions on the landscape and the some from where the matter is carried by flowing water determine the texture as a function of topography. As a result of rapid downward movement of rain water, the finer particles were readily carried away toward the lower areas while coarse particles remain. This could be attributed as a main reason for the behavior of texture with change of slope. Similar results were also observed by Gupta et al. (1999), Sarkar et al. (2001) and Maji et al. (2005). Soil structure Single grain structure was the feature of soils associated with the hill top of Aravali mountain ranges. The structure become medium coarse weak sub angular blocky in the soils of pediment and upper horizon of pedon P 3 while the soils of pedon P 4 had medium moderate sub angular blocky in upper horizon and in subsequent horizons the structure become medium moderate angular blocky. The structure of lower horizons of pedon P 3 exhibit medium moderate fine sub angular blocky arrangement. The soils of Malwa plateau associated with the hill top had single grain structure. The structure become moderately fine sub angular blocky in the soils of pediment. It was found medium weak fine sub angular blocky in upper layers and in subsequent layers it become medium moderate fine sub angular blocky in soils of valley. The soils of plain (P 8 ) had medium moderate to strong angular blocky structure. Lime concretions and effervescence The extent of distribution of calcareousness (reaction to dilute HCl) over different physiographic units is shown in Table 2. The data reveal that calcareousness was uniform and throughout in soils of plain pedon (P 4 ) in Aravali mountain ranges and valley and plain pedons (P 7 , P 8 ) in Malwa plateau soils evident by the reaction with dilute HCl being slight to violent effervescence in all these pedons. While in pedon P 1 it was seen non-effervescent in the surface layer that is, A1. In rest of the pedons which occurring on elevated topographic position, no reaction was observed with dilute HCl, indicating that these soils were either free of calcareousness or the content was too low to be detected by dilute HCl. Horizon designation Absence of cultivation over the surface was designated as Al (P 1 , P2, P 7 and P 8 ). A2 was allocated to the horizon, having slight improvement in soil structure in term of grade and angularity (P 3 ). Those soils which were under cultivation and leaves of stubble at the surface which are ploughed before sowing of every crop. As such, surface horizons were therefore designated as Ap horizon that is, plough layer. Bw is given to layer which show some evidence of development either due to increase in clay content , formation of well defined peds or some reddening of hue (P 3 , P 4 , P 6 and P 7 ,). Bss horizons are the mark of slickenside presence (P 4 , and P 8 ). Slickensides are formed as a result of the swelling of clay minerals and shear failure, commonly at angle of 20 to 60 degree above horizontal. C horizon is marked for weathered material in pedons P 2 -P 8 except P 4 and P 7 where it was designated as Ck due to accumulation of carbonates. PHYSICAL PROPERTIES Results pertaining to mechanical composition, bulk density, porosity and water retention characteristics of soils associated in both transect namely Aravali mountain ranges and Malwa plateau, are described in this section while data on these features are elucidated in Tables 3 to 5. Mechanical composition Relative proportions of sand, silt and clay in soils of both Table 3. Sand Total sand content of all pedons in Aravali mountain ranges ranged from 8.09 to 51.67% with a weighted mean value of 35.95%. The content was highest in the soils associated with hill (P 1 ) followed by valley (P 3 ), pediments (P 2 ), while it was lowest in the soils of plain (P 4 ). However, in general, fine sand fraction dominates over the different sand fractions of all the profiles. Generally, soil texture was coarser on the higher landforms or sloping landforms, because the fine materials like silt and clay are removed from the relatively higher location to those portion where slopes become gentler attain heavily level relief. In soils of Malwa plateau transect, total sand content ranges from 12.09 to 62.51% with a weighted mean value of 35.75%. The sand content was higher in the soils on hill top (P 5 ) and pediments (P 6 ), while it was lower in the soils of plain (P 8 ). However, valley (P 7 ) and had intermediate amount of sand. By and large fine sand fraction on higher elevation, dominates over other fraction while on lower elevation, very fine sand fractions were dominant over fine sand. Similar results were also reported by Singh (2004). Silt The content of the silt in the soils of Aravali mountain ranges varied between 31.47 to 43.87% with a weighted mean value of 35.75%. Highest average silt observed in soils of plain (P 4 ). While lowest average found in soils of hill top (P 1 ) followed by valley (P 3 ). While the soils of pediment (P 2 ) exhibited intermediated trend of percent silt contribution toward the mechanical composition of these soils. In the soils of Malwa plateau transect, it was observed that the silt content varied between 19.21 to 60.00% with a mean value of 33.27% with highest average value in valley (P 7 ) followed by plain (P 8 ) and lowest average value in hill top (P 5 ) followed by pediment (P 6 ) soils. An increasing trend of higher silt content from elevated topographic position to lower topographic position as well as along with down the depth of pedons was observed which could be because of movement of silt particles along with downward movement of water as well as due to erosion agents. Clay The clay content varied from 16.07 to 49.75% in soils of Aravali mountain ranges with a mean value 28.22%. The highest average value of clay content was observed in the soils of plain (P 4 ). Whereas lowest value of clay content was in the soils of hill top (P1). The soils of pediment (P 2 ) and valley (P 3 ) were found intermediate in clay content. While in case of soils of Malwa plateau, clay content varied from 18.46 to 44.95% with mean value 30.89%. The highest average value of clay content was observed in the soils of plain (P 8 ) followed by pediment (P 6 ), and valley (P 7 ) whereas lowest value of clay content was in the soils of hill top (P 5 ). The higher clay content down the slope was also reported by Sarkar et al. (2001). Bulk density Bulk density is a reliable index for determining the presence of compact layers particularly in the subsoil. An examination of data, presented in Table 4 indicates that values of bulk density increase with the depth of the soil. Bulk density of soils in Aravali mountain ranges between 1.45 to 1.65 mg m -3 with a weighted mean value of 1.53 mg m ) in soils of pediment (P 6 ). In generally an increase in bulk density in the subsurface horizons was observed in all the pedons examined along both transect. Chaudhary et al. (2005) while studying the soils of Himalayas also found similar results of increasing trend of B.D. with depth. Particle density It is evident from the data presented in Table 4 that the particle density of the soils of Aravali mountain ranges was found to range between 2.52 to 2.65 mg m -3 . While in case of soils of Malwa plateau, particle density varied from 2.55 to 2.64 mg m -3 . It was found to increase with depth but such an increase was only up to a certain depth of various landforms followed by decrease. Particle density was found higher in the soils of Malwa plateau as compared to the soils of Aravali mountain ranges which suggest heterogeneity in profile development in Malwa plateau. Similar trend was also recorded by Veerpal (1976), Datta et al. (1990) and Singh et al. (1999). Porosity The packing pattern of soil fragment determines the porosity of the soils. The porosity of various pedon ranged between 35.79 to 43.35% with a weighted mean value of 40.13 in soils of Aravali mountain ranges. While in soils of Malwa plateau, it was ranged between 37.93 to 48.85%. The maximum value of porosity was recorded in A1 layer of hill top (P 1 ) while minimum value was recorded in ck horizon of plain soils in Aravali mountain ranges. In case of Malwa plateau soils the maximum value was observed in the Ap horizon of hill top soils while minimum value was recorded in C layer of plain (P 8 ). This difference was mainly due to variation in silt and sand fractions and their arrangement or shape of orientation at different locations of the transect. Generally the porosity was high in surface horizons and decreases with depth (Table 4) which may be due to higher value of bulk density in subsurface soils. Similar observations were also reported by Wick and Whiteside (1959); Rathore (1993) and Sharma (1994). Further, the bulk density and porosity were inversely related which is also supported by the findings of Kolarkar et al. (1974), Rathore (1993) and Sharma (1994). Moisture retention characteristics The data on the effect of different landform on water retentions characteristics are presented in Table 5. It was seen from the data that the amount of the water retained at 0.03 MPa ranged from 0.25 to 0.49 m 3 /m 3 with a weighted mean value of 0.35 m 3 /m 3 in soils of Aravali mountain ranges. Water retention was recorded highest, Singh and Rathore 2523 ranging from 0.43 to 0.49 m 3 /m 3 with a weighted mean value 0.46 m 3 /m 3 in the soils of plain (P 4 ) followed by valley (P 3 ). Lowest water retention was recorded in soils of hill top (P 1 ) followed by pediments (P 2 3 ) in soils of plain (P 8 ) followed by valley (P 7 ). The water retention in remaining pedon was in pediment>hill top soil sequence. The water retention at 1.5 MPa also followed the similar trend, ranging from 0.10 to 0.26 m 3 /m 3 with a weighted mean value of 0.16 m 3 /m 3 in soils of Aravali mountain ranges whereas corresponding value are slightly higher 0.05 to 0.29 m 3 /m 3 with a weighted mean value of 0.14 m 3 /m 3 in soils of Malwa plateau. indicated that an increase in clay and silt content had contributed marginally towards the available water capacity due to corresponding increase in water retention at both levels that is, 0.03 and 1.5 MPa suction pressures. The amount of water retained was found to be higher in subsurface layer in comparison to surface layer in both transects. Similar observations were made by Nagar et al. (1995) and Balpande et al. (2007). Available water capacity (AWC) volume basis Available water capacity is an important indicator for sowing seed/or crop planning, irrigation scheduling and crop selection under rainfed conditions/areas like Pratapgarh district. Available water capacity is the difference of water content at 0.03 and 1.5 MPa suction pressures. Available water capacity was varying from 0.14 to 0.24 m 3 /m 3 with a weighted mean value of 0.19 m 3 /m 3 in the Aravali mountain ranges. Maximum available water capacity observed in plain (P 4 ) followed by valley (P 3 ) and hill top (P 1 ), whereas lowest value comes under pediments (P 2 ). While in case of Malwa plateau, available water capacity was ranged from 0.13 to 0.24 m 3 /m 3 with weighted mean value 0.16 m 3 /m 3 . Maximum available water capacity in this transect was observed in plain (P 8 ) followed by valley (P 7 ) and whereas lowest value comes under pediments and hill top (P 1 and P 2 ). It was observed that soils of Malwa plateau had more available water content compare to soils of Aravali mountain ranges which could be attributed to the relatively higher finer fraction in these soils. Water holding capacity (WHC) The water holding capacity ranges from 23.56 to 40.11% where maximum water holding capacity found in plain (P 4 ) and minimum in pediments (P 2 ) in Aravali mountain ranges. In case of Malwa plateau maximum water holding CHEMICAL PROPERTIES In order to understand the specificity of relationship between the soils and physiography it is imperative to analyze the results of various chemical properties in light of micro-topographical variations imposed by variations in landforms. Soil reaction (pH) Soil reaction (pH) is one of the important parameter controlling availability of plant nutrients in the soils. In the present investigation the pH of the soils in Aravali mountain ranges was between 6.92 to 7.72 with a mean value of 7.43 indicating that the soils are near neutral to slightly alkaline (Table 6). In soils of Malwa plateau, the pH ranged between 6.03 to 7.75 with a mean value 6.92. It was near neutral in the soils of hill top pedon P 1 (6.03) and moderately alkaline in the soils of plain pedon P 8 (6.92 to 7.59). A critical examination of data indicates that soil pH in most cases was found to increase with depth in both transects. This increase level of pH down the depth of pedons was mainly due to movement of soluble salts and increased content of calcium carbonate. The higher pH values in soils of lower slopes and its increased value with soil depth could be attributed to the deposition of illuviated bases from surrounding upper slopes. Similar results were also observed by Dutta et al. (1990), Deshmukh and Bapat (1993), Rathore (1993), Sharma (1994) and Chamuah et al. (1996). Electrical conductivity (EC) EC is a measure of concentration of soluble salts in the soil at any temperature and it was determined in soil: water, 1:2 suspension and data presented in Table 6. The electrical conductivity was found range between 0.32 to 1.26 dSm -1 with a mean value of 0.56 dSm -1 in soils of . The general trend of soluble salt distribution was found increasing from upper rolling topographic position to lower elevation, indicates that the appreciable amount of salts moved down the slope along with flowing water. The findings are in line with the results of Gaikawad et al. (1974), Dutta et al. (1990) and Sharma (1994). Organic carbon The organic carbon content of the soils is an indication of nitrogen status. In the soils of the Aravali mountain ranges, organic carbon content was found to range between 1.35 and 7.85 g kg 1 with a weighted mean value of 4.31 g kg -1 . It was minimum in soils of plain P 4 followed by valley P 3 and pediments P 2 (weighted mean value 2.42, 2.70, and 4.29 g kg -1 ) respectively, and maximum in soils of hill top (7.85 g kg -1 ). In the soils of Malwa plateau, the organic carbon content was found to range between 2.35 to 9.50 g kg -1 with a weighted mean value of 5.01 g kg -1 . No specific trend of distribution has emerged out with respect to the topography in the soils selected for the study. In general the content of organic carbon is higher at the surface, decreasing down the depth in soil profile. This was mainly due to accumulation of plant residues on the soil surface and very less opportunity to move it down the depth due to rapid decomposition at higher temperature and inadequate pedoturbation. Almost all soils were low in organic carbon (<5 g kg -1 ) content except soils of pedon P 1 in Aravali mountain ranges and pedon (P 5 -P 7 ) in Malwa pateau, and it was mainly due to rapid rate of mineralization at higher temperature and adequate soil moisture level. The organic carbon distribution is mainly associated with physiography and land use. Similar results were also observed by Sharma et al. (1999), in soils of Haldi-Ghati region of Rajasthan (Walia and Rao, 1996). Calcium carbonate In present investigation, the content of calcium carbonate of both transect is shown in Table 6. It can be seen that the content of calcium carbonate in Aravali mountain ranges ranged between 0.00 to 175.40 g kg -1 with a weighted mean value 86.14 g kg -1 . While in Malwa plateau, its content ranged between 0.00 to 146.40 g kg -1 with a weighted mean value 109.12 g kg -1 . It was observed that calcium carbonate content in higher topographic positions that is, hill and pediment (P 1 , P 2 and P 5 , P 6 ) in both transects was found absent throughout whole profile which could be ascribed to the completely leached out of the profile as well removal of calcium carbonate and its subsequent deposition in lower topographic positions. The increasing trend for calcium carbonate content was found to be associated with decreasing topographic positions. A specific regular trend of distribution of calcium carbonate has emerged out with respect to the topography (hill top to plain) in the soils of Aravali mountain ranges and Malwa plateau. In cases of both transect, the surface layer contained lower/ nil values of calcium carbonate which gradually increased down the depth of profile. This was due to downward movement of calcium ions and precipitated in subsurface layers at higher pH level. An increasing trend of calcium carbonate with depth was registered in some soils of Udaipur and Chittaurgarh districts of Rajasthan by Singh et al. (1999). Similar observations were also recorded by Maji et al. (2005) and Kumar and Prasad (2010). Exchangeable cations A critical examination of data from Table 7 revealed that exchangeable calcium was the dominant cation followed by magnesium, potassium and sodium in soils of both transect. Exchangeable calcium, magnesium, potassium and sodium ranged between 5.86 to 22.05, 4.08 to 12.60, 0.18 to 0.89 and 0.44 to 0.95 C mol (p + ) kg -1 with a weighted mean value of 12.65, 7.09, 0.60 and 0.72 C mol (p + ) kg -1 , respectively in soils of Aravali mountain ranges. The soils of plain (P 4 ) had maximum average exchangeable calcium, magnesium, sodium and potassium. The soils of pediments (P 2 ), valley (P 3 ) hill top (P 1 ) come next with respect to these cations. In case of Malwa plateau, exchangeable calcium, magnesium, sodium potassium and ranged between 7.04 to 23.35, 6.07 to 12.05, 0.35 to 1.55 and 0.42 to 1.32 C mol (p + ) kg -1 with a weighted mean value of 15.98, 8.62, 0.8 and 0.84 C mol (p + ) kg -1 respectively. Exchangeable calcium was higher (23.35 C mol p + kg -1 ) in soils of plain (P 8 ) followed by pediments pedon P 6 [20.60 C mol (p + ) kg -1 ]. The exchangeable magnesium was followed the same trend of distribution. No specific trend of distribution has emerged out with respect to the exchangeable potassium and sodium. Calcium was dominant cation followed by magnesium, potassium and sodium also reported by Maji et al. (2005). The higher content of exchangeable calcium in soils of lower plains of Aravali mountain ranges had been attributed to the presence of dolomitic parent materials which contributes most of the calcium ions in runoff water which carry them to deposit in the soils of lower landforms and to the movement of bases from the upper part of transect to lower one, which are carried with the moving finer fractions of soils under influence of erosion, Walia and Chamuah (1994) also recorded higher base saturation at the lower topographical position. Cation exchange capacity Data pertaining to cation exchange capacity of soils are presented in Table 7 . The soils of plain (P 8 ) have maximum value of cation exchange capacity followed by the soils of Pedimant (P 6 ). While the soils of hill top (P 5 ) have the lowest value of CEC. A critical examination of data indicated that the cation exchange capacity of soils was found to be closely related to the clay content. The drifting of clay along with the bases down the slope might be the factor for the increased level of cation exchange capacity in subsurface layer of soils (Bhatia et al., 2005;Maji et al., 2005). It can be inferred that increase in clay content provide more exchange sites to get the cations adsorbed on it. Base saturation The data on per cent base saturation are presented in Table 7 Base saturation is by and large uniform in the soils of transect under study. It was found to range from 84.40 to 97.95% with a weighted mean value of 93.58% in soils of Aravali mountain ranges. While in soils of Malwa plateau it was found to range from 89.94 to 97.40% with a weighted mean value 93.92%. The higher base saturation in the soils of the study area could be attributed to the basic nature of the parent materials and also to the semi arid climatic conditions which allows bases to accumulate in soil matrix. It was uniform in all the soils of the area irrespective of the landforms. Exchangeable sodium percentage (ESP) Sodium saturation in the soils is expressed as ESP in the soil solution, because of its significance in deteriorating physico-chemical properties of soil and adversely affecting the growth of plant. The values of exchangeable sodium percentage ranges between 1.47 to 4.30% with a weighted mean value of 2.95% in soils of Aravali mountain ranges. The highest value of exchangeable sodium percentage was found in the soils of hill top P 1 (4.26%) followed by valley soils (3.10%) while the lowest value of exchangeable sodium percentage was recorded in plain P 4 (2.08%) and in pediments (2.39%) exhibit intermediate pattern with respect to exchangeable sodium percentage. In case of soils of Malwa plateau, exchangeable sodium percentage ranges between 0.88 to 5.65% with a weighted mean value 3.1%. The soils of Malwa plateau followed the similar pattern of distribution of exchangeable sodium in all topographic positions as in the soils of Aravali mountain ranges. Highest value of exchangeable sodium percentage is observed in soils of hill top pedon P 5 (4.58%) followed by pediment soils P 6 (4.31%). While lowest value of exchangeable sodium percentage observed in soils of plain P 8 (1.69%). The variation in exchangeable sodium percentage in surface soil with respect to slope was similar to exchangeable sodium. Generally, its content was less than 5.17 percent in all the pedons. Similar results were also reported by Saxena and Singh (1982). SOIL CLASSIFICATION The key diagnostic properties used for the classification of soils of both transects are listed in the Tables 8 and 9 at the different categorical levels and briefly described prior the taxonomic description of the soils. The classification of the soils up to the family level is elucidated in the Table 9. The brief description about the classification is given in diagnostic characteristics Diagnostic characteristics The following diagnostic characteristic are used in the classification of soil in both transects under investigation. Cambic horizons The improvement in soil structure in terms of angularity and grade in the subsurface horizon and/or richer in organic carbon and clay and darker in chroma and value as compared to the overlying or underlying horizons characterized the presence of cambic horizon in soils of valley (P 3 ) and plain (P 4 ) in case of Aravali mountain ranges soils while in case of Malwa plateau it was found in the soils of valley (P 7 ) pedons. The presence of cambic horizon is used as a diagnostic characteristic to separate the soils of inceptisols from Entisols after giving the due weight age to other features essentially needed for the other orders. Slickensides A layer of 25 cm or more thick with an upper boundary within 100 cm of the mineral soil surface that has either slickensides close to intersect or wedge shaped aggregates, which have their long axis tilted 10 to 60° from horizontal. This is one of the chief diagnostic characteristic required to mark a soil in Vertisols. In the present investigation, soils of pedon P 8 (plain) have more than 25 cm thick horizon within 100 cm of the surface, which have the above mentioned characteristics. Soil moisture regime The soils of both transects throughout the moisture control section remain dry for more than 90 days during the year and therefore quality for ustic soil moisture regime. The criterion was used as differential characteristics for classifying the soils at the suborder level within soil great group level (Table 8). Lithic contacts The presence of rock within 50 cm from the surface in the soil profile is characterized as lithic contacts in the soils of hill (P 1 ), pediments (P 2 ) of Aravali mountain ranges and in soils of hill (P 5 ) and pediment (P 6 ) of the Malwa plateau ( Figure 1). The criteria is used to classify the soils of both transects at subgroup level for separating the soil bearing this trait from their typic counter part. Soil temperature regime The soil temperature was more than 20°C within the control section and therefore qualifies for hyper thermic soil temperature regime. The difference between MWST and MSST is greater than 5°C and therefore does not qualify for isohyperthermic soil temperature regime. The criteria are used to classify the soils at the family level. Particle size class Coarse loamy skeletal, loamy skeletal, fine loamy, fine loamy mixed calcareous, calcareous, particle size (Table 9) characterized the soils of both transects. These together with soil temperature regime are used to classify the soils of both transect at the family level. Depth, texture and coarse fragments The characteristics of the soils are not directly visible in the classification. However, these together constitute the particle size class of the soils. Taxonomic descriptions The taxonomic classification of the soils of Pratapgarh district has been worked out based on morphological, physical and physico-chemical properties and climatic data according to Soil Taxonomy (Soil Survey Staff, 2000) into the order Entisols (Pedons P 1 and P 2 of Aravali mountain ranges and pedon P 5 and P 6 of Malwa plateau), Inceptisols (Pedons P 3 and P 4 of Aravali mountain ranges and Pedons P 7 of Malwa plateau) and Vertisols (Pedon P 8 of Malwa plateau). No diagnostic horizon and decrease in organic carbon was observed in the soils of hill (P 1 ) and pediment (P 2 ) of the Aravali mountain ranges and in the soils of hill (P 5 ) and (P 6 ) of the Malwa plateau. While cambic horizon is a key characteristics to mark as alteration in the original parent material in soils of valley (P 3 ), plain (P 4 ) of the Aravali mountain ranges and in the soils of valley (P 7 ) of the Malwa plateau. Slickenside was the important feature in the soil of plain (P 8 ) of the Malwa plateau. Based on these diagnostic features, soils of pedon P 1 , P 2 , and P 5 were classified in Entisols soil order, while the soils of pedon P 3, P 4 , and P 7 , were put under Inceptisols soil order. The soils of pedon P 8 was classified in Vertisol soil order. The soil orders are further taken down to the suborder level, using the other differential characteristics. Since the soils of P 1 , P 2 and P 5, P 6 could not qualify for psamments and fluents suborder, consequently placed in orthents. Based on the soil moisture regime, the soils of inceptisols and vertisol soil order are classified as a member of ustepts and usterts suborder, respectively. The Ustic moisture regime is considered for bringing down the soil of orthents to the Ustorthents at great group level. Since ustepts and usterts do not qualify for any other great group within suborder, consequently, these have been placed in Haplustepts and Typic Haplusterts great group of their respective suborder. Presence of rock within 50 cm of the soil profile was the criteria to separate the soils of pedon P 1 , P 2 , P 5 and P 6 from other Ustorthents to Lithic Ustorthents and Ustepts to Lithic Ustepts at the subgroup level, respectively. The soils of pedon P 3, P 4 , P 7 , P 8 , and P 9 , represent the central concept of Haplustepts subgroup. As a result, these have been classified in Typic Haplustepts subgroup. The soils of pedon P 8 belonging to the Haplusterts great group also represent the central concept, consequently qualify for Typic Haplusterts subgroup of vertisols soil order. Based on the particle size class, hyperthermic soil temperature regime, mixed mineralogy class, soils of both transects are further taken down to the lowest taxa of soil taxonomy, soil family. According these features, soils of pedon P 1 and P 2 have been classified as a member of loamy skeletal mixed hyper thermic family of Lithic Ustorthents subgroup under Entisols soil order. While soils of P 5 has been classified as a member of fine loamy mixed hyperthermic family of Typic Ustorthents subgroup. The soils of pedons P 6 in Lithic Haplustepts subgroup are classified as a member of fine loamy calcareous mixed hyper thermic soil family of Entisols soil order. In case of Pedon P 3 and P 9 soils have been classified as a member of fine loamy hyper thermic family of Typic Haplustepts in Inceptisols order. While the soils of Pedon P 4 , have been classified as a member of fine loamy calcareous hyper thermic family of Typic Haplustepts in Inceptisols order. Soils of Pedon P 8 have classified as a fine mixed calcareous hyperthermic family of Typic Haplusterts subgroup of Vertisols soil order.

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Historical Use of Cultivars as Parents in Florida and Louisiana Sugarcane Breeding Programs Sugarcane (Saccharum L. spp. hybrids) growers depend on breeding programs for new, high-yielding cultivars that have resistance to abiotic and biotic stresses, so breeders continually seek out widely adapted, high yielding germplasm to be used as parents for their programs. Cultivars are sometimes used for this purpose, but their use may be minimized to prevent genetic diversity erosion. The purpose of this study was to determine the importance of cultivars as parents in three USA (one in Florida and two in Louisiana) sugarcane breeding programs by quantifying the percentage of cultivars that had these parental groupings based on published registrations and crossing records. The percentage of cultivars with at least one commercial parent for each program was 81.8%, 77.5%, and 64.3% for the Houma (Ho), Louisiana, Canal Point (CP), Florida and Louisiana State University (LSU) programs, respectively, but cultivars were recently used as parents in only 11.8% (Ho), 16.39% (CP), and 34.3% (LSU) of crosses. The results indicate that the CP and Ho programs should consider increasing the use of cultivars as parents in their breeding programs to increase the probability of selecting potential commercial genotypes, but this should be balanced with high diversity crosses to avoid the loss of diversity. Introduction Mainland US sugarcane breeding programs employ recurrent selection principles that continually identify the highest yielding germplasm with acceptable stress resistance and make the majority of crosses in their breeding programs using these genotypes as parents. Throughout this paper, sugarcane genotype is used to denote a genetically unique individual derived from sexual recombination. Genotypes are most often tested in different stages of selection programs and are generally named in an early stage of selection. Later, once more of the data are available for a given genotype, some are used as parents in crosses. Cultivar denotes a genotype that has been publicly released for commercial use. We use the term commercial parent to describe a cultivar that is used as a parent in a cross. Three breeding programs were researched in this study. The two USDA-ARS breeding programs are based in Houma, LA, Canal Point, FL, and the third Louisiana State University program in St. Gabriel, LA. Each breeding program has different breeding strategies that address the different regions they serve. Florida has a subtropical climate where cold tolerance is a concern, although damaging freezes do not occur every year. However, Louisiana has a colder climate with regular damaging freezes, and cold tolerance is more important in Louisiana than in Florida. The Louisiana sugarcane industry is comprised of a relatively large number of growers with smaller farms while the Florida industry has large corporations and generally much greater land areas per farm owner than in Louisiana. For example, a representative farm size for the Louisiana industry would be 404.7 ha [1] while a representative farm size in Florida would be 2000 ha [2]. Such industry characteristics result in programmatic differences in the number and type of crosses made, type of plants selected, and numbers of cultivars released. Houma and LSU programs, whose origin can be traced to 1885 [3], are highly collaborative and mutually strive to improve productivity of the Louisiana sugarcane industry [4]. Both Louisiana programs conduct sugarcane breeding and selection activities [5]. Crossing through early stage clonal testing and selection are conducted independently by each of the two institutions, and midway through clonal selection stages promising genotypes from the two are jointly evaluated by the three Louisiana organizations. The American Sugar Cane League (ASCL) assists with the final multilocation testing stage and has primary responsibility for seed increase and distribution to growers upon varietal release. In accordance with the three-way agreement, all three Louisiana organizations jointly decide on a cultivar release [5]. Sugarcane crosses have been made in Louisiana since 1948 [6] by Louisiana State University. The current Louisiana breeding program is conducted by Louisiana State University Agricultural Center (LSU) at St. Gabriel, LA, along with the USDA-ARS Sugarcane Research Unit at Houma, LA, in cooperation with the ASCL located at Thibodaux, LA, under a three-way cooperative agreement [7]. The ASCL is Louisiana's sugarcane grower and processor organization and contributes professional expertise and financial support [7]. The Houma program emphasizes germplasm enhancement through basic breeding [8]. Basic breeding is a scheme to intercross the desirable alleles from diverse wild germplasm into a composite breeding material that will be used to backcross into elite cultivars [9]. The Florida and Louisiana programs have long been closely affiliated. Prior to 1972, basic-and commercial-type sugarcane crosses for the Houma program were made only at the USDA-ARS Research Station at Canal Point (CP), Florida. Since then crossing efforts specifically targeting germplasm enhancement were initiated at Houma [8]. Crossing facilities at Houma are primarily for basictype crosses, while CP serves as the main source of Houma's commercial-type crosses using Louisiana germplasm. Houma cultivars from CP 65-357 to CP 79-318 received the CP name designation even though germplasm had been selected at Houma. This included the BC 1 cultivar TucCP 77-42 which was selected in Argentina from a cross made at Houma [10]. Cultivars released from the LSU program have an "L" name designation while those from the Houma program have an Ho name designation. A compound naming designation was developed to assure that a cultivar name appropriately reflected the breeding groups involved in its development [9]. For example, HoCP 85-845 indicates that the cross was made at CP and the clone was selected at Houma [11]. Sugarcane breeding in the United States for the Louisiana breeding program began in Canal Point Florida in 1919 [12] but from 1960 breeding efforts were also directed for Florida region [13]. The current Canal Point program consists of a three-way cooperative agreement between the USDA-ARS Research Field Station at Canal Point (CP), the University of Florida Everglades Research and Education Center located at Belle Glade, and the Florida Sugarcane League located at Clewiston, Florida. Cultivars are recommended for release by the vote of a committee with representatives from each of these three organizations and carry a CP name designation. Florida and Louisiana programs also share germplasm between themselves resulting in combined LCP, HoCP, and LHo name designations. Deren [14] traced the genealogy of US sugarcane germplasm and found that 10 clones were the source of 90% of Florida germplasm and that two ancestors were the source of 90% of Louisiana germplasm. He noted that the diversity of sugarcane was buffered due to multiple interspecific crosses in the respective breeding programs. These include wild species and early generation intergeneric and interspecific crosses used to broaden genetic diversity and increase disease resistance. This is exemplified by the Houma, LA, breeding program which maintains two general types of germplasm: basic germplasm, typically F1 to BC2 generations, and elite or nearly elite commercial germplasm (typically BC3 and higher generations) [5]. There are also basic crosses made in Canal Point which are also evaluated for cold tolerance and in the energycane program, but the main focus of the Canal Point program is sugarcane cultivar development. In most crops, including sugarcane, new cultivars are selected from the progeny of crosses of commonly grown cultivars and other elite materials [5]. There is some evidence of past successful use of cultivars as parents in other programs including POJ2878 which was the parent of 181 sugarcane varieties worldwide and Co419 that was the parent of 81 sugarcane cultivars including two of China's most widely grown cultivars [15,16]. Other popular cultivars in China include ROC22 which has been used as a parent in crosses since 2002 and at least five of its progeny have been released as cultivars including Guitang 04-1001, Guitang 29, [17], Dezhe 03-83 [18], and Liucheng 03-182 [16]. Cultivars are sometimes used in crosses, but their importance as breeding parents in sugarcane breeding programs has not been documented. The purpose of this study was to determine the importance of cultivars as parents in Florida and Louisiana public sugarcane breeding programs by quantifying the percentage of cultivars derived from crosses with commercial parents. Materials and Methods Three public sugarcane breeding and selection programs were included in the study: CP, Houma, and LSU. Records of cultivar pedigrees were collected using existing pedigree dendrograms and published cultivar registrations. Information for some cultivars was obtained from [19,20]. The search was limited to cultivars grown for sugar and biofuel. Genealogies for the Florida program began with cultivar CP 63-306 which was released in 1971. Earlier cultivars in the CP program are not evaluated, but they are noted as parents thereby covering 52 years of breeding. Some CP cultivars developed by the Houma breeding program, before the compound naming system, were often planted in Florida. Early CP cultivars selected for release to the Louisiana industry were counted as Houma cultivars and those selected for release in Florida were counted as CP cultivars. Cultivars developed by LSU in this study began with L 62-96 which was released in 1969. Crossing records from CP for 2000-2006 and 2010-2012 and LSU for 2003-2012 and Houma from 1992 to 2012 were obtained from crossing records of each program to compare the number of cultivars that was used as parents of other cultivars and the use of cultivars as parents in the total number of crosses generated. Within the CP records all crosses are recorded including those made for CP and Houma and the crosses made for both. The years 2007-2009 were not included in CP counts because the difference between crosses used in Florida and crosses used for Houma, Louisiana was not recorded and the crosses for Florida and Louisiana could not be distinguished. Ten years were assumed to be sufficient for CP program based on the large number of crosses (6945 total crosses) included in the study. Additional years were included for the Houma program (6630 total crosses) to approximate the number crosses of the CP program. The number of crosses for LSU was restricted to 4423 total crosses as additional crossing records for this program were not available. To calculate the percentage of cultivars with at least one commercial parent, the presence of one or two commercial parents was counted as one per cultivar progeny then divided by the total number of cultivar progeny (at least one parent cross/total number of crosses * 100). The percentage of all commercial parents was calculated by adding all the commercial parents and dividing them by the total number of parents, including those with unknown parents. The percentage of cultivars calculated from crossing records was calculated by dividing the total number of times cultivar parents were used in the cross by the total number of times all parents were used and the selfs were treated as one parent use (cultivar parents/all parents * 100). Two types of Chi-square tests were conducted using SAS PROC Freq: a test between the breeding locations and a proportion test of cultivar versus noncultivar within breeding locations ( < 0.05). Results and Discussion There were 71 cultivars from the CP program reviewed (Table 1). Of these 71 cultivars 77.5% of them had at least one cultivar parent, and 54.6% of the total parents were cultivars ( Table 2). There were 42 cultivars used as parents in the crosses that developed these 71 cultivars, and each of these cultivars was a parent of another cultivar at an average of 1.83 times (sum of the times a particular cultivar was used as parent divided by the total number of cultivar parents). Cultivars were used as female parents of cultivars 47 times and as male parents 30 times including two selfs; CP 89-2143 [21] was the parent of CP 00-1101 and HoCP 91-552 [22] which was the parent of CP 00-2180 [23]. The most successful commercial parent was CP 68-1067 [24], which was used as a parent six times from which new cultivars were selected. The female and male parents of CP 68-1067 are CP 52-68 [20] and CP 57-603 [25], respectively, and they were also cultivars. Cultivar progeny of CP 68-1067 are CP 75-1082 [26], CP 75-1632 [27], CP 77-1776 [28], CP 78-1628 [29], CP 80-1743 [30], and CP 81-1384 [31]. Out of six cultivars five were produced when CP 68-1067 was used as a female parent and one (CP 80-1743) when used as male parent. CP 80-1743 was the most widely grown sugarcane cultivar in Florida for several years and CP 78-1628 was the most widely grown sugarcane cultivar on sand soils in Florida for several years [32]. Most of the cultivar progeny of CP 68-1067 had high or moderate commercial recoverable sucrose, except CP 75-1082 which had high cane tonnage and low commercial recoverable sucrose. Some of the cultivar progeny of CP 68-1067 also had cultivar grand-progeny. CP 75-1082 was the female parent of CP 86-1633 [33] and CP 80-1743 was the female parent of CP 88-1762 [34], CP 97-1944 [35], and CPCL 02-0926 [36]. CP 88-1762 became the most widely grown cultivar in Florida in 2010 [37]. The parents of 22 Houma cultivars were reviewed (Table 3). Of these 22 cultivars 81.8% had at least one cultivar parent, and 56.82% of the total parents were cultivars ( Table 3). The most successful cross in the Houma program was between CP 65-357 [50] and L 65-69 [51]. The hugely successful Louisiana cultivar, CP 65-357, was grown on more land area (71%) than the sum of all other cultivars in 1980 [52]. It took two decades before another cultivar, LCP 85-384, assumed an equally prominent lead above all other cultivars, with 91% of the acreage in 2004 [53]. The cross of cultivars CP 65-357 × L 65-69 produced four cultivars: CP 73-351 [54], CP-74-383 [55], CP 76-331 [56], and CP 79-318 [57]. None of the cultivars from the L 65-69 × CP 65-357 crosses parented other cultivars. CP 65-357 was not a parent of any LSU cultivar (Table 4). More cultivars and parents were shared between the two Louisiana programs (Tables 3 and 4) than between Louisiana and Florida programs (Table 1), probably reflecting the different cultivar specific requirements of their respective industries. The parents of 14 LSU cultivars were reviewed and of these 64.3% had at least one cultivar parent, and 42.86% of the total parents were cultivars. Of these, three cultivars were repeated as parents. CP 52-68 [20] was a parent of three commercial progenies. CP 48-103 [20] had two commercial progenies and LCP 85-384 [58] had three commercial progenies (Table 4). It is likely that these programs actually have higher percentages of cultivars with commercial parents. For example, 4 International Scholarly Research Notices if we evaluate the parents in the CP program that were not identified as cultivars, we find that there were six unknown genotypes because of labeling issues and 12 unknown males because they were in polycrosses. Thus, there is a chance that some of these 18 unknown parents were cultivars. The paternity of these polycrosses could be determined with further study using molecular markers [59]. A total of 46 genotypes that were parents of commercial progeny in the CP program were noncommercial mostly elite germplasm. The most successful of these parents was CP 84-1322; it was the male parent of three cultivars. For two of three cultivars the female parent was also a cultivar (crosses that developed CP 92-1641 and CP 92-1666). Also, the parents of CP 84-1322 were both commercial cultivars (CP 52-068 [20] and CP 72-2086 [60]). CP 72-2086 was the most widely grown cultivar in Florida in 1994 [43]. Most noncommercial germplasm only parented commercial progeny one or two times. The Houma program had two noncommercial elite parents producing commercial progeny two times each CP 83-644 and CP 61-39, and the LSU program also had two noncultivar elite parents producing commercial progeny two times each CP 77-310 and L 93-365. Four cultivars from Houma were derived from basic parental clones (Table 3) including SES 234 (S. spontaneum L.) and three US clones. Houma clones selected in basic breeding were previously assigned US [United States] breeding numbers, but basic selections are now given Ho designations. Basic clones typically were used in the breeding program as nonrecurrent parents for backcrossing with selected interspecific hybrids used as recurrent parents [10], and cultivars were often the recurrent parent. In practice, crosses usually were constructed taking into consideration the parental pedigrees to minimize potential effects of inbreeding. For example, LCP 85-384 [58] has occurred frequently as a parent in the Louisiana programs (Tables 3 and 4), so caution is needed to minimize full-and half-sib crosses with this cultivar. The high number of noncommercial crosses in the Houma program (Table 2) indicates that the basic program is strong. But since the released cultivars have ancestry which is primarily commercial, new methods should be sought to incorporate basic germplasm in such a way to develop successful cultivars that utilize better the diversity of the basic program. The crossing records at CP were gathered from recent records to see how the programs have acted recently and see how they might change. Programs differed significantly in the percentage of cultivars used in crosses in the order LSU > CP > Houma, 34.3, 16.4, and 11.8%, respectively (Table 2). LSU had nearly a 2-to 3-fold greater use of cultivars as parents than the other programs. Overuse of cultivars in crosses could cause a reduction in genetic diversity. Of the 71 CP program cultivars surveyed, 55 (77.8%) had at least one commercial parent. Over half of the parents (77/141 * 100 = 54.6%) of cultivars in the CP program were cultivars. According to the crossing records examined, cultivars were used in only 16.4% of the crosses in this program 6 International Scholarly Research Notices (Table 2). Houma used cultivars as parents in significantly (Chi-square < 0.05) fewer crosses (11.8%) but had a larger percentage of parent cultivars (56.8%) and cultivars with at least one parent (81.8%) but these differences were not significant. But the proportions of cultivars with at least one cultivar parent to those with no cultivar parentage in the CP (77. 46) and Houma (81.82) programs were significantly unequal. The proportion of at least one cultivar parent in the LSU (64.29) program was not significantly unequal indicating that the CP and Houma programs have released proportionally more cultivars with commercial parents. The LSU breeding program used cultivars in a greater percentage of their crosses (34.3%) than CP and Houma. However, there were fewer cultivars with parent cultivars (42.9%) and few cultivars with at least one cultivar parent (64.3) compared to the CP or Houma programs. The crossing percentages from the different programs do not directly relate to the percentages of cultivars with cultivar parents because the crossing period examined does not correspond with the time periods when most of the older cultivars were developed. The current crossing records do indicate that commercial cultivar crossing is not a priority and could be increased. The difference between programs for total commercial parents and cultivars, at least one cultivar parent, was not significantly different between the breeding programs, but the percentage of cultivars used in crossing was significantly different between programs. Our results indicate that cultivars are an excellent source of successful parents in all three programs and even though the programs have different breeding strategies, the cultivars they release are not significantly different in their proportions of total cultivar parents. LSU uses significantly more cultivars in crosses, but the proportion of at least one cultivar parent is proportional to noncultivars unlike the other programs ( Table 2). The Houma program had percentages similar to CP even though it differs somewhat from the other two programs due to its emphasis on germplasm enhancement through basic breeding [5], with lesser importance placed on commercial crosses. Basic and commercial germplasm gradually merge through recurrent selection to the point at which cultivars are released for sugar or bioenergy. Cultivars also were used in 16.4% (CP), 11.8% (Houma), and 34.3% (LSU) of total crosses demonstrating their importance for contributing superior genes in the breeding pool at each location. The lower frequency of cultivars in total crosses at Houma probably reflects the greater use of basic germplasm in this program compared to that at LSU. But the genotype crosses at CP include mostly elite germplasm crosses and a few basic and energycane crosses. Energycane breeding which represented 13% of the CP crosses (159 crosses) in 2012 overlaps somewhat with basic breeding at CP and the industry for energycane is relatively new so not many cultivars have been released. There is also proportion of basic crosses shared between Houma and CP programs. The choice of germplasm for breeding is biased in subtropical climates by the problem of unreliable flowering [61]. Often promising germplasm or cultivars are not selected for crossing because they will not flower under the conditions at the breeding station. Some promising foreign germplasm at Canal Point is not selected for crossing because they will not flower (personal communication). This could explain why there is less exotic material in the pedigrees of cultivars. Also lack of flowering synchrony will limit the types of crosses that can be made [61]. This could have skewed the use of cultivars in crossing at each breeding program. If breeding programs increase the percentage of cultivars in their crossing programs, it should be done strategically to ensure that robust sources of diversity are maintained. Loss of diversity could hamper long-term breeding progress. Broadness of germplasm among the parental pool is an important measure of genetic diversity [62], and restricting parents to cultivars would narrow the parental gene pool. Maintaining a diverse germplasm and having the flexibility to import new germplasm are also important because of the constant need to incorporate new disease resistance genes into the parental pool, but the selection of these for crossing could be affected by the ability of these parents to flower. A possible reason why cultivars with cultivar parents are chosen is adaptability. The short cold Louisiana season and the muck organic soils in Florida are unique environments that probably favor particular gene combinations within existing cultivars. A future challenge will be to find efficient ways to quickly incorporate diversity and disease resistance into high performing germplasm like commercial cultivars. There is a group of cultivars ( Table 5) that did not produce commercial progeny. This could be due to not being crossed or deficiencies in their progeny. This should be investigated in the future to clarify better what useful germplasm is. It could be that the yield of some commercial and elite germplasm is due to heterosis and nonadditive genes. As molecular breeding technology becomes more practical and economically feasible it would be interesting to locate quantitative trait loci (QTL) of good parents then trace them within a lineage and compare them to cultivars that did not have successful progeny. This would allow us to understand better how QTLs from diverse backgrounds interact [63]. Conclusions Sugarcane cultivars have been found to be acting as successful parents in all three breeding programs. A large percentage of the total number of cultivars in all programs has at least one commercial parent. In the LSU sugarcane breeding program the proportion of commercial parents to noncommercial parents was not significantly different but in the more recent crossing time period analyzed cultivars were crossed significantly more frequently than in the other programs. The Canal Point and Houma programs did have significantly higher proportion of their cultivars with at least one commercial parent. Programmatic differences reflect, to some extent, philosophical approaches and responses to their respective industries. For example, the Florida program is larger than the two Louisiana programs combined, serves a subtropical climate with a longer growing season, and releases more cultivars to larger growers. Conversely, the Louisiana industry serves many growers with smaller farming operations with a shorter growing season who prefer that cultivars demonstrate early maturity prior to release.

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2019-04-01T13:12:44.539Z

2015-08-01T00:00:00.000Z

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PEDOTRANSFER FUNCTIONS FOR WATER RETENTION IN THE MAIN SOILS FROM THE BRAZILIAN COASTAL PLAINS Pedotransfer functions (PTFs) are equations used to estimate soil characteristics difficult to determine from other easily obtained ones. Water retention in soil is used in several agronomic and environmental applications, but its direct determination is time consuming and onerous, therefore PTFs are alternatives to obtaining this information more quickly and economically. The aims of this study were to generate a database and develop PTFs for water retention at potentials of -33 kPa (field capacity) and -1500 kPa (permanent wilting point) for Yellow Argisol and Yellow Latosol from the Brazilian Coastal Plains region. The Coastal Plains soils are mostly developed from Barreiras formation (pre-weathered sediments) and their main uses are sugarcane, livestock, forestry and fruticulture. The database to generate the PTFs was composed from the selection of information derived from scientific works and soil survey reports of the region. Specific PTFs were generated for each soil class, in their respective A and B horizons and for solum, through multiple regression by stepwise package of R language programming. Due to the small pedological variability (small number of soil classes containing great geographical expression) and mineralogical uniformity, usually observed in this environment, non-stratification of soil classes to create general PTFs presented similar or superior results compared to equations for each soil class. The adjustment of data demonstrated that water retention values at -33 kPa and -1500 kPa potentials can be estimated with adequate accuracy for the main soils of the Brazilian Coastal Plains through PTFs mainly from particle size distribution and secondarily from organic matter data. INTRODUCTION The importance of knowledge about hydrical characteristics of soil is related to its direct influence on water reservoir and availability to plants. These characteristics are difficult to be determined due to the high cost analyses, time and personal demand. In order to facilitate the obtaining of data on water content at specific matric potentials, many researchers have proposed mathematical models for estimating water retention from easily obtained and fast determination attributes. These models are denominated Pedotransfer Functions (PTFs). In Brazil, the major difficulty to the development of such models is the relative scarcity of data bases in different regions. The PTFs are commonly utilized in Soil Science, Hydrology and Agrometeorology for estimating water conductivity, water retention curve and parameters related to soil water infiltration (Medeiros et al, 2014). Many models utilize PTFs for simulating soil water, air and solutes transport, compaction, structure stability, and penetration resistance (Botula et al., 2014). For different soils of Rio Grande do Sul State, Santos et al. (2013) verified that data of water retention at -33 and -1500 kPa matric potentials can be estimated from particle size distribution and organic matter data and the stratification by soil classes increases the PTF accuracy due to the great pedological and mineralogical variability. The Brazilian Coastal Plains environment represents a 20 million hectares area (Souza, 2010, personal information). The soils are mainly derived from pre-weathered sediments of Barreiras Formation and the more expressive uses are sugarcane, cattle raising, forestry (eucalyptus) and fruticulture (Fonsêca et al., 2007). A remarkable characteristic of those soils are the cohesive subsuperficial horizons, which difficult the root system deepening and maximize the water stress mainly for perennial crops (Carvalho Filho;Fonseca, 2013). The subsoiling practice at greater depths has been applied to minimize such constraints. According to Curi and Ker (2004) and Carvalho Filho, Curi and Fonseca (2013), the main soil classes are Yellow Argisols (Ultisols), followed by Yellow Latosols (Oxisols). The objectives of this study were to generate a soil database from the selection of soil profiles data of scientific works and soil survey reports from the Brazilian Coastal Plains region and to develop PTFs for estimating the water retention for the main soils of this environment, taking into account particle size distribution and organic matter as independent variables, keeping in mind the known interdependence among these characteristics. MATERIAL AND METHODS The database for PTFs generation was composed of soil profiles data from scientific works and soil survey reports of the Brazilian Coastal Plains region. 69 soil profiles were utilized, being 55 of Yellow Argisol (YA) and 14 of Yellow Latosol (YL), making up a total of 138 soil horizons (A and B). In Table 1, it is presented the bibliographical source, the number of soil profiles and horizons utilized in this study. It were selected scientific works that employed disturbed samples, considering that, in the vast majority of soil surveys, soil water retention is measured using disturbed samples, and that the collecting of undisturbed samples is more time-consuming and more expensive, besides the fact that the disturbed samples present lower variability in such measurements. It is known that in higher matric potentials such as -33 kPa (Field Capacity -FC), the water retention is influenced by structure (Oliveira et al., 2002) and the FC is overestimated for most soils, except for sandy soils (Bell;Van Keulen, 1995). However, this methodology is justified considering two main reasons: the development of these PTFs seeks maximum efficiency with simple and low cost input data, and most of the potential users of these PTFs are in countries with low amount of data, case of Brazil and other tropical countries. In addition, papers such as Oliveira et al. (2002) and Santos et al. (2013) obtained adequate PTF fitting utilizing disturbed soil samples. The database was stratified by soil class (YA and YL), soil horizon (A and B) and solum. The data were submitted to multiple regression in R programming language, using the stepwise package. The independent variables for generating the PTFs were particle size distribution, organic matter and water content at -33 kPa and -1500 kPa, corresponding to field capacity and permanent wilting point. Ciênc. Agrotec., Lavras, v. 39, n. 4, p. 331-338, jul./ago., 2015 In addition, PTFs were generated without soil classes and horizons stratification in order to test the need of data separation. The PTFs performance was evaluated by means of the following statistical parameters: determination coefficient (R 2 ), estimative standard error (ESE) and 1:1 relation of estimated versus observed data. The R 2 measures how well the model can explain the observed values. The ESE measures the average deviation between the Y real and estimated values. It provides approximate information on the error extension between the estimated values and the values obtained by PTF functions. Table 2 presents the minimum, maximum and average values of samples from the database utilized for generating PTFs, in which can be observed a great data range. RESULTS AND DISCUSSION The organic matter ranged from 0.1 to 8.5 dag kg -1 . Clay, silt, fine sand and coarse sand ranged from 3 to 78, 1 to 43, 4 to 72, and 1 to 84 dag kg -1 , respectively. The water content retained at -33 kPa (FC) and -1500 kPa (PWP) varied from 2.6 to 25.8, and from 0.9 to 18.4 dag kg -1 , respectively. Table 3 presents the PTFs for Yellow Argisol (YA), Yellow Latosol (YL) and for both soils (General) considering A and B horizons separately and together (solum), for water content at -33 kPa and -1500 kPa, with their respective R 2 and ESE. The multiple regression equations presented significative R 2 at 5% probability. For -33 kPa potential, R 2 values ranged from 0.32 to 0.73. For -1500 kPa, these values ranged from 0.12 to 0.75. The highest R 2 values occurred when there was not an A and B horizons stratification (solum), being this behavior applied for individual soils as well as for the general model for both soil classes. This fact can be explained by the great mineralogical uniformity in the soils (Duarte et al., 2000;Resende et al., 2011;Carvalho Filho;Fonseca, 2013) and in the parent material of soils from this environment (Nascimento et al., 2010). The lower R 2 values for YL can be related to this soil class being in much smaller number in the database, in agreement with its much smaller geographical expression in the Brazilian Coastal Plains (Carvalho Filho, Curi;Fonsêca, 2013). The general PTF presented R 2 very close or superior to that involving data stratification by soil classes due to the smaller pedological variability (small number of soil classes having great geographical expression) of this environment. The general PTFs with the junction of A and B horizons (solum) revealed high predictive power for the estimation of water content at -33 kPa and -1500 kPa, 73% and 75%, respectively. The higher R 2 values, followed by the lower ESE (Table 3), observed in the predictive equations of water content at -1500 kPa matric potential, are indicative of the better expressed relationships between water retention and particle size distribution at more elevated tensions, besides the presence of stable microaggregates, according to Oliveira et al. (2002). Figures 1, 2 and 3 illustrate the correlation results between the observed and predicted data for the YA, YL and General models, respectively. In Table 3, the junction of soil classes presented high predictive power of equations, allowing a higher data entry for generation of PTFs. The results found by Santos et al. (2013) for Rio Grande do Sul State soils demonstrated that for regions where the pedological and mineralogical variability is expressive (Streck et al. 2008), the stratification by soil classes for generating PTF results into higher coefficients than PTFs in which a small number of data of all soils is compiled in a sole model. Due to the smaller pedological and mineralogical variability in the Coastal Plains environment (Resende et al. 2011;Carvalho Filho;Fonseca, 2013), the junction of different soil classes (Yellow Argisol and Yellow Latosol) and soil horizons (A and B) did not cause a deleterious effect on coefficient of PTFs. It constitutes a recent support for future scientific and practical works.

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2016-05-12T22:15:10.714Z

2015-05-22T00:00:00.000Z

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Two Fixed Ratio Dilutions for Soil Salinity Monitoring in Hypersaline Wetlands Highly soluble salts are undesirable in agriculture because they reduce yields or the quality of most cash crops and can leak to surface or sub-surface waters. In some cases salinity can be associated with unique history, rarity, or special habitats protected by environmental laws. Yet in considering the measurement of soil salinity for long-term monitoring purposes, adequate methods are required. Both saturated paste extracts, intended for agriculture, and direct surface and/or porewater salinity measurement, used in inundated wetlands, are unsuited for hypersaline wetlands that often are only occasionally inundated. For these cases, we propose the use of 1:5 soil/water (weight/weight) extracts as the standard for expressing the electrical conductivity (EC) of such soils and for further salt determinations. We also propose checking for ion-pairing with a 1:10 or more diluted extract in hypersaline soils. As an illustration, we apply the two-dilutions approach to a set of 359 soil samples from saline wetlands ranging in ECe from 2.3 dS m-1 to 183.0 dS m-1. This easy procedure will be useful in survey campaigns and in the monitoring of soil salt content. Introduction Salts commonly occur across the Earth's surface. Most salts needed for life are imbibed by plants from the soil. Thus, fertile soil provides an adequate content of salts needed by plants. Contrariwise, highly soluble salts are biocides at high concentrations. Few organisms, called extremophiles, are adapted to hypersaline conditions. Soil salts at the Earth surface are dissolved and redistributed across the landscape, leading either to salt leaching or accumulation at specific geomorphic positions. Saline soils are more frequent in regions where evaporation exceeds rainfall. Together with natural factors, human actions can salinize or desalinize soils, sometimes in only a few years. Examples of such include the clearing of lands in Australia, or irrigation with brackish water pervasive in some countries. In some cases, changes in soil salt contents have been measured or surmised at several temporal scales [1][2][3][4]. The conservation of saline enclaves comes up as the demands for environmental protection become more elaborate, e.g., [5], especially in arid lands as reviewed by Williams [6][7]. Many saline wetlands around the world are included in the Ramsar Convention signed by 167 conductivity of the saturated paste extract (ECe) for expressing soil salinity, with the extracts at other fixed soil to water ratios considered auxiliary or less important. The amount of water needed to prepare the saturated paste is reported as cm 3 of water per 100 g of soil, i.e., weight to weight (w/w). This amount, i.e., the saturation percentage (SP), depends on the textural composition of the soil, clay mineralogy, and organic matter contents. Saturated paste was intended to enable soil solution to be extracted with simple equipment, overcoming the practical impossibility of extracting the actual soil solution used by crop roots. ECe has become a standard measure of soil salinity, widely used to compare the salt tolerance of cultivated plants, and for irrigation and drainage engineering. The purpose of soil salinity monitoring in hypersaline soils of protected wetlands is not to determine the effects of dissolved salts on plant growth, as is the case in agricultural or in plant physiology research. The monitoring of protected wetlands will watch and ward the salt contents associated with the prevalence of the halophytes and other valuable organisms adapted to hypersalinity. The available methods to measure the electrical conductivity (EC) in-situ are influenced by temperature and soil moisture, and are unpractical in these intermittently inundated wetlands. Measures of soil salinity by a lumped parameter, EC, at standard and repeatable conditions of temperature and dilution will be needed for years to come. 3.1 Extracts at fixed soil to water ratios, with 1:5 and 1:10 as the more popular, have been used in soil science. The disclosure of the relationships of their EC with ECe [15] has garnered the attention of many scientists using different approaches as shown by [16]. However, the United States Salinity Laboratory Staff ( [14], page 13) recommended fixed ratios for determining the change in salinity with time; in the same way, [17] recommend 1:10 for evaluation of the total soluble salts of soil and for the assessment of reclamation procedures. The search for such an equation can be unpractical for soil survey or monitoring operations due to the need for either supplementary data to be included in the equation or chemical considerations often applied with ionic speciation software. However, if the interest is focused on the off-site effects of soil salinity (i.e., salt discharge to surface and underground waters, rather than on the effects of salinity on plants), a fixed soil to water ratio more diluted than the saturation extract can better estimate the content of salts in soils. 3.2 The non-occurrence of ion-pairing is a condition to have a simple relationship of EC with the content of salts in solution. If ion-pairing occurs, the measure of the salt content would require either gravimetry of the total dissolved salts or the individual titration of soluble ions. Ion-pairing does not occur in saturation extracts of low EC; e.g., in the experiments of [35] with a nonsaline soil of ECe = 0.66 dS m -1 . However, the reservations [36][37] for soil salinity estimates from ECe in non-saline calcareous soils can be extended to gypsiferous soils [29]. 3.3 The correspondence between ECe and salt content diminishes at high salinities, with the classical olifant-shaped silhouette in the scatter diagrams of ions vs. ECe [34] needing logarithmic transformations to approach linear relationships. Many soil scientists [38,39] have shown the problems of relating ECe with total salt content for hypersaline soils. Similar concerns were expressed by [40] for ECe > 8 dS m-1 when relating ECe with the electrical conductivity measured in the saturated paste. Moreover, [36] found that for non-saline calcareous soils, saturation extracts require dilution by a factor of 1000 to accurately predict soil salinity. The aforementioned considerations jeopardize the meaning of ECe and also the additivity of this magnitude from different samples, and can question the meaning of averaged ECe for pedons sampled at several depths, or for multiple samples when estimating soil salinity of broad extensions. These constraints can be dramatically reduced if no ion-pairing happens, as would be the case for extracts more diluted than those from saturated paste. 3.4 Most ECe measurements and subsequent determinations in the saturation extracts are used for agriculture or for plant salt-tolerance appraisal e.g., [41][42][43], and for the study of saline habitats, e.g., [44,45]. Most of these works do not report saturation percentage (SP). However, when ECe is to be used for comparison of salinity in the same soil, evaluation of the deviations in SP between different times or laboratories [27,34,46] is compulsory. Yet often SP is not reported in comparisons of salinity, e.g., [2,47,48]. A 5% SP deviation has been suggested as an allowable difference ( [49], page 265). This is a key issue for comparisons involving different technicians or laboratories, especially for long-term monitoring. The measurement of changes in ECe and ion concentrations can be biased not only because of the different criteria in the end-point of the paste but also by the grinding or extraction methods [50][51]. Moreover while centrifugation of the saturated paste is used by many labs for obtaining the extract, many others use vacuum extraction, a technique that concentrates the extract, as evidenced by the descent of temperatures in the flasks produced by evaporation under vacuum. In practice, extract concentration achieved by evaporation cannot be compensated by calculations, and will variably affect the extraction depending on the intensity and time of vacuum, lab temperature, or the whole volume of air evacuated by the pump. The total effects will be associated with the granulometry and other properties of the soil sample, affecting each sample at different degrees even within the same extraction set. 3.5 The saturation extract cannot be obtained in field labs. Moreover, saturated paste preparation is made by hand, with no prospects for automatization, and the extraction of the solution by vacuum can last more than one hour. The process is lengthy and hard to perform under the current conditions of dismantlement of many soil labs. This, plus the scarcity of technicians trained in the preparation of saturation extracts endorses the use of extracts at fixed water to soil ratios for surveys or other works of great spatial or temporal span needing serialized determinations. 3.6 Natural hypersaline soils occur both in the coasts and inland with salinity levels and other circumstances precluding the growth of common profitable crops. Moreover environmental regulations of some countries forestall agriculture in these wetlands. Very often, the appraisal of soil salinity by EC in such environments is needed for applications other than agriculture. Most of these soils are intermittently inundated, or at least saturated, by salty water or brine. For salt-tolerance evaluation, it seems trivial to relate ECe or EC at other dilution ratios with the natural composition of the soil solution that would limit the growth of plants. Under these conditions, to appraise the content of salts is more interesting than to approach the salinity affecting plant growth as intended by the saturated paste method. Thus, application of a fixed ratio should be better than saturation extracts. 3.7 Very often, the saturation extracts from saline wetlands make ion-pairs, while in most cases the 1:5 and 1:10 extracts will not do that. Thus, the relationships between the ionic concentration and EC will be simpler than the saturation extract. A noteworthy advantage of these extracts for hypersaline soils would be to start in the lab from concentrations more convivial with the conventional chemistry labs titrating ions with methods or equipment not devised for high ionic concentrations. It will reduce or eliminate the need for time consuming dilutions; a classical source of mistakes and errors in serial analytical determinations. 3.8 Extracts at 1:5, 1:1, or other fixed soil to water ratios are often used in different scientific domains . Regrettably, sometimes the dilution ratio is missing or not clearly stated in reports or articles [78][79][80][81] or maybe this ratio, saturation or other, is assumed, e.g., [82][83][84][85][86], compromising or precluding future comparisons and generalization. As saturated paste is unlikely to be adopted as a standard in many of these scientific domains, and the SP is very often not reported, fixed soil to water ratios would be easier and more repeatable. In short, should the methods of soil science depart from other scientific disciplines like biology or ecology that also conduct studies on soil salinity? 3.9 Even though in our experience no ion pairing happens in 1:5 extracts, this circumstance merits checking in hypersaline soils. The method of diluting the saturation extract until EC reaches a value between 0.1-0.3 dS m -1 [87] overcomes ion-pairing to achieve a linear relationship with the salt content of the saturated extract. This method could be used for extracts at fixed ratios (e.g., 1:5), where the occurrence of ion-pairing was suspected, but regression with a lower ratio extract (e.g., 1:10) is easier and ancillary to a duplicate determination of the EC1:5. [88] successfully used paired 1:5 and 1:10 soil to water extracts in seven saline wetlands. Should the relationships between EC1:5 and EC1:10 depart from the linearity pointing to ion-pairing, an extraction at more diluted ratios would be needed. The Two Dilutions Extract Approach Our proposal for soil salinity expression for monitoring purposes is to prepare both 1:5 and 1:10 extracts. The regression between EC1:5 and EC1:10 will be a check for the absence of ionic pairs. This easy procedure is free of chemical models and permits a choice of the extract to be used for further titration of ions. The second extracts can be considered as surrogates for duplicated determinations. Depending on the desired confidence level, the second dilution can be limited to a reduced subset of soil samples if only a statistical check of no ionic-pairing is desired. The effects of the preparation procedures of the 1:5 extract using a set of 20 samples ranging in ECe from 0.96 to 21.20 dS m -1 , and in SP from 32.4% to 68.1%, were studied by [89]. After selecting the appropriate procedure, they stressed the need to report its detailed description. Our approach involving two different soil to water ratios can be transposed to the methods based on volume ratio extracts instead of weight ratios, as proposed by [90] for glasshouse soils. More recently, upon the establishment of the suborders Wassents and Wassists in US Soil Taxonomy [48], a method for measuring the EC of subaqueous soils has been defined by Soil Survey Staff ( [91], page 292). This method measures the EC from a fresh, field wet sample using a mixture (not extract) of soil to water at the ratio of 1:5 by volume (EC1:5 vol), instead of the conventional 1:5 ratio by weight, and measures the EC of the supernatant. If, for taxonomic or characterization purposes the salinity is to be calculated from EC1:5 vol, the eventual occurrence of ion-pairing could be checked with the extract of 1:10 vol. Materials and methods We studied several hypersaline wetland "saladas" protected under Spanish environmental regulations and located in the Monegros desert, Spain (Fig 1). The geological framework of the distribution of these wetlands was addressed by [12]. The soils of the saladas were Gypsic Aquisalids and Typic Haplogypsids per US Soil Taxonomy [49]. Permissions for sampling were granted by the Authority in charge of protected areas: Instituto Aragonés de Gestión Ambiental (www.aragon.es/inaga). The soils at the floors of ten of these saladas: Amarga Alta, Amarga Baja, Camarón, Gramenosa, Guallar, Muerte, Pez, Piñol, Rebollón, and Rollico (Fig 1), were hand-augered at 59 sites, taking cores by depth increments of 20-25 cm. The resulting 359 soil samples were air-dried first at room temperature and then in a ventilated oven for 2 weeks at 40°C in order to avoid the destruction of gypsum (CaSO 4 •2H 2 O). Samples were ground to pass Table. The insert shows the location of the Monegros area within the Ebro Basin, Spain. a 2-mm sieve; no coarse fragments occurred. The saturated pastes were prepared with their saturation percentage (SP) recorded. The electrical conductivity (EC) was measured from the saturation extract (ECe) and from extracts with soil to water ratios in weights of 1:5 (EC1:5) and 1:10 (EC1:10). All the electrical conductivities were expressed in dS m -1 at 25°C. All samples contained gypsum, a common mineral in saline wetlands. Then, sufficient time was allowed to guarantee the maximum dissolution of gypsum to attain stable and reproducible EC measurements. The saturated paste was left to stand overnight before extraction. For the 1:5 and 1:10 extracts, we applied 30 min of reciprocal shaking at 175 oscillations per minute followed by overnight standing before filtration for EC measurement. The Clwas titrated in 288 samples from eight of the studied saladas on extracts at 1:10 dilution using a potentiometer, and expressed in milliequivalents per liter (meq L -1 ). Gypsum content was determined by thermogravimetry per [92] and calcium carbonate equivalent was measured by gasometry with a Bernard calcimeter. After exploratory data analysis of the data shown at S2 Table, ordinary least squares (OLS) regression was applied with the coefficient of determination (R 2 , expressed as %) and the standard error (SE) calculated. Results The sampled soils were either bare or supported halophytes at the less inundable positions. This is in agreement with the hypersalinity of the studied soils expressed by the mean ECe = 72.3 dS m -1 of the 359 samples studied and their range from 2.32 dS m -1 to 183.00 dS m -1 (Table 1). Ten samples were below the ECe threshold 4 dS m -1 for saline soils, while 300 samples had ECe > 16 dS m -1 ,5 the threshold for very strongly saline soils. Only 14 samples had EC1:10 2.25 dS m -1 , the approximate electrical conductivity produced by calcium sulfate saturation marked with a dashed line in Table 1 shows the high contents of gypsum and calcium carbonate. The gypsum content ranged from 1.9% to 96.4%, with a mean of 49.3%. Fig 3 shows the samples ranked by gypsum content, with 180 samples having > 50% gypsum. Only three samples with gypsum ranging from 1.9% to 2.1% had their gypsum content below the thresholds for saturating the 1:5 or the 1:10 dilutions, i.e., 1.2% and 2.4% gypsum, respectively, assuming that saturation is attained at 2.4 g of gypsum per liter of pure water, a solubility that increases if Clor other non-common ions are present. Most samples qualify as gypseous [93], but their high salinity supersedes other limitations to life [94]. Fig 3 also shows the contents of calcium carbonate equivalent (CCE). The sum of CCE plus gypsum ranged from 37.8% to 99.6%, with a mean of 71.2%; this sum was > 50% for 338 samples of the 356 having both gypsum and CCE titrated. The average SP = 39.1% falls within the range of the SP reported for coarse or sandy textures [14,95]. It agrees with the field textures of these soils, controlled by the abundance of visible-sized gypsum crystals. The Clconcentration (Cl1:10) in the 1:10 extracts of the 288 samples analyzed showed a linear distribution against the EC of the same extracts, in spite of the inflection in the scatterplot at EC~2.25 dS m -1 (Fig 4). The OLS adjustment is given in Eq 1: The relationship between the EC at the two soil to water dilutions of 1:5 and 1:10 was studied by scatterplot (Fig 5) and by OLS regression using the 359 soil samples. The OLS adjustment is given in Eq 2: Discussion We have reviewed the shortcomings of ECe for the study of soil salinity in hypersaline environments with no agricultural purposes, and the advantages of using more diluted extracts. Our proposal of assessment of salinity is based on two extractions at different soil to water ratios, 1:5 and 1:10. The preparation of these extracts is much easier, less time-consuming, and equipment-demanding than saturation extracts. Also, further titration of individual ions would be easier than from the saturation extract, which is much more concentrated. Another advantage of the 1:5 extract is the coincidence with the common practice in Australia of expressing salinity by EC1:5 [31,96] and by the Clconcentration in this extract [97]. The quality of the analyses in our example is supported by the scatterplot of Clon EC in the 1:10 extracts (Fig 4) and by the linear relationship between these determinations shown by Eq 1 with R 2 = 98.8%. The same support is provided by the scatterplot of EC1:5 on EC1:10 ( Fig 5) and by the R 2 = 99.1% attained by the OLS regression (Eq 2). Straight line adjustments indicate that both extracts have the same degree of ionization, i.e., no ion-pairing happens. Systematic departures from the adjusted lines occur for the few soil samples which lack sufficient gypsum or other soluble minerals for achieving an EC1:10 of 2.25 dS m -1 . If these samples are eliminated from the adjustments (Eqs 1A and 2A) the slight improvements are below the average allowable errors of most routine lab analyses. The ubiquity of gypsum in the studied samples, a frequent setting in athalassohaline wetlands, and their specific ionic composition prevents comparisons of the above adjustments with other soils. The purpose of the adjustments was not predictive, but a check of both the full ionization in the 1:5 dilution and the analytical quality. Thus, depending on circumstances like the confidence in the lab performance, the allowable lab workload, and the number of samples, the determinations of EC1:10 could be limited to a reduced number of samples covering all range of salinity determined at 1:5 dilution. The scatterplot of EC1:5 over EC1:10 ( Fig 5) shows the classical inflection due to gypsum. The samples having either EC1:5 or EC1:10 2.25 dS m -1 are located under the inflection in the figure, showing in this region a systematic departure from the OLS regression line. The presence of gypsum was early recognized as a source of troubles in the conversion of EC between extracts at different soil to water ratios ( [98], page 14). That notwithstanding, the effect of gypsum producing an EC of~2.25 dS m -1 at saturation, can be neglected in these conversions for hypersaline soils if the gypsiferous and non-saline samples are a minority. These samples, grouping around the point ECe = EC1:5 = 2.2 dS m -1 [99], can compromise the homocedasticity of the distribution if they are numerous. If a significant number of extracts with EC < 2.25 dS m -1 occurs, or if specific consideration is wanted for these extracts, a separate regression could be undertaken. However, categorizing soils into groups with or without any measurable amount of gypsum to improve the prediction of ECe [29] with a separate determination of gypsum would be cumbersome for long series of determinations and for simple labs. A similar procedure based on a qualitative assessment of gypsum by precipitation with acetone in 410 soil extracts at 1:5 dilution was used in gypseous soils by [99]. Several authors have used EC1:5 as an auxiliary variable for soil mapping with electromagnetic induction (EMI) measurements [100], or have calibrated EMI measurements with EC1:5, as is the case of [52,[101][102] and the examples mentioned by [103]. In our experience, the calibration of EMI measurements with EC1:5 was possible, even if the attained coefficients of determination were lower than with ECe [99,104]. EMI devices, as other sensors, respond to both variable and invariable soil characteristics and their distribution along the soil profile such as moisture, temperature, mineralogy, pore size and architecture, etc. Therefore, it is requisite to take some soil samples for calibrating the sensor signal with the target characteristic. EMI has been used to map soil salinity in areas with shallow water tables in the central Ebro valley, e.g. [105][106][107], and in saline coastal wetlands, e.g. [33,108,109]. The adoption of EC1:5 as the standard measure of soil salinity would simplify the calibrations and eliminate the time consuming preparation of the saturation extract. Other developing technologies will be able to measure salinity in the field. One example is portable x-ray fluorescence spectrometry whereby Clor other elements are used as a proxy for soil salinity, but it also requires correction for soil moisture when levels are above 20% [110,111]. It shows promise for soil salinity appraisal, and could overcome the measurements of ECe on water extracts. These matters fall out of the scope of this article and merit discussion and effort both for comparisons and for long term monitoring of soil salinity. The multitude of models and adjustments proposed in the literature for predicting ECe from EC at a fixed soil to water ratio do not provide a conclusive, universal, and non-sophisticated procedure. For the studied soils, Fig 6A shows the inflection due to gypsum plus the classical dispersion at higher values; both features entangling the response model. For large sets of samples, one can undertake an adjustment of EC1:5 or other fixed ratios versus a reduced number of ECe determinations. However, it must be noted that the "gypsum inflection" lasts ( Fig 6B) even after the transformation that includes SP [46,112] and enables for decreasing the dispersion. Moreover, the shortcomings mentioned at Sections 3.3 and 3.4 strongly question the estimation of total salt content from ECe, at least for soils containing calcium carbonate or gypsum as well as for hypersaline soils. Conclusions The study of inland wetlands located in arid climates requires the development of new concepts to be incorporated into mainstream wetland science. With the proposed approaches, we try to simultaneously assess "agricultural" and "environmental" methods that are studying the same natural objects, saline wetlands. The degrees of salinity of the studied soils plus frequent flooding preclude their agricultural use. This setting is frequent in saline wetlands around the world that are often protected by environmental rules. Conservation of these valuable habitats requires monitoring the salt contents of the soils, diminishing the usefulness and meaning of saturated paste. The expression of total salt content by the electrical conductivity in the extracts at 1:5 dilution, with checking for no ion-pairing with the 1:10 extract, has shown to be easy, unsophisticated, and robust. Our two dilution approach bypasses the agricultural salinity expression by saturation extract by adopting the 1:5 extract as a standard for soil salinity expression instead of the saturation extract. Supporting Information S1 Table. UTM coordinates (European Datum ED 50) of the 59 sampling sites with their vegetation and the depth of the augerings. (PDF) S2 Table. Analytical data of the soil samples. (PDF)

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Biological Control of Phytophthora palmivora Causing Root Rot of Pomelo Using Chaetomium spp. Phytophthora diseases have become a major impediment in the citrus production in Thailand. In this study, an isolate of Phytophthora denominated as PHY02 was proven to be causal pathogen of root rot of Pomelo (Citrus maxima) in Thailand. The isolate PHY02 was morphologically characterized and identified as Phytophthora palmivora based on molecular analysis of an internal transcribed spacer rDNA sequence. This work also presents in vitro evaluations of the capacities of Chaetomium spp. to control the P. palmivora PHY02. As antagonists, Chaetomium globosum CG05, Chaetomium cupreum CC3003, Chaetomium lucknowense CL01 inhibited 50~61% mycelial growth, degraded mycelia and reduced 92~99% sporangial production of P. palmivora PHY02 in bi-culture test after 30 days. Fungal metabolites from Chaetomium spp. were tested against PHY02. Results showed that, methanol extract of C. globosum CG05 expressed strongest inhibitory effects on mycelial growth and sporangium formation of P. palmivora PHY02 with effective dose ED50 values of 26.5 µg/mL and 2.3 µg/mL, respectively. It is interesting that C. lucknowense is reported for the first time as an effective antagonist against a species of Phytophthora. Mycobiology Biological Control of Phytophthora palmivora Causing Root Rot of Pomelo Using Chaetomium spp. Citrus are important fruit crops, being widely and commercially grown in Southeast Asia. However, under prevailing wet climatic conditions, Phytophthora is a major impediment in the citrus production and has caused annual loss of about 6~12% of yield in Thailand alone [1]. Phytophthora palmivora (Butl.) is a ubiquitous and prominent plant pathogen with a wide host range, which infects various important crops in Southeast Asia [1,2]. On citrus, this fungal-like organism has been reported to infect almost every part of the plant at any stage of its growth [3]. The spread and pathogencity were reported to be faster and more aggressive to roots of citrus than P. parasitica and P. citrophthora [4,5]. Since Phytophthora and other oomycetous organisms have different biochemical pathways compared to the true fungi, they are insensitive to many fungicides [6]. In addition, the resistance of Phytophthora species to an important group of fungicides such as phenylamides (metalaxyl and related compounds) has become a serious problem in their chemical control [2]. In order to develop eco-friendly management of Phytophthora diseases and to reduce the costly applications and harm of fungicides, screening of bio-control agents against citrus Phytophthora has become a vital research aspect and is being carried out all over the world [3]. Chaetomium Kunze is a large genus of saprophytic ascomycetes with more than 350 species [7]. Relying on lytic enzymes, they decompose cellulose and other organic materials [8,9]. Some Chaetomium species are reported to act as antagonists against various plant pathogens. Even commercial bio-product has also been developed from potent strains of Chaetomium spp. [10]. Furthermore, over 200 metabolites with a wide range of bioactivities have been found from the genus Chaetomium and many of which exhibited antifungal activity against plant pathogens [7]. However, there is a significant limitation of research on the abilities of Chaetomium species and their metabolites to control the Phytophthora. Recently, we have isolated a single species of Phytophthora (denominated as PHY02) from roots of pomelo (Citrus 64 Hung et al. maxima) in orchards having serious root rot problems in Chang chen Sao province, Thailand. This study reports characteristics and identification of this isolate as well its in vitro bio-control using Chaetomium species as antagonists and their metabolites. To identify the pathogen at molecular level, universal primers ITS 5 (5'-GGAAGTAAAAGTCGTAACAAGG) and ITS 4 (5'-TCCTCCGCTTATTGATATGC) were used to amplify an internal transcribed spacer (ITS) rDNA region of isolate PHY02, with previously described PCR conditions [11], then was directly sequenced with the same primers at First BASE Laboratories Sdn Bhd (Selangnor, Malaysia). The full-length ITS rDNA nucleotide sequence of PHY02 was used as a query to search the GenBank DNA database of Phytophthora Database (http://www. phytophthoradb. org/blast/) using BLAST tool. Reference ITS rDNA sequences of related taxa were also downloaded from the Phytophthora Database. Subsequently, the sequences of PHY02 and related taxa were aligned and performed phylogenetic analysis using MEGA ver. 5.2. Finally, phylogenetic tree was constructed using the neighbor-joining method with 1,000 bootstrap replications [12]. Pathogenicity test. Pathogenicity was proved by artificial inoculation PHY02 into roots of pomelo seedlings (Citrus maxima) var. Khao nam Pueng (the same affected variety where PHY02 was obtained). Root inoculation was done using the infested soil method of Zitko et al. [4]. Six-month-old pomelo seedlings were thoroughly washed to be free of potting mix and then planted in plastic tubes (10 × 15 cm) containing sterilized clay soil-sand (1 : 1) with 5 chlamydospores of PHY02 per cubic centimeter. Controls were prepared by planting the seedlings in same size tubes, but containing sterilized clay soil-sand (1 : 1) only. All pots including the controls were maintained in the green house at temperature of about 25~30 o C and flooded with water for 24 hr each week. After 6 wk, the plants were carefully removed from plastic tubes, the soil was washed out and rating was done based on degree of the root rot symptoms. The pathogen then was re-isolated from newly infected roots symptom and morphology characteristics were compared with the isolate PHY02. Fungal isolates. C. globosum strain CG05, C. cupreum strain CC3003, and C. lucknowense strain CL01 (from collection of Dr. Soytong K., KMITL, Thailand) were used as antagonists and produced antagonistic substances. The isolate PHY02 was represented as the target control in this study. Bi-culture test. A mycelial disc of PHY02 (5 mm diameter) was placed singly (as controls) or oppositely to a mycelial disc of each above antagonist on 9-cm-diameter Petri dishes, which contained PDA. After incubation at 25 o C for 30 days, data were collected as colony diameter and number of sporangia produced by PHY02 in both bi-culture and control plates. Numbers of sporangia were counted by using haemacytometer. Data were computed in a form of inhibition percentage of mycelial growth and sporangial production of the pathogen by using the formula below: , where A = colony diameter or numbers of sporangia of PHY02 in control plates; B = colony diameter or numbers of sporangia of PHY02 in bi-culture plates. Finally, variance and the treatment means were analyzed and compared by using Duncan's multiple range tests at 0.05. In vitro test of crude extracts from Chaetiomium spp. to inhibit P. palmivora PHY02. C. globosum CG05, C. lucknowense CL01, and C. cupreum CC3003 were separately cultured in potato dextrose broth (500 plates per antagonist) at room temperature in 45 days. Fungal biomass of each antagonist was separately collected as fresh biomass, then was dried out at room temperature. Subsequently, the extraction of dried biomass from each antagonist was performed by the method described by Kanokmedhakul et al. [13]. Each dried biomass was ground and extracted with hexane (1 : 1 v/v) and incubated by shaking for 72 hr at room temperature. The filtrate was separated out the marc by filtration through filter paper (Whatman No. 4). The hexane filtrate was performed through rotary vacuum evaporator to yield crude hexane extract. The marc from hexane extraction was further extracted with ethyl acetate and followed with methanol using the same procedure as hexane to yield crude ethyl acetate and methanol extract. The three different crude extracts of each antagonist were conducted in a factorial experiment to test for inhibition of mycelial growth and sporangium formation of P. palmivora PHY02 by using poisonous food method. A mycelial disc (5 mm in diameter) of PHY02 placed on PDA plates (5 cm in diameter), which contained different concentrations of each crude extract (0, 10, 50, 100, 500, and 1,000 µg/mL). To obtain the desired crude extract concentrations, stock crude extract was weighted then dissolved in 2% dimethyl Biological Control of Phytophthora palmivora Causing Root Rot of Pomelo Using Chaetomium spp. 65 sulfoxide and added to molten PDA before autoclaving at 121 o C (15 psi) for 20 min. After incubating at 25 o C for 10 days, colony diameter and numbers of sporangia production of the pathogen were collected and then expressed as inhibition percentage using the same formula above. Effective dose ED 50 values on mycelial growth and sporangial production were calculated by probit analysis using the software SPSS Statistics ver. 19.0 (IBM Co., Armonk, NY, USA). RESULTS Pathogenicity of Phytophthora PHY02. After 6 wk of inoculation with chlamydospores of PHY02 on roots, the pomelo seedlings had an average of 47.6% root tips rotted and produced very few new roots (data not shown). Cortex of feeder roots turned soft and was sloughed to leave only steles, which were similar to symptoms of root rot in the affected orchards in Chang chen Sao, Thailand (Fig. 1). Meanwhile, non-inoculated seedlings produced abundantly new roots and no symptom of rot was observed. A single species of Phytophthora was re-isolated from disease symptoms on newly infected roots, which all had identical morphology characteristics with the isolated PHY02. As a result, PHY02 was proven to be the causal pathogen of the pomelo root rot in Chang chen Sao, Thailand. Characteristics and identification of Phytophthora PHY02. Colonies of PHY02 showed stellate pattern, with aerial mycelia on V8A and PDA whereas nearly no aerial mycelium on CMA (Fig. 2). The isolate PHY02 produced lumpy-branching hyphae with hyphal swellings. Zoospores were directly released from sporangia when flooded in distilled water. Sporangia produced abundantly when grown on PDA and V8A after 3~5 days, occurred in groups on sympodium or irregularly, were papillate and caducous with short pedicels up to 6 µm long (mean 3.3). Sporangial shape varied from ellipsoid, ovoid, pyriform, obpyriform to near spherical, and mean of sizes were 53.8 × 33.3 µm with a length to breadth ratio of 1.2~2.2 (mean 1.6) ( Table 1). Most of chlamydospores were globose in shape, produced abundantly from mycelia when incubated in dark. No sexual organ was observed in cultures of this isolate since it was a heterothallic species. The sequence with 951 bases of the ITS rDNA PCR product of PHY02 was determined and used as a query to search the Genbank DNA database of the Phytophthora Database using the BLAST search. It was found that there was 99.75% (809/811), 99.87% (782/783) to identity with Phytophthora palmivora accession number PD_00627 and PD_02505, respectively. The phylogenetic tree, which illustrated relationships between PHY02 and related taxa was constructed as shown in Fig. 3. The isolate PHY02 was identified as Phytophthora palmivora (Butl.), based on its morphology and the molecular analysis. Bi-culture test. As shown in Table 2, the tested antagonists led to 50~61% growth inhibition and reduced 92~99% sporangial production of P. palmivora PHY02 in bi-culture plates at 30 days, when compared to the controls. Moreover, CG05 and CL01 were seen to grow rapidly over PHY02 colony after 10~15 days. In all bi-culture plates, both antagonists degraded mycelia of the pathogen, resulting in change of color from white to light yellow-brown and a part or entire colony death (Fig. 4). Differently, C. cupreum CC3003 was a slow growing fungus, as it grew over gradually and resulting in degradation of colony of PHY02 in some cases. Mycelia of all the tested antagonists were observed to penetrate mycelia of PHY02 in some cases. In both inhibitions of mycelial growth and sporangial production, CC3003 was significantly less efficient than CG05 and CL01. In vitro tests of crude extracts from Chaetomium spp. to inhibit P. palmivora PHY02. Total nine crude extracts from CG05, CL01, and CC3003 were tested at different concentrations to evaluate their capacities to inhibit mycelial growth and sporangium formation of P. palmivora (PHY02). The effective doses (ED 50 ) of each crude extract on mycelial growth and sporangial production were also examined to determine their fungicidal spectrum. As shown in Table 3, the three crude extract of CG05 exhibited more antifungal activities against mycelial growth of PHY02 as compared to crude extracts of CL01 and CC3003. Particularly, the tested pathogen did not grow at all in the presence of higher 500 µg/mL methanol extract and 1,000 µg/mL ethyl acetate extract of CG05. At tested concentrations from 10~500 µg/mL, the methanol extract was more effective than the others (Figs. 4 and 5). Conversely, all crude extracts of CC3003 were less effective on mycelial growth of PHY02 with higher ED 50 values (596.8~2,495 µg/ Fig. 3. Phylogenetic relationship between Phytophthora palmivora PHY02 and related taxa inferred using a neighborjoining method with internal transcribed spacer (ITS) rDNA sequences. Bootstrap value based on 1,000 replications is shown above the branch. All the tested crude extracts exhibited stronger inhibitory effects on sporangium formation than on mycelial growth of PHY02 with much lower ED 50 values. The sporangium formation of PHY02 was most sensitive to crude extracts of CG05 with ED 50 values of as 5.1, 3.0, and 2.3 µg/mL for the hexane, ethyl acetate and methanol extract, respectively. Meanwhile, all three crude extracts of CC3003 were least effective on sporangial production of PHY02 with higher ED 50 values. Of which, the methanol extract was more significantly effective than the others with ED 50 value of 93.6 µg/mL, and gave an inhibitory rate of 96.5% at 1,000 µg/mL. The hexane and ethyl acetate extract gave the highest ED 50 values among nine tested crudes, which were 307.9 and 145 µg/mL, respectively. To reduce 50% sporangial production of PHY02, the hexane, ethyl acetate and methanol extract of CL01 required concentrations of 16.7, 3.5, and 4.0 µg/mL, respectively. However, none of them could reduce 90% sporangial production of PHY02 at 50 µg/mL. At concentrations from 10~500 µg/mL, the ethyl acetate extract of CL01 was more significantly effective than the others, showing an inhibitory rate of 94.0% at 100 µg/mL. While at concentration of 500 µg/mL, the hexane and methanol extract reduced 82.3% and 93.8% sporangial production of PHY02, respectively. All the hexane extracts of tested antagonists were less effective than the ethyl acetate and methanol in both inhibitions of mycelia growth and sporangium formation DISCUSSION We have designated the species isolated from root rot symptoms of pomelo (Citrus maxima) in Thailand as Phytophthora palmivora (Butl.). The highly pathogenic of P. palmivora PHY02 to roots of pomelo here is supported by previous studies which demonstrated that this species is more aggressive and damages even larger root than P. parasitica on citrus [4,5]. Serious root rot disease of citrus caused by P. palmivora has been recorded in India, America [3,14]. The antagonistic activity of bio-control microorganisms is often demonstrated by the inhibition of growth, infection or reproduction of pathogen [15]. The tested strains of Chaetomium spp. as antagonists here not only inhibited colony growth and degraded mycelia but also reduced 929 9% sporangial production of P. palmivora PHY02. The degradation of mycelia of tested pathogen probably resulted from the lytic enzymes which are commonly secreted by Chaetomium species [8,9]. On other hand, every tested antagonist in this study was demonstrated to produce antibiotics exhibiting antifungal activities against different plant pathogens [10,13,16]. It refers that the inhibitions of P. palmivora here were resulted from both antibiotics and lytic enzymes secreted by the antagonists. C. globosum and C. cupreum have ever been reported to control Phytophthora spp. in dual culture test [17,18]. C. lucknowense was concluded to control Fusarium oxysporum causing tomato wilt [16]. However, to best our knowledge, this is the first report of C. lucknowense as an effective antagonist against a species of Phytophthora. Chaetomium species are known as producers of many different bioactive metabolites which play important roles in their biological control activities [7,9]. One Chaetomium species may produce many metabolites with different bioactivities and molecular weight, this lead to differences in antifungal activity of its crude extracts [16,19]. That explains why there were variations in responses of P. palmivora PHY02 to different crude extracts in this study. The less efficiency of hexane extracts of Chaetomium spp. in comparison with the ethyl acetate and methanol against fungal pathogens were also confirmed by previous authors Fig. 5. Inhibitory effects of crude extracts of Chaetomium spp. on mycelial growth (the charts on the left site) and sporangial production (the charts on the right site) of Phytophthora palmivora PHY02 at different concentrations. The same letter above columns in each chart represents no significant difference between treatments, based on the Duncan's multiple range test at p = 0.01. [16,20]. C. globosum CG05 has been known to produce many bioactive compounds such chaetoglobosin (A, G, C, V, etc.) and chaetoviridins (A, B) [10]. Of which, the chaetoglobosin C is usually referred as antifungal principle of this strain and was reported to inhibit colony growth, sporangium formation of P. parasitisca and other fungal pathogens [10,19]. However, the chaetoviridins A produced from C. globosum F0142 also has strong antifungal activities against P. infestans, P. capsici [21]. Thus, it suggests that chaetoglobosin C and also other bioactive compounds involved in crude extracts of C. globosum CG05 caused the high inhibitions of P. palmivora PHY02 in this study. C. cupreum CC3003 has been known to produce compound rubrorotiorin which inhibited growth of Candida albicans at low concentration (EC 50 = 0.6 µg/mL) [13]. However, crude extracts of C. cupreum CC3003 here showed least inhibitory effects on P. palmivora with high ED 50 values. Similarly, crude extracts of C. cupreum have been known to be less effective against Pythium aphanidermatum [20]. Both the crude extracts and chaetoglobosin C produced by C. lucknowense CL01 have been reported to inhibit colony growth and spore formation of Fusarium oxysporum causing tomato wilt [16]. However, this is the first time the inhibitory effects of metabolites from C. lucknowense on a species of Phytophthora are reported. Sporangium formation is the most sensitive stage in life cycle of Phytophthora species [2]. This study demonstrated that sporangium formation of P. palmivora PHY02 was much more sensitive than mycelial growth to all tested crude extracts. The high inhibitory effects of crude extracts from Chaetomium spp. on spore formation of Fusarium oxysporum and P. parasitisca have been noted by previous authors [16,19]. It is clear that only metabolites which were not decomposed through the extraction and autoclaving of the crude extracts resulted in the inhibitions of P. palmivora PHY02. All enzymes and many antibiotics will be decomposed because of heat and pressure when autoclaving [2]. That why mycelia of the pathogen were not degraded when growing in crude extracts. Therefore, the antifungal activities of crude extracts here probably did not account for all biocontrol activities of the tested antagonists against P. palmivora PHY02. Beside the known metabolites, degrading enzymes should be considered as effective factors involved in biocontrol activities of the tested antagonists against the pathogen. The roles and secretion of lytic enzymes of Chaetomium species under antagonism conditions with Phytophthora need to be investigated in the future. The effective crude extracts may possible develop to be microbial elicitors to induce immunity in citrus plants against P. palmivora.

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2017-06-03T15:43:07.303Z

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Row Ratios of Intercropping Maize and Soybean Can Affect Agronomic Efficiency of the System and Subsequent Wheat Intercropping is regarded as an important agricultural practice to improve crop production and environmental quality in the regions with intensive agricultural production, e.g., northern China. To optimize agronomic advantage of maize (Zea mays L.) and soybean (Glycine max L.) intercropping system compared to monoculture of maize, two sequential experiments were conducted. Experiment 1 was to screening the optimal cropping system in summer that had the highest yields and economic benefits, and Experiment 2 was to identify the optimum row ratio of the intercrops selected from Experiment 1. Results of Experiment 1 showed that maize intercropping with soybean (maize || soybean) was the optimal cropping system in summer. Compared to conventional monoculture of maize, maize || soybean had significant advantage in yield, economy, land utilization ratio and reducing soil nitrate nitrogen (N) accumulation, as well as better residual effect on the subsequent wheat (Triticum aestivum L.) crop. Experiment 2 showed that intercropping systems reduced use of N fertilizer per unit land area and increased relative biomass of intercropped maize, due to promoted photosynthetic efficiency of border rows and N utilization during symbiotic period. Intercropping advantage began to emerge at tasseling stage after N topdressing for maize. Among all treatments with different row ratios, alternating four maize rows with six soybean rows (4M:6S) had the largest land equivalent ratio (1.30), total N accumulation in crops (258 kg ha-1), and economic benefit (3,408 USD ha-1). Compared to maize monoculture, 4M:6S had significantly lower nitrate-N accumulation in soil both after harvest of maize and after harvest of the subsequent wheat, but it did not decrease yield of wheat. The most important advantage of 4M:6S was to increase biomass of intercropped maize and soybean, which further led to the increase of total N accumulation by crops as well as economic benefit. In conclusion, alternating four maize rows with six soybean rows was the optimum row ratio in maize || soybean system, though this needs to be further confirmed by pluri-annual trials. Introduction Northern China has a very intensive agriculture with high inputs of seeds, irrigation and chemicals, because of high pressure of food security. This has caused severe environmental problems [1], including pollution of groundwater by nitrate from soils [2], gas emission to air [3], and soil acidification [4]. Loss of nitrogen (N) during maize (Zea mays L.) growth season is an especial concern, as excessive application of N is often combined with heavy summer rains in this region [5]. To ensure both food security and environmental quality, it is essential to seek best management practices, which include appropriate cropping systems that can efficiently utilize solar and soil resources with minimum nutrient inputs. Intercropping, one type of a multiple cropping system, is recommended to be used in many parts of the world for food or fibers productions, because of its overall high productivity, effective control of pests and diseases, good ecological services and economic profitability [6][7][8][9]. In an intercropping system, there are often two or more crop species grown in the same field for a certain period of time, even though the crops are not necessarily sown or harvested simultaneously. In practice, most intercropping systems involve only two crops, as inclusion of more crops results in higher labor costs [10]. An intercropping system often consists of three phases: (1) one crop grown for a short time, (2) two intercropping crops grown simultaneously for a long time, and (3) the other crop grown for a short time [11]. The second phase is essential (or even the only phase), and it is the key phase for formation of intercropping advantage. The success of intercropping systems is due to an enhanced temporal and spatial complementarity of resource capture, for which both above-ground and belowground parts of crops play an important role [12]. Cereal crops intercropping with legumes are a popular option in intercropping. Even though the two crops compete for soil N as they both need it for the growth, the competition drives legumes to fix atmospheric N 2 in symbiosis with Rhizobium [13]. This actually results in complementary utilization of N by the crops, which is of particular importance in soils where inorganic N is limited or over-fertilized. However, negative intercropping productivity due to interspecific competition has also been reported [14], especially when the fields are managed inappropriately [11]. Therefore, only reasonable use of competitive and facilitative interactions between crops in intercropping systems can enhance crop productivity and nutrient use efficiency [15][16][17]. In China, intercropping is regarded to be an important agronomic practice, given the high pressure of food security due to the already large and increasing population with limited and decreasing area of arable land [15]. Among different kinds of intercropping systems, strip intercropping has the greatest advantage in terms of convenience in field management of sowing and harvest [18]. Several previous studies have reported that intercropping can increase crop yield [19], due to efficient utilization of nutrients [20] and light [21], and enhanced positive interactions between crops [22,23]. However, most of these studies were focused on effects of different intercrop species [14,24]. Rare studies have been made to investigate effects of ratio of rows between crops within a specific intercropping system. There is neither report in literature about optimum row ratio of maize intercropping with soybean (Glycine max L.), nor explanation of the processes behind. In this study, we carried out two field experiments to firstly screen the optimal intercropping system of maize and legume crops, and secondly to identify the optimum ratio of rows of maize and the legume (soybean) selected in the first experiment. Specifically, we evaluated intercropping effects on crop yields, economic benefits, crop N uptake, soil nitrate-N accumulation, and its residual effects, and we investigated the reasons for advantages of intercropping systems. Study area The field experiments were conducted during 2010 to 2012 at Liucun, Xushui (38°09-39°09 N, 115°19-115°46 E), Hebei Province, North China Plain. This region has a temperate continental monsoon climate, with four distinct seasons. The site has annual mean temperature of 11.9°C, annual precipitation of 567 mm and evaporation of 1,200 mm. Annual sunshine duration is 2,745 h and the frost-free period is 184 days. Wheat (Triticum aestivum L.)-maize rotation is a common cropping system in this region and this system had been used during 1990-2010 on the experimental site. Each year, the field was tilled with a disk plough before sowing of wheat in October. The experimental site had a Haplic Luvisol soil (FAO classification). Physical and chemical properties of the experimental soil were determined before the start of this study (Table 1). Ethics Statement The experimental field used in this study belongs to the Institute of Agricultural Resources and Regional Planning (IARRP) of Chinese Academy of Agricultural Sciences (CAAS), which is a national comprehensive research institution, and it has a research ethics review committee to ensure the experiment does no harm to crops, animals and humans. Our study was approved by this committee, so no specific permissions were required for the described field experiments. The sampling locations were not privately-owned or protected in any way, and this field study did not involve any endangered or protected species. In addition, there was also no vertebrate in this study. Experimental design Experiment 1: screening of optimal intercropping system of maize and legume crops. This experiment was conducted from June 21st, 2010 to June 22nd, 2011. The experiment used a randomized complete block design, and it included five treatments with different cropping systems: (1) monoculture of maize, (2) monoculture of soybean, (3) monoculture of red bean (Vigna angularis (Willd.) Ohwi et Ohashi L.), (4) maize intercropping with soybean (Maize ǁ soybean), and (5) maize intercropping with red bean (Maize ǁ red bean). Each treatment was replicated three times. After harvest of these crops in autumn, a subsequent winter wheat crop (a 0.15 cm inter-row distance) was planted at all the plots. The experimental plot size ranged from 175 m 2 (25 m × 7 m) to 225 m 2 (25 m × 9 m) in different treatments, to aid practical identification of optimum ratio of rows of maize and soybean. Experiment 2 was conducted to identify optimum ratio of rows of maize and soybean, as maize ǁ soybean was identified as the best intercropping system in Experiment 1. A randomized complete block design, which included five treatments with different planting patterns in three replicates, was used in summer, 2011. The treatments were: (i) monoculture of maize, (ii) monoculture of soybean, (iii) alternating two maize rows with six soybean rows (2M:6S), (iv) alternating four maize rows with six soybean rows (4M:6S), and (v) alternating six maize rows with six soybean rows (6M:6S) (Fig 1). Detailed planting pattern of every treatment was shown in Table 2. Maize and soybean were sown on June 24 th , 2011, and harvested on October 6 th , 2011. A subsequent winter wheat crop was sown on October 7 th , 2011 with a 15 cm inter-row spacing, and it was harvested on June 17 th , 2012. In both experiments, maize, winter wheat, soybean and red bean were supplied with 225, 225, 45 and 45 kg N ha -1 in urea (46% N), respectively, and all crops were supplied with 33 kg P ha -1 in calcium superphosphate (12% P) and 62 kg K ha -1 in potassium sulphate (52% K). For maize and wheat, half of the N was incorporated into the top 20 cm soil as base fertilizers at sowing, and the rest half was applied during the jointing stage, which was 40 days after planting (DAP). All N for beans, and all P and K fertilizers for all crops were applied as basal fertilizers. Five flooding irrigations were applied to wheat, with one irrigation of 50 mm water at wheat growth stages of sowing, overwinter, erecting, booting and filling, respectively. Sample collection and measurement Before the start of the study in June 2010, soil was sampled randomly in the field by using a soil auger for every 20 cm soil depth until 200 cm deep, to determine basic physical and chemical properties of each soil layer. In Experiment 1, soil samples were collected from every crop strip with the same procedure as described above, both after harvest of summer crops and after harvest of winter wheat. In Experiment 2, soil samples (0-20 cm and 20-40 cm) were collected in inter-row (Fig 1) [20]. Soil organic matter was determined by the potassium dichromate method, total N by the automatic Kjeldahl method after wet digestion [25], and soil water pH by using a standard calomel electrode [26]. In Experiment 1, five plants of maize or bean were sampled from each crop strip after harvest in summer. In Experiment 2, plant samples of maize and soybean were collected to determine dry matter content at maize pre-jointing stage (26 DAP), tasseling stage (57 DAP) and ripe stage (104 DAP), respectively. At each stage, 10 plants of each crop were sampled from every row (Fig 1). Wheat plant samples within an area of 2 m 2 were taken separately for the previously established maize or soybean strip. Stalks and grains of the crops were harvested separately at ripe stage. All the samples were oven-dried at 105°C for 30 min and then at 85°C until constant weights. Thereafter, dry plant samples were ground to determine total N concentrations by the automatic Kjeldahl method, after wet digestion of the samples with H 2 SO 4 and H 2 O 2 [27]. Photosynthetic characteristics of maize were measured in each row using an infrared gas analyzer-based photosynthesis system (LI-6400, Li-Cor., Lincoln, NE, USA) at tasseling stage (57 DAP) and filling stage (74 DAP). The measurements took place at the middle of the ear leaf during normal weather conditions with sunshine and no rain in summer, between 9:00-12:00 in the morning. Based on the literatures [28][29][30] and the actual circumstances, the conditions supported were as follows: photosynthetic photon flux density of 1800 μmol m -2 s -1 , CO 2 concentration of 400 μmol mol -1 , air temperature of 30°C and relative air humidity of 0% (0% moisture from air to the leaf chamber). Calculation and Statistical analysis Land equivalent ratio (LER), which is often considered as an indicator of intercropping benefit [31], was calculated according to: where Y im (kg ha -1 ) and Y is (kg ha -1 ) are respective yields of intercropped maize and soybean or red bean per ha intercropping area, and Y sm (kg ha -1 ) and Y ss (kg ha -1 ) are yields of maize and bean in monoculture treatments. If LER is greater than 1.00, there is a yield advantage by intercropping; otherwise there is no yield advantage. Total N accumulated by the crop (Nacc, kg ha -1 ) was calculated according to: where M is the amount of dry matter (kg ha -1 ) and C is the N concentration in the plant (%). Economic benefit (E, USD ha -1 ) was calculated according to: where Y is yield (kg ha -1 ), P is grain price (USD ha -1 ), LF is labor fees (USD ha -1 ), FF is fertilizer fees (USD ha -1 ), SF is seed costs (USD ha -1 ) and MF is machinery expenses (USD ha -1 ). USD is U.S. dollar. Nitrate-N accumulation in soil (R, kg ha -1 ) was calculated according to: where T is the thickness (cm) of a soil layer, B is soil bulk density (g cm -3 ), and C is concentration of soil nitrate-N (mg kg -1 ). Analysis of variance (ANOVA) was conducted using the SPSS19.0 software package and mean values (n = 3) were compared by least significant difference at the 5% level. Results Screening of optimal intercropping system of maize with legume Compared to monoculture, both maizeǁsoybean and maizeǁred bean cropping systems had intercropping advantages in yield and economy (i.e., promoted crop yields and farmers' income) ( Table 3). Both intercropping systems had a LER value greater than 1. Maizeǁsoybean had the highest economic benefit among all the cropping systems, but it did not significantly differ with that of maizeǁred bean. In addition, both maizeǁsoybean and maizeǁred bean cropping systems significantly reduced soil (0-200 cm) nitrate-N accumulation compared to monoculture of maize (P<0.05). Maizeǁsoybean had soil nitrate-N accumulation 56 kg ha -1 lower than monoculture of maize, and 18 kg ha -1 lower than maizeǁred bean. In addition, compared to monoculture of maize, both maizeǁsoybean and maizeǁred bean did not significantly affect yield of subsequent wheat, but they reduced nitrate-N accumulation in soil (0-200 cm) after harvest of subsequent wheat (Table 4). Considering all the aspects above, maizeǁsoybean performed better than maizeǁred bean and all monocultures, and thus maizeǁsoybean was investigated with further details. Effects of row ratios on agronomy and environmental benefits of intercropping systems Overall effects of row ratios. Despite being applied with less N, intercropping significantly increased crop yields, economic benefit and crop total N uptake (P<0.05), and reduced nitrate-N accumulation in soil at harvest, compared to crop monocultures on equivalent land areas. Specifically, intercropping increased maize yield (per hectare maize growing area) by 42.2-92.3%. Soybean yield was slightly decreased by 6.5% in 2M:6S and by 4.4% in 6M:6S, but it was increased by 3.0% in 4M:6S. As a result, the LER values of the three intercropping systems were all greater than 1, and the land utilization rates in these systems were 24-30% higher than the rates in the two monocultures (Table 5). 4M:6S had the best economic benefit, which was 26.0% higher than maize monoculture, 45.4% higher than soybean monoculture, 8% higher than 2M:6S, and 5% higher than 6M:6S. Amounts of total N accumulation in crops were similar in 4M:6S (258 kg ha -1 ) and 2M:6S (257 kg ha -1 ), which were significantly higher than those in the other treatments (P<0.05). Soil nitrate-N accumulation (0-40 cm) at harvest was smaller at lower proportion of maize growing area. Compared to maize monoculture, intercropping significantly reduced soil N accumulation after crop harvest, by 46.6% in 6M:6S, 57.6% in 4M:6S and 65.1% in 2M:6S. Photosynthetic characteristics of maize at different growth stages. Intercropping enabled an efficient utilization of environmental resources. At maize tasseling and filling stages, photosynthetic gas-exchange parameters, including net photosynthetic rate (P n ) and transpiration rate (T g ), collectively showed a border row effect [32]. That is, values of photosynthetic characteristics were larger at a smaller distance to the border row within a maize strip (Table 6). At both stages, the P n of maize in row 1 of 4M:6S and 6M:6S were significantly (P<0.05) higher than that in the inner-row in these treatments as well as that in maize monoculture. The T g of maize in row 1 did not significantly differ between intercropping systems, but they were all significantly (P<0.05) higher than that in maize monoculture. Moreover, T g of maize in the innerrow in intercropping systems was also slightly higher than that in maize monoculture. Uptake of N by crops at different growth stages. Intercropping systems did not reduce N concentrations in maize or soybean compared to monocultures (Table 7). At each growth Table 5. Total N accumulation in crop (Nacc), crop yield, economy, soil nitrate-N accumulation at 0-40 cm soil depth (NA) and land equivalent ratio (LER) in different cropping systems at harvest in the summer of 2011 (Experiment 2). Note: Different letters represented significant differences between means of replicates (n = 3) at the 0.05% level within a column. doi:10.1371/journal.pone.0129245.t005 Table 6. Net photosynthetic rate (P n ) and transpiration rate (T g ) of maize at tasseling and filling stages under different cropping systems. Growth stage/Cropping system P n (mmol m -2 s -1 ) T g (mol m -2 s -1 ) stage, N concentration of maize in all intercropping systems did not significantly differ from that of maize monoculture (P<0.05), except that N concentration of maize in 2M:6S was significantly higher than that in maize monoculture at pre-jointing stage. The N concentration of soybean monoculture was significantly lower than that of the intercropped soybean (P<0.05) at the early growth stage of pre-jointing, but they did not differ at tasseling and ripening stages. At pre-jointing stage, the N concentration of the intercropped soybean descended from row 1 to row 3, while the concentration did not significantly differ between rows at tasseling and ripening stages. At ripening stage, the soybean straw had much lower N concentration (3.64-4.77 mg kg -1 ) than the grains (60.71-66.54 mg kg -1 ). For the entire growth period, intercropping increased total N accumulation of the whole system compared to monocultures (Table 5). Crop biomass at different growth stages. Crop biomass per plant gradually increased from seeding to ripening stage (Table 8). At pre-jointing, biomass of intercropped maize and soybean (except maize row 1) in 2M:6S was significantly lower than biomass of monoculture (P<0.05). 2M:6S and 6M:6S showed a negative border row effect on biomass per plant, but the effect was positive in 4M:6S. Positive border row effect on maize biomass started to appear at tasseling stage (Table 8), when biomass per soybean plant in intercropping showed an advantage of inner rows and gradually increased from row 1 to row 3. At ripening stage, row 1 of all intercropped maize had an obvious advantage and its biomass per plant was significantly higher than that of monoculture of maize (P<0.05), but this was not observed for the maize inner rows (row 2 and row 3). The biomass per intercropped soybean plant gradually increased from row 1 to row 3, but all biomass except in 4M:6S were significantly lower than that of soybean monoculture (P<0.05). Overall, 4M:6S had an advantage in row 2 and row 3 of the intercropped soybean compared with monoculture, but there was always a disadvantage in 2M:6S and 6M:6S. Soil nitrate-N concentration at different growth stages. Nitrate-N concentration in topsoil (0-20 cm) was obviously higher than that in sub-soil (20-40 cm). In the soybean strip, nitrate-N concentration in both soil layers gradually decreased from pre-jointing to ripening stage of soybean, while for maize the concentration decreased from pre-jointing to tasseling stage and increased after topdressing N until ripening stage (Fig 2). Before N topdressing for maize, soil nitrate-N concentration (0-20 cm and 20-40 cm) in rows of maize monoculture was significantly lower than that of all intercropping rows. After N topdressing, nitrate-N concentration at 0-20 cm soil depth of all maize rows gradually increased until harvest. In particular, the nitrate-N concentration in maize monoculture had a dramatic amplification until reaching the highest point at ripening stage. However, N topdressing did not substantially affect soil N concentration at the depth of 20-40 cm (6.03-6.47 mg kg -1 ). Soil nitrate-N concentration (0-20 cm and 20-40 cm) in rows of soybean monoculture gradually decreased until reaching the lowest point at ripening stage compared to intercropped soybean. Except at prejointing stage, soil nitrate-N concentration in all soybean rows had a small variation, especially Soil nitrate-N concentration in the junction row (between maize strip and soybean strip) also differed with intercropping systems, but with no clear trends. In the later period of crop growth especially after filling stage of maize, soil nitrate-N concentration in junction rows was higher than that of soybean row 1, but lower than that of maize row 1. Yield of subsequent wheat and soil nitrate-N accumulation. Intercropping in summer did not significantly affect yield of winter wheat, but it significantly reduced soil nitrate-N accumulation (0-100 cm) after harvest of wheat, compared to monoculture of maize in summer (Table 9). 4M:6S produced 12-263 kg ha -1 higher wheat yields than the other four cropping systems, which indicated that intercropping systems in summer could even slightly increase yield of the subsequent crop. Compared to maize monoculture, intercropping systems reduced soil nitrate-N accumulation during wheat season by 21.9-51.7%. Discussion This study clearly demonstrated that intercropping systems presented advantage over maize monoculture. Intercropping system of maize with legumes reduced N application in the same planting area compared to maize monoculture, probably because of the enhanced biological N fixation by legumes [6]. Both maizeǁsoybean and maizeǁred bean systems showed intercropping advantages in yield, economy, land utilization ratio and reducing soil nitrate-N accumulation, as well as better residual effect on the subsequent wheat crop. Previous studies had also reported beneficial effects of intercropping systems on yield, economy and the environment [6,33], which stresses the importance of using intercropping in sustainable agriculture to alleviate pressure in intensive farming systems with high inputs and outputs [14]. Soybean is more important than red bean in China, with more consumption and relying on import [34], and decreasing planting area year by year [35]. Therefore, considering maize intercropping with soybean as the best cropping system in summer in the present study is reasonable and necessary. In particular, the optimal intercropping system was strip intercropping of 4 maize rows with 6 soybean rows (4M:6S), which had positive effects on yield, economy and environment in this study. Its LER value, crop N uptake, yields of both maize and soybean and economic benefit were even greater than those of 2M:6S and 6M:6S. This confirmed the previous finding that row ratio can influence intercropping efficiency [36,37]. In addition, 4M:6S significantly reduced soil nitrate-N accumulation compared to maize monoculture after harvest of summer crops. The LER values greater than 1 in all intercropping systems in the present study indicated high land-use efficiency compared to monoculture of maize or soybean [36]. Advantage of intercropping is probably derived from high light use efficiency above-ground and nutrients (e.g., N) below-ground [22]. Ability of maize to capture sunlight was enhanced at border rows, while there was small difference in photosynthetic rate and transpiration rate between inner-rows within a strip. The best light use efficiency was obtained in 4M:6S with narrow strips and a high proportion of border rows. Enhanced photosynthesis existed only in the two side rows, which indicated that four maize rows consisted of the optimal maize strip for light utilization. As a result, maize yield of intercropping systems was linearly correlated with photosynthetic efficiency, and light transmission was affected by between-row spacing, which supported previous findings by Prasad and Brook [38]. Intercropped soybean probably facilitated growth of maize by transferring the N fixed [39]. However, more N fertilization would inhibit N fixation of legumes [40], thus N was applied as basal fertilizer to both maize and soybean but only topdressed for maize in this study. Results showed that intercropping could provide enough nitrate for crops during the whole growth period. Top-soil was the main source of nitrate for crops, with significantly higher nitrate concentration than that of subsoil. Soil nitrate had different patterns in different intercropping systems from seeding to ripening stage. Soil nitrate-N in maize monoculture gradually increased to the highest value at ripening stage compared to intercropped maize, while soil nitrate-N in soybean monoculture gradually decreased to the lowest value at ripening stage compared to intercropped soybean. Elsewhere, Ossom et al. (2009) also observed significant differences in soil nitrate-N between different intercropping systems. Our results showed that soil nitrate-N accumulation increased gradually with the increasing maize area, probably partly because the N fertilizer rate for per unit area of maize was higher than that for soybean after topdressing. Intercropping advantage was most obvious in 4M:6S, but the advantage did not emerge at the beginning of growth. At the early stage, biomass per plant of most intercropped crops was smaller than the corresponding monoculture, probably because maize suffered border row effect and soybean growth was also negatively affected [32]. In the middle stage with two intercropping crops grown simultaneously, different cropping systems probably had different extents of interactions between interspecific competition and facilitation [13], which led to different growth rhythm. Intercropping advantage of maize started to emerge at tasseling and lasted until ripening stage, by showing a border row effect. In contrast, intercropping advantage did not appear in soybean during the entire growth period, except in 4M:6S which had a slightly increased soybean yield at ripening stage. A previous study showed that intercropping increased N concentration in junction crops [41], but this was not observed after maize tasseling stage in our study. Thus, the increase in total N accumulation of crops in intercropping systems was mainly because intercropping promoted biomass production. Our finding that accumulation of N by crops in intercropping systems was higher than that in maize monoculture was consistent with the conclusion of Li et al. [20], but it was contradict with Zhang et al. [42] who observed a higher amount of N accumulation in monoculture than intercropping. An optimal cropping system should aim at having a positive residual effect and increasing or at least not reducing yield of a subsequent crop. A promoted yield production of a subsequent crop by intercropping systems was observed in some studies [43,44], but not in others [45]. In the present study, yields of wheat following 4M:6S was the highest among all the treatments and its soil (0-100 cm) nitrate-N accumulation was significantly lower than that following maize monoculture. This provides additional proof of advantage existing in 4M:6S intercropping. Conclusions Compared to conventional monoculture of maize, both maize ǁ soybean and maize ǁ red bean had significant advantage in yield, economy, land utilization ratio and reducing soil nitrate-N accumulation, as well as better residual effect on the subsequent wheat crop. In particular, maize ǁ soybean performed best, and was thus identified as the optimal summer cropping system in this study. Intercropping systems could reduce N fertilizer use and increase relative biomass of intercropped maize, as a result of high photosynthetic efficiency of border rows and sufficient nitrate supply during symbiotic period. Noticeably, intercropping advantage was not inherent but began to emerge at tasseling stage after N topdressing for maize. 4M:6S was the best intercropping system in this study, as it had the largest LER, crop total N accumulation and economic benefit. In addition, compared to maize monoculture, 4M:6S significantly reduced nitrate-N accumulation in the soil after harvest of both summer crops and winter wheat, and it even slightly increased wheat yield. The most important advantage of 4M:6S was to increase biomass of intercropped maize and soybean, which further led to the increase of total N accumulation by crops as well as economic benefit. In conclusion, alternating four maize rows with six soybean rows is the optimum row ratio in maize || soybean system, though this needs to be further confirmed by pluri-annual trials.

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2018-04-03T01:39:17.429Z

2015-10-01T00:00:00.000Z

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Relation between antioxidant status and postpartum anestrous condition in Murrah buffalo Aim: Objective of the present study was to investigate the relation between antioxidant status and postpartum anestrous (PPA) condition in Murrah buffalo. Materials and Methods: Jugular blood samples were collected from two different groups of Murrah buffaloes each group consisting of 20 animals. Group I was of PPA and Group II were of cyclic buffaloes. The animals selected were examined for confirmation for cyclic and acyclic condition (>120 days) after calving by routine transrectal ultrasonography. Heard record was also used for cross confirmation. Results: The analysis of antioxidants in plasma and hemolysates revealed that the levels of vitamin E, β-carotene and reduced glutathione in plasma and superoxide dismutase (SOD) in hemolysate were significantly higher in cyclic animals than PPA animals. The levels of vitamin C, SOD and glutathione peroxidase in plasma did not show any significant difference among the two groups studied. The low antioxidant level in affected animals may predispose them toward PPA condition. Conclusion: Stress imposed by pregnancy and lactation affected the reproductive performance in PPA animals which might be inherently more susceptible to these stressors than those who were normal cyclic as all the animals were maintained under similar feeding and management practices. Introduction As the second largest source of milk in the world and about 56.5% of total milk production in India, Buffalo bear premier importance in dairy industry along with its significant contribution in foreign exchange earnings by export of meat [1]. The low reproduction efficiency in buffalo is the major constraint in obtaining maximum production potential set a perfect platform for the current research input. Among the various factors reducing its reproductive efficiency, postpartum anestrous (PPA) is a vital anomaly [2,3]. Many factors contributing individually or in concert were recognized to be responsible for postpartum infertility and anestrous making it a complex phenomenon. In ruminants, these factors include nutrients, mineral deficiencies [4], season [5], suckling [6], parity [7], infection [8,9], and dystocia [10], etc. Stress responses in heat, pregnancy and milk production lead to formation of reactive oxygen and nitrogen species (ROS and RNS). These ROS and RNS include hydroxyl radicals, superoxide ion, hydrogen peroxide, nitric oxide radicals and are involved in free radical chain reaction affecting lipid peroxidation, apoptosis, and fertility [11]. The biological consequences of these ROS and RNS mediated free radical chain reaction leads to infertility by affecting folliculogenesis, steroidogenesis, and preimplantation of an embryo which are sensitive to free radical damage [12]. Antioxidant defense system mitigates the free radical damage by disposing these ROS and blocking the free radical chain reaction to keep the animals healthy. The enzymatic components of this defense system mainly comprises of superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX) whereas the non-enzymatic counterpart includes reduced glutathione (R-GSH), vitamin C, vitamin E, βcarotene, and different macro and micro elements. So, hypothesizing that the pro-oxidant and antioxidant balance underlining the animal reproductive efficiency; the present study was designed to explore the relation between antioxidant status and PPA condition in Murrah buffalo. Ethical approval The experiments on animals including all procedures of this study were approved by Institutional Animal Ethics Committee. Available at www.veterinaryworld.org/Vol.8/October-2015/1.pdf Experimental animals The present investigation was carried out at the animal farms of Central Institute for Research on Buffaloes, Hisar; and Lala Lajpat Rai University of Veterinary and Animal Science, Hisar. Twenty PPA and twenty normal cyclic Murrah buffaloes were selected on the basis of their reproductive history obtained from farm records. According to the herd records, the buffaloes that had shown anestrous for more than 120 days were selected in postpartum group (PPA) and animal coming in estrous before 65 days of postpartum for more than three consecutive lactations including present lactation were selected in normal cyclic group for conducting the study. The animals selected in the current lactation were having average postpartum anestrous period of 191.47±13.37 days and those in normal cyclic had 60.64±5.38 days. Current status of reproductive organs of all animals in the study was also examined and verified by per rectal examination and ultrasonography. The animals were maintained as per the standard feeding and management practices followed at the farms and were fed for body maintenance and according to the level of milk production, so that the green and dry fodder were appropriately supplemented with concentrate mixture containing mineral mixture. Buffaloes were loose housed in open and closed paddock. Collection and transportation of blood samples Approximately,10 ml of jugular blood sample was collected from each experimental animal in 15 ml sterile polypropylene centrifuge tube containing ethylenediaminetetraacetic acid as anticoagulant in the month of November and December. Plasma was separated in refrigerated centrifuge at 3000 rpm for 15 min and stored in aliquots at −20°C until analysis of vitamins and antioxidants. Following separation of plasma from blood samples by centrifugation, the white blood cells layer was separated, and the remaining erythrocytes were washed thrice with a cold normal saline solution. Then distilled water was added to erythrocyte pellet slowly and with constant stirring up to 1:1 dilution to prepare hemolysate. It was stored at −20°C for estimation of SOD. All chemicals used in this study were procured from Sigma-Aldrich chemicals, USA. Nitric oxide scavenging activity of plasma was measured by the method of Sreejayan and Rao [13]. R-GSH assay was performed by the method of Beutler [14]. Plasma GPX-3 was estimated by kit provided by Cayman Chemical Company, U.S.A. The activity of SOD in red blood cells (RBC) hemolysate and plasma was measured by the method of Madesh and Balasubramaniam [15]. Vitamin E was estimated in plasma by method of Kayden et al. [16]. Estimation of vitamin C in plasma was done by 2,4-dinitrophenylhydrazine. Plasma βcarotene was estimated by the method of Baker et al. [17] and plasma protein by commercially available kit (Total Protein Kit, Coral Clinical System, India). Statistical analysis All the data were expressed as mean±standard error values. Statistical analyses were carried out using GraphPad Prism v6.0 (GraphPad Software, San Diego, CA, USA) software implementation of Student's t-test. Vitamins Plasma vitamin E, β-carotene, and vitamin-C levels were compared between PPA and normal cyclic animals. Vitamin E and β-carotene levels were observed to be significantly (p<0.05) higher in normal cyclic animals in comparison to PPA animals whereas vitamin-C level did not differed significantly between the two groups ( Table-1). Antioxidants The mean plasma nitric oxide scavenging activity, plasma R-GSH, plasma GPX-3, plasma SOD and SOD in RBC between PPA and normal cyclic animals along with their standard error has been depicted in Table-2. Plasma R-GSH concentration was found to be significantly higher (p<0.05) higher in normal cyclic animals than PPA animals. In terms of enzymatic antioxidants, SOD activity in RBC was found to be significantly (p<0.01) higher in normal cyclic animals but the plasma SOD activity and GPX-3 activity did not differed significantly (p<0.05) between the two groups of animals while the difference in nitric oxide scavenging activity was also found to be non-significant (p<0.05) between the two groups. Plasma protein The total plasma protein concentration was observed to be significantly (p<0.01) higher in normal cyclic animals (Table-2). Discussion In the present study, vitamin E was significantly (p<0.05) higher in normal cyclic animals than PPA group which is in corroborate the finding of Kahlon and Singh [18], Surapaneni and Vishnu [19]. This indicates that animal showing PPA is under oxidative stress resulting in decreased fertility. Similarly, analysis of data revealed significantly (p<0.05) lower level of β-carotene in PPA than normal cyclic animals. This may be due to that β-carotene is consumed during scavenging of free radicals. Similar reports such as Weiss [20] Jukola et al. [21] and Derar et al. [22] in cattle are also in accordance with the current findings. The non-significant variation in vitamin C level in both groups is in agreement with Serpek et al. [23]. The higher level of plasma GSH concentration in normal cyclic animals is also corroborate with the experiments of Ahmed et al. [24], Hanafi et al. [25] and Ahmed et al. [26], who reported similar observation in anestrous or non-cyclic animals. Similarly, Ahmed et al. [26] reported significant decrease in erythrocytic R-GSH in non-cyclic heifer buffalo than normal cyclic. Surapaneni and Vishnu [19] observed significantly lower level of erythrocytic GSH in polycystic ovary syndrome compared to normal cycler female in human. This indicates that PPA animals were in oxidative stress, and decrease in the levels of these non-enzymatic antioxidants values were probably due to increased turnover for prevention of oxidative damages because the role of the antioxidants is to prevent the generation of free radicals and to nullify the effect of free radicals [27]. SOD converts ROS generated by cell to hydrogen peroxide by spontaneous dismutation [28]. In our study, significantly higher level (p<0.01) of erythrocytic SOD in normal cyclic animals than PPA group animals was observed. Whereas plasma SOD did not differ significantly between normal cyclic and PPA group animals. The results are in accordance with related reports in buffaloes suffering from heat stress [24,26,29]. Whereas Kahlon and Singh [18] in anestrous buffaloes and Surapaneni and Vishnu [19] in polycystic ovary syndrome animals reported opposite effect. This indicates PPA animals were in oxidative stress, and decrease in the levels of erythrocytes SOD values were probably due to increased turnover for prevention of oxidative damages. Whereas plasma SOD showed non-significant variation between both the groups as plasma SOD indicates short-term stress condition. The current study revealed that plasma GPX-3 level was not significantly different in both group. Similar reports for dairy cow are also documented in related experiments [30]. Significantly higher protein in normal cyclic animals than PPA buffaloes has been reported by Amanullah et al. [31] and Kumar et al. [32]. The present study also confirms these previous findings. So, our results indicate that amelioration of production stress is essential to maintain reproductive efficiency in female animals [33]. Conclusion The vitamin E, β-carotene, reduced glutathione and protein investigated in plasma of buffaloes suffering from PPA were significantly (p<0.05) lower than normal cyclic buffaloes. The SOD level in hemolysate was also significantly (p<0.01) lower in PPA group animal than normal cyclic animals. Whereas plasma vitamin C level, SOD, GPX-3, and nitric oxide scavenging activity (%) were non-significantly (p<0.05) different in both groups. This indicates buffaloes under PPA condition are suffering from stress which may be because of production or reproduction. It also indicates that these animals are more susceptible to stress and apart from normal feeding additional supplementation of antioxidant vitamins can be beneficial to them at time of stress to optimize the performance in buffaloes. Authors' Contributions RK, MG, and AKB have designed the study and planned the research experiment. RK, MG, and SK performed the research experiments. IS, AKB, and MG supervised the research and performed manuscript preparation. All the authors read and approved the final manuscript.

v3-fos

2019-04-01T13:11:50.411Z

2015-11-30T00:00:00.000Z

88794687

s2

Leaf Proteome Analysis in Brassica rapa L. (Inbred line ‘Chiifu’) using Shotgun Proteome Approach Through high throughput shotgun proteomics approach, the proteome of seedling leaf of Brassica rapa L. was identified. From three biological replications, a total of 2,122 non-redundant proteins of Brassica rapa L seedling leaf were identified, with a wide range and unbiased physiochemical properties. Their pI values ranged from pH 4.27 (Bra004590) to pH 11.81 (Bra013905). Their molecular weight (MW) ranged from 5.6 kDa (Bra006908) to 534.5 kDa (Bra028068). Gene ontology enrichment analysis revealed that these proteins were associated with cellular process, metabolic process, and enriched catalytic activity compared to whole brassica proteins. The highest presented protein in Brassica rapa seedling leaf was RuBisCO, accounting for 11.56% of total leaf proteins. Also, many ribosomal proteins were identified. The relative amount of all ribosomal proteins comprised 8.47% of total leaf proteins. The relative amount of two RuBisCO and ribosomal proteins was about 20% of total leaf proteins. Thus to detect proteins presenting low abundance, additional fractionating procedure to remove RuBisCO and ribosomal proteins is required. INTRODUCTION Brassica is an economically important vegetable in the world. Some brassica such as Brassica napus L, Brassica Carinata, and Brassica rapa produce fatty acids widely used as vegetable oil (Harvey and Downey 1964) or biodiesel-fuel (Cardone et al. 2003). Brassica rapa is intensively consumed as a fresh vegetable in East Asia (Crawford 2006). Research on Brassica rapa L. (Inbred line 'Chiifu') has been progressed (Vanjildorj et al. 2009;Wang et al. 2010). However, its full genome sequence is currently unknown. Recently, the whole genome sequencing of Brassica of a Chinese cabbage has been completed (Wang et al. 2011). Omics research such as proteomics using its genome data is ongoing (Liu et al. 2013). For proteomic analysis, two -dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique has been used over 30 years since its introduction (O'Farrell 1975). It is simple and fast. However, it has some weakness in identifying proteins that are less abundant, hydrophobic, or very basic (pI >10). In addition, proteins with very small or very large molecular weight are hardly identified (Harry et al. 2000). Nowadays, advanced technique has been emerged to identify proteins. Shotgun proteomic method (Haynes and Roberts 2007), also known as multidimensional protein identification technology (MudPIT), is a non-gel based method to overcome the limitation of protein resoling on a gel. This method requires a much shorter time than 2D-PAGE (Lee and Cooper 2006). It can identify proteins in large scale with high throughput (Agrawal et al. 2009). Therefore, shotgun proteomics is regarded as complementary to 2D-PAGE. The benefit of shotgun proteomics in crops has been only applied for rice because shotgun proteomic analysis highly relies on a complete genome. Proteomic analysis with 2D-PAGE for Brassica rapa are also limited (Giavalisco et al. 2006;Wang et al. 2010). In this study, we identified the proteome of Brassica rapa leaf in large-scale with high throughput shotgun proteomic analysis using the complete genome database of Brassica rapa L. (Inbred line 'Chiifu'). Plant materials Brassica rapa L. (Inbred line 'Chiifu') seeds were used to identify seedling leaf proteins. Seeds were germinated in water and transplanted to soil at the density of one plant in one pot (90 nm inner diameter X 90 nm height). Brassica rapa L. plants were grown for 3 weeks in a growth chamber at 25 o C under 16 h day/8 h night with 40-70% relative humidity (RH) and sufficient water provided daily. Leaves of 3 weeks old seedling were harvested. Protein extraction Collected samples were ground in liquid nitrogen and added to 1.5 ml Eppendorf tube. Proteins were extracted with extraction buffer (8M Urea/5mM DTT/1% LDS/100 mM Tris-HCl pH 8.5). The suspension was incubated at room temperature for 30 minutes with vortex. After centrifugation at 14,000 g for 15 minutes, the supernatant containing extracted protein was collected. Proteins were precipitated overnight with 20% (v/v) trichloroacetic acid (TCA), washed several times with cold acetone until pigments (including chlorophyll) were removed. The protein pellet was dissolved in 8M Urea/100 mM Tris-HCl pH 8.5. Protein concentration was assayed with 2D-Protein Quant Kit (GE Healthcare, Piscataway, Nj, USA) using published method (Lee et al. 2007). One-dimension LDS-PAGE and in-gel digestion One-dimension LDS-PAGE and in-gel trypsin digestion were performed using published method (Lee et al. 2007 with alkylation buffer (55 mM iodoacetamide in 25 mM NH4HCO3) at room temperatures under dark. Gels were dried with SpeedVac. Dried gels were mixed with trypsin (trypsin 12.5 ng/l in 50 mM ABC) and digested at 36 o C overnight. Tryptic peptides were harvested from gels using harvest buffer (5% formic acid in 50% ACN). After vortexing for a few seconds, samples were centrifuged with 21,000 g at room temperature for 20 minutes. Supernatants were then dehydrated with SpeedVac. Samples were then desalted with Pierce Ⓡ C18 spin columns (Thermo Scientific, Rockford, IL, USA). Desalted samples were then subjected to LC MS/MS. LC MS/MS analysis with Q exactive Nanoflow HPLC instrument (Easy nLC, Thermo Fisher Scientific, San Jose, CA, USA) was connected on-line to a Q Exactive mass spectrometry (Thermo Fisher Scientific, Bremen, Germany). Columns (12 cm, 75 m inner diameter) used for analysis were packed in-house with Alltima C18-AQ 5 m resin. For reversed phase chromatography, binary buffer system consisting of 0.1% formic acid (Buffer A) and acetonitrile in 0.1% formic acid (buffer B) was used. Chromatography was performed for each sample with a linear gradient of 3-50% buffer B at a flow rate of 270 nL/min. The total run time for one LC MS/MS sample was 120 minutes. MS data were acquired using a data-dependent top 8 method that dynamically chose the most abundant precursor ions from survey scan (300-2,000 Da) for higherenergy collisional dissociation (HCD) fragmentation. Dynamic exclusion duration was 60 seconds. The isolation window of precursors was 4. Survey scans were acquired at a resolution of 70,000 with m/z at 200. The resolution for HCD spectra was set at 17,500 with m/z at 200. Analysis of proteomic data Proteome Discoverer (version 1. 3) software (Thermo Fisher Scientific) was used to identify proteins and spectral count for each identified protein with pI value and MW. Fragmentation spectra were searched against protein database of Brassica rapa (Brassica v 1.2, BRAD). Precursor and fragment mass tolerances were set at 10 ppm and 0.8 Da, respectively, with up to two missed cleavages. Carbamidomethylation of cysteine was set as a fixed modification. Oxidation of methionine was set as a variable modification for data searching. Both identifications were filtered at false discovery rate of 1%. Comparative analysis of relative protein abundances Proteome Discoverer (version 1.3) data of LC MS/MS analysis were exported to Microsoft Excel to calculate normalized spectral count (NSpC). NSpC of each protein k was calculated with the following formula: where the total number of MS/MS spectra matching peptides from protein k (SpC) was divided by protein's length (L) which was then divided by SpC/L for all N proteins in the experiment. Bioinformatics analysis of proteomic data Gene Ontology annotations for Brassica rapa seedling leaf proteins were retrieved from BRAD Brapa genome data V1.2. Gene ontology (GO) enrichment analysis was performed with agriGO (http://bioinfo.cau.edu.cn/agriGO/) using Brassica rapa whole genome as background/ reference. Identified proteins from Brassica rapa seedling leaf A total of 2,122 non-redundant proteins were identified by MudPIT runs. The physiochemical properties of these 2,122 identified proteins were analyzed. Their pI values and molecular weights were compared to those of whole brassica genome (Fig. 1). Bra004590 (disproportionating enzyme 2) had the lowest pI value at pH 4.27. Bra013905 (fibrillarin 2) had the highest pI value at pH 11.81. Based on the pI value, the distribution of identified proteins was similar to that of the entire brassica proteins, although more proteins were identified from Brassica rapa seedling leaf in the range of pH5-6 and pH6-7 compared to those in the whole genome. The molecular weight (MW) of identified proteins from Brassica rapa seedling leaf ranged from 5.6 kDa (Bra006908: dual specificity protein phosphatase or DsPTP1 family protein) to 534.5 kDa (Bra028068: GF14 protein phi chain). Based on the MW, the distribution of identified proteome was similar to that of whole genome. However, for proteins whose MW was 20 kDa, less proteins were identified from Brassica rapa seedling leaf compared to those from the whole genome. However, for proteins with MW in the range of 80 kDa to over 120 kDa, more proteins were identified from Brassica rapa seedling leaf compared to those from the whole genome. Gene Ontology (GO) analysis To study the proteome feature of seedling leaf of Brassica rapa, Gene Ontology (GO) enrichment analysis was performed for these 2,122 identified proteins. Twenty two GO terms of biological processes (Fig. 2), 8 GO terms of cellular components, and 8 GO terms of molecular functions were enriched in these 2,122 proteins. For the GO term of biological processes, proteins associated with cellular process and signaling processes were present in high proportions while proteins associated with regulation of biological process and biological regulation had less proportion. For cellular components, proteins associated with organelle, macromolecular complex, cell parts, and organelle part had high proportions. In molecular functions, proteins associated with catalytic activity had high proportion (Fig. 2). Highly presented protein in Brassica rapa seedling leaf To quantify protein amounts, spectra counts in MudPIT have recently been developed (Liu et al. 2004;Washburn et al. 2002). The spectra counts of the 2,122 proteins were estimated from the MS/MS spectra. The relative amount of proteins in one sample was estimated and the NSpC from the three replicates was averaged (Supplementary file 1). Averaged NSpC from 3 biological replications. The most abundant protein was RuBisCO (Fig. 3). In addition, most proteins in the top 20 highly presented proteins were subunits of RuBisCO and other proteins associated with photosynthesis (Table 1). Excluding these highly abundant photosynthesis associated proteins, tubulin and histone were abundant. One unknown protein (Bra 037380) was presented in high amount. Based on BLAST search, most Bra 037380 homologue proteins were uncharacterized in a few species as a hypothetical protein. DISCUSSION In this study, we identified 2,122 non-redundant Brassica rapa seedling leaf proteins through shotgun proteomic approach. Some of the identified proteins were not identified in all three replicates. This could be due to the fact that only a portion of samples were analyzed from the highly complex mixtures of peptides. A single analytical run may only recognize a fraction of total peptides in random (Wilkins et al. 2006). This result might also represent the complexity of leaf proteome. Proteins identified in this study may not completely cover all proteins in the leaf. However, there was no bias in the identification. The distribution of proteins identified in the seedling leaf of Brassica rapa was similar to that of the entire Brassica rapa proteome encoded by the whole genome. Unlike 2D-PAGE gel where basic proteins are hardly resolved or detected, ∼50 % of proteins identified in this study showed over pH 7. The GO enrichment analysis results revealed that proteins associated with organelle, macromolecular complex, cell parts, and organelle part were presnt in the seedling leaf in high proportion. This result implies that the status of seedling leaf is in active growth stage when proteins for constructing cell components are highly activated. Of the identified leaf proteins, RuBisCO proteins had the highest amount. High abundance of RuBisCO proteins are common features of green leaf in plant (Krishnan and Natarajan 2009). Our results revealed that the relative amount of all RuBisCO proteins comprised of 11.56% of total amount of leaf proteins. In addition, many ribosomal proteins were identified. The relative amount of all ribosomal proteins comprised 8.47% of total leaf proteins. The relative amount of two RuBisCO and ribosomal proteins was about 20% of total leaf proteins, making it difficult to identify low abundant proteins. Besides proteins associated with photosynthesis, histone and tubulin proteins were also present in high amounts. Histone is a nucleoprotein located in the nucleus while tubulin is a monomer of microtubule as components of cytoskeleton (D E Fosket and Morejohn 1992;Hansen et al. 1998). Because harvested leaf contained whole part of the shoot, our result indicated active cell division in seedling leaf. In conclusion, with high throughput shotgun proteomic approach, we identified Brassica rapa seedling leaf proteins in high depth. We analyzed cellular status based on GO enrichment analysis. We also determined the relative amounts of these proteins based on spectra counts.

v3-fos

2017-08-03T02:53:40.426Z

2015-02-28T00:00:00.000Z

13117467

s2

Effect of Allelic Variation in Triticin on Bread- and Chapati-Making Qualities of Wheat (Triticum aestivum) Triticin, a legumin-like storage protein of wheat endosperm, was discovered nearly three decades ago but so far there is no report on its effect on the processing quality of wheat that is thought to be determined primarily by prolamins, its major seed storage proteins. To investigate the effect of different classes of seed proteins on wheat quality using a genetic reconstitution approach, we produced 31 near-isogenic lines (NILs) with different alleles of triticin, high molecular weight glutenin subunits (HMW-GS), low molecular weight glutenin subunits (LMW-GS), gliadins and albumins in a common genetic background of wheat variety HD2329 and analysed different quality parameters over a period of 4 years. The NILs did not differ in their flour protein content, but showed significant differences in SDS-sedimentation volume, Farinograph dough stability, bread loaf volume and chapati quality score. Main focus was on triticin for which two NILs with alleles Tri-A1a and Tri-D1a derived from a high-quality Indian wheat variety K68 were analysed. Positive effects of these triticin alleles on dough physical properties, bread loaf volume and chapatti quality score were quite large, comparable to the widely known effect of HMW-GS 5 + 10. Specific alleles of HMW-GS, Glu-A1a (subunit 1), Glu-B1b (subunits 7 + 8), Glu-B1i (subunits 17 + 18) and Glu-D1d (subunits 5 + 10) showed strong positive effects, whereas null allele Glu-A1c showed negative effect on the quality of recipient variety HD2329. Similarly, different alleles of LMW-GS showed varying effects with Glu-A3d, Glu-A3e and Glu-D5a showing positive effects, Glu-A3c showing negative effect and Glu-A3a showing no significant effect. Gliadin alleles generally showed negative effects, whereas albumins showed no significant effect. While results with glutenin and gliadin alleles were as expected, we show here for the first time a significant effect of triticin on the wheat flour quality, suggesting that end-use quality of wheat varieties can be improved by combining specific alleles of triticin. Introduction The end-use quality of wheat grain is determined by its protein, starch and lipids constituents of whom gluten proteins play a pivotal role. At the turn of the twentieth century, wheat seed proteins were grouped based on their solubility properties into four classes namely albumin, globulin, gliadin and glutenin [16]. While albumin and globulin are minor proteins of the wheat endosperm and are not known to greatly influence its end-use quality; gliadin and glutenin, the two major components of gluten, are the key determinants of wheat flour quality for making bread, biscuit, noodle and other products [24,26,27]. Fractionation and reconstitution studies with wheat flour have shown that polymeric glutenin is responsible for the strength or elasticity of wheat flour dough, whereas monomeric gliadin is responsible for its viscosity [14,15]. Native glutenin fraction is a complex polymer mainly composed of high molecular weight (HMW) and low molecular weight (LMW) subunits whose allelic differences are known to affect the bread-making quality of wheat [5,11,19]. The native glutenin fraction also contains small proportion of globulins and albumin but their role in determining wheat end-use quality is not known [15]. Apart from the role of individual glutenin subunits, studies on native proteins without reduction of their disulphide bonds have shown that dough strength and bread-making quality are positively correlated with the proportion and molecular size distribution of polymeric proteins in the total flour protein [5,9,33,34]. Large glutenin polymers are formed by inter-polypeptide disulphide bonds, which give wheat flour dough its unique visco-elastic properties. The HMW subunits of glutenin are encoded by Glu-A1, Glu-B1 and Glu-D1 genes located on the long arm of wheat chromosomes 1A, 1B, and 1D, respectively [11,18]. The Glu-D1 locus is shown to have the single largest effect on bread-making quality, followed by Glu-B1 and Glu-A1 loci [11]. The LMW subunits of glutenin are coded by Glu-A3, Glu-B3 and Glu-D3 genes located on the short arm of chromosomes 1A, 1B and 1D, respectively, tightly linked to the Gli-1 loci coding for gliadins [31]. The LMW subunits have been quite difficult to study by electrophoresis due to their overlapping size with gliadin polypeptides but after development of a simplified SDS-PAGE procedure, it was shown that allelic differences at Glu-A3, Glu-B3 and Glu-D3 loci coding for LMW glutenin subunits are equally important in determining the dough properties and breadmaking quality [5,8,13,35]. Gliadins are monomeric proteins and when fractionated by acidic starch or polyacrylamide gel electrophoresis (APAGE), they separate into four groups, namely a, b, c and x gliadins [40]. Gliadin synthesis is controlled by several related genes of a limited number of multi-gene families located on the short arm of group 1 and 6 chromosomes [41]. Combined studies of HMW glutenin subunits and gliadin composition in different wheat cultivars and progenies have revealed their relative contribution to dough properties [14,15]. It has been suggested that the effect of gliadins on dough quality should be attributed to their tight genetic linkage with LMW glutenin subunits genes [17]. Purified gliadin is known for its negative effect on dough strength and bread-making properties, and therefore, positive effect of specific Gli-1/Glu-3 complex on dough resistance and extensibility is most likely due to the genetically linked Glu-3 alleles due to their polymerization properties [5,8,14,15]. Triticin is a minor seed storage globulin of the wheat endosperm first identified by Singh and Shepherd [28] and subsequently characterized in much detail at the genetic, biochemical, physiological and molecular level. [3,25,28,29,31,32,36]. Triticin genes are located on the short arm of wheat chromosomes 1A and 1D, near the centromere far away from the major Gli-1/Glu-3 loci [28,31]. Triticin is synthesized specifically during the wheat seed development and is deposited in the electron dense inclusion bodies within the main storage protein bodies of the endosperm [3,29]. Unlike the glutenin and gliadin which are prolamin type proteins (rich in proline and glutamine amino acids), triticin shows homology to the 11-12S legumin-like storage globulins of leguminous species [32,36]. However, the role of triticin in determining wheat dough properties and bread-making quality has not yet been investigated. The aim of this study was to develop a set of near-isogenic lines (NILs) with different alleles of triticin, HMW and LMW glutenin subunits, gliadins and albumins in a common genetic background of Indian bread wheat variety HD2329 and analyse their quality parameters. The variety HD2329 was chosen for its medium bread-making quality so that both positive and negative effects of individual alleles can be observed easily. Plant Material and Field Experiments A set of 31 NILs with different alleles of HMW-GS, LMW-GS, gliadin, triticin and albumin were used (Table 1). NILs were produced by crossing a highly adaptable bread wheat variety HD2329 with donor wheat varieties having different seed storage protein alleles followed by three backcrosses coupled with phenotypic and AFLP marker-based background selection and protein electrophoresis-based foreground selection [22,23]. Segregating populations for triticin alleles were developed by crossing triticin NILs with the recipient variety HD2329. Two such segregating populations were developed, one each for the Tri-A1 and Tri-D1 loci. Homozygous lines for the two triticin alleles were selected from the F 2 progeny by SDS-PAGE on endosperm half of the seed, while embryo half was grown to obtain F 3 seeds for quality analysis. Seeds of all the NILs were multiplied in the experimental fields of IARI, New Delhi, using a completely randomized block design in two replicates with a plot size of 100 plants each sown in a 6 9 6 grid design of 3 m 9 3 m during the Rabi seasons of 2007-2010. The field managements were carried out according to standard practices for wheat, and mature grains were harvested for analysis of quality traits. F 3 families of triticin segregating lines were grown in a net house to obtain enough F4 seeds for the quality analysis using SDS sedimentation and extensigraph tests. Protein Extraction and SDS-PAGE Sequential extraction of seed albumin, gliadin and glutenin were done from crushed endosperm half of single seed (*15 mg) or 20 mg of four samples in 1.5 ml Eppendorf tubes. First, albumin was extracted in 200 ll of RO water (18 Ohm) at 25°C for 30 min, centrifuged at 150009g for 10 min, and then 100 ll of the supernatant was mixed with equal volume of 29 sample buffer [2 % SDS, 20 % (w/v) glycerol, 82.5 mM tris-base, 0.2 % bromophenol blue, pH 8.0] containing 1 % (v/v) dithiothreitol. The residue was washed with 0.5 ml of RO water and then extracted with 200 ul of 50 % (v/v) propan-2-ol, centrifuged at 15,0009g for 10 min as reported earlier [22,23]. Except for albumins which were extracted at room temperature, all other seed storage proteins were extracted by incubation at 60°C for 10-15 min just before loading in the gel. Glutenin (HMW-GS and LMW-GS) extraction and separation were done according to Singh et al. [35]. Albumins (30 ll), glutenins (25 ll) and gliadins (20 ll) extracts were separated in a 10 % polyacrylamide gels with 1.5 % crosslinking. Electrophoresis was performed in 1.5 mm thick slab gels of 20 9 20 cm dimension using Hoefer SE600 electrophoresis system at a constant current of 40 mA/gel for 2.5 h. Triticin was extracted from single seeds in 1 M NaCl and precipitated with acetic acid as described in Singh et al. [30]. The triticin gels were run for a longer period of 3.5 h for better resolution of high molecular weight triplet protein bands. After electrophoresis, gels [38]. Grain Protein Content and Kernel Characteristics Grain protein content (GPC) was measured by near infrared reflectance spectrometry (NIRS) from grain samples according to the AACC method [1]. White flour protein content and moisture level were also measured by NIRS and used together with grain hardness index for calculating the amount of water required for Farinograph test [Brabender 1965]. Grain hardness (GH), moisture content (MC), grain diameter (GD) and thousand kernel weight (TKW) were measured on 300 kernels for each sample using Perten Single Kernel Characterization System (SKCS) 4100 system, following manufacturer's protocol (Perten Instruments North America Inc., Springfield, IL). SDS-Sedimentation Volume Test Flour samples were evaluated for bread-making quality using SDS-sedimentation volume (SDS-SV) test [2]. In this method, the volume of material which sediments after mixing flour with a solution of SDS and lactic acid is measured. Milling of grains was performed in a Brabender Junior mill to obtain flour extraction rate of 60 %. Farinograph Test Farinograph curves (C.W. Brabender Instruments, Inc., South Hackensack, NJ, USA) were generated according to the AACC method [1]. The 50 g mixing bowl was used in conjunction with the standard operating speed of 63 rpm. The curves were read manually, and different parameters were recorded, including Farinograph water absorption (FAB, 14.0 % moisture basis), the amount of water required to centre the curve on the 500 BU line; dough stability (STA), the difference in time from when the top of the curve first reaches the 500 BU line (arrival time, AT) to when it first leaves the 500 BU line (departure time, DT); mixing tolerance index (MTI), the drop in the curve 5 min after peak development, measured in BU units; dough development time (DDT), the time required to reach peak dough development; and time to breakdown (TTB), the time from the start of mixing to the time at which the consistency decreases 30 BU from the peak. Dough Resistance and Extensibility The segregating lines of triticin were analysed using texture analyser for extensigraph properties. This was done by TA.XTplus Texture Analyser from Stable Micro Systems using Kieffer extensibility rig. It uses the same principle as Brabender Extensograph, except that the sample is stretched upwards and the dough requirement is low. It provides information about dough resistance to stretching and extensibility by measuring the force to pull a hook through a cylindrically shaped piece of dough [43]. Bread Loaf Volume Baking performance was evaluated by doing an optimized straight-dough bake test (Approved Method 10-10B, AACC 1995) using 100 g of flour (14 % moisture basis). Optimum bake water absorption (%WA) and mixing time (min) were those resulting in dough with optimal handling characteristics as judged by bakers. Loaf volume (cm 3 ) was determined by rapeseed displacement method on fresh loaves. Chapati-Making Quality Chapatis were prepared from whole-wheat flour according to the method developed by Haridas Rao et al. [10]. Flour (200 g, 14.0 % moisture basis) and water as determined from a 500 B.U. Farinograph trace were mixed in a Hobart dough kneader (HL 120 Hz 50/60) for 5 min. The dough was rested for 10 min before being cut into four equal sections of 40 g each. A section of the dough was then placed on a rolling board with a thickness guide of 1.5 mm. The dough was rolled in one direction, inverted, rotated at 90°and rerolled. The sheeted dough was cut with a circular die to get a 12-cm diameter uniform chapati (Fig. 1a). The raw chapati was placed on a preheated griddle at 215°C. The chapati was cooked for 70 s on one side, flipped, and then cooked for 85 s on the second side. The cooked chapati was quickly transferred (\10 s) to an adjacent heater and allowed to puff for 20 s before removal and cooling at room temperature for 10 min. Puffing height was recorded by a scale with sliding bar (Fig. 1b). Puffing height of chapati between 0 and 5 cm chapati was given 5 points. Chapati was also evaluated by a trained panel of four judges and scored (0-10) subjectively for the following quality parameters; appearance, tearing strength, pliability, aroma and eating quality (0-15). After taking one set of observations, chapati was placed in a resealable plastic bag and stored for 4 h before next round of evaluation. Again after 4 h chapattis were evaluated for tearing strength and pliability (score 0-10). The higher the score, the better the quality of chapatti. Assessments were made in duplicate, and scores of all the panellists were averaged. Statistical Analysis Analysis of variance (ANOVA) was done using SPSS software package ver. 16. The seed samples of all 4 years were taken for the analysis of SDS sedimentation and protein content, while only two-year seeds were used for Farinograph and baking quality tests. T test was performed for assessing the significance of differences among the means for the NILs and triticin segregating lines at 0.5 % P level of significance. Characterization of Seed Protein NILs Total thirty-one NILs were developed with different seed storage protein alleles in the common genetic background of bread wheat variety HD2329. Different seed protein genes, the allele for which individual NIL differed from the recipient variety HD2329, donor variety and corresponding allele in the recipient variety are shown in Table 1. Genes and alleles for which no recognized symbols are available, e.g. albumin polypeptides, were assigned new temporary symbols [23]. Field observations over 4 years showed that the NILs were quite similar in appearance and yield performance to the recipient variety HD2329, except for white glume colour in some of the NILs for Gli-B1/Glu-B3 loci, namely [GLI-1,3,4,7 and LMW-7], and the rest of NILs were brown in glume colour like recipient variety HD2329. This was due to a tight genetic linkage between gene for red glume colour and Gli-B1/Glu-B3 locus on the short arm of chromosome 1B [12]. The NILs were characterized for their complete seed protein profile by SDS-PAGE to check for similarity of non-target protein loci with the recipient parent HD2329. There were two NILs with different alleles of triticin, namely TRI-1 and TRI-2, corresponding to triticin alleles Tri-A1a and Tri-D1a, respectively (Fig. 2). The recipient variety HD2329 had Tri-A1b and Tri-D1b alleles at these loci resulting in a narrow triplet band compared to NIL TRI-1 which had a faster moving Tri-A1 band and NIL-2 which had a slower moving Tri-D1 band resulting in wider triplet bands in the two NILs. The intensity of triticin bands was also consistently darker in the two NILs as compared to HD2329. Both the triticin NILs showed identical electrophoretic profiles for HMW-GS, LMW-GS, gliadin and albumin fractions. Only two of the seven LMW-GS NILs namely LMW-2 and LMW-6 showed gliadin patterns identical to HD2329, and the remaining five LMW-GS NILs showed differences in the xand c-gliadin regions as marked in Fig. 4b. Interestingly, NILs LMW-3 and LMW-4 both have the same LMW Glu-A3d allele but they differ for gliadin patterns. LMW-3 NIL has different x-gliadin pattern which can be due to some insufficient backcrossing or rare recombination between Glu-A3 and GliA1 loci while LMW-4 NIL gliadin pattern is similar to HD2329. There were 12 NILs with different gliadin alleles; seven of these (Gli-1 to Gli-7) differed in the x-gliadin region, while remaining five (Gli-8 to Gli-12) differed in the cgliadin region (Fig. 5a). SDS-PAGE analysis showed that similar to the LMW-GS NILs, gliadin NILs showed differences in their LMW-GS profiles due to tight genetic linkage between the two loci (Fig. 5b). The LMW-GS are divided into two groups, B and C subunits, based on their size distribution. Variation in the LMW-GS of these NILs was mainly in the slower moving B group of subunits, variation in the C group of subunits was limited to absence in the c-gliadin NILs of one of the three subunits of HD2329. HMW-GS, albumin and triticin profiles of the gliadin NILs were identical to the recipient variety HD2329. There were four NILs with different albumin alleles for which temporary new symbols Alb-mc, Alb-mf, Alb-mb and Alb-mg were assigned as there were no gene symbols available for these in the literature (Fig. 6). Effect of Allelic Variation in Seed Proteins on Quality The thirty-one NILs were analysed for a range of grain quality parameters considered important for the end-use products namely bread, biscuit and chapati. A single kernel characterization system (SKCS) was used for the analysis of grain hardness, TKW and grain moisture content but these traits did not show significant variation among the NILs, except for TKW which varied significantly between 33.5 and 39.5 g, which is important for flour yield during milling of the wheat grains (Table 2). Further there was no significant difference for grain protein content among the NILs and recipient variety HD2329 (Table 2). This shows that different alleles of seed storage proteins had no relationship with the above quality parameters. However, there were significant differences among NILs for SDS-sedimentation volume, Farinograph dough development time, dough stability and bread loaf volume, showing that these parameters were affected by the seed storage protein allelic composition. Thirty-one NILs analysed in this study represented all four classes of seed proteins. The major storage proteins glutenin and gliadin showed significant positive or negative effect on the dough and bread-making quality of the base wheat variety HD2329, whereas albumin NILs showed no significant effect as also described in the published literature [21]. The most important novel finding of our study was significant positive effect of triticin on wheat quality parameters. Effect of allelic variation in seed protein on different wheat quality parameters are described below. SDS-Sedimentation Volume Sodium dodecyl sulphate-sedimentation volume (SDS-SV) measures degree of sedimentation of wheat flour suspended in a lactic acid-SDS medium during a standard time of settling [2]. The SDS-SV value depends on the protein quality and provides an indication of wheat gluten strength. Triticin NILs, with alleles Tri-A1a and Tri-D1a, showed highly significant positive effect on SDS-SV, which was comparable to the effect of HMW glutenin subunits 5 ? 10 known for their strong positive impact on bread-making quality [17]. Effect of Tri-D1a was comparatively more pronounced than Tri-A1a ( Table 2). The HMW-GS NILs with alleles Glu-A1a (subunit 1), Glu-B1b (subunit 7 ? 8), Glu-B1b* (subunit 7* ? 8), Glu-B1i (subunit 17 ? 18) and Glu-D1d (subunit 5 ? 10) showed significant positive effect on SDS-SE of HD2329, while Glu-A1c (Null allele) showed significant negative effect in 4 years of evaluation ( Table 2). All the HMW-GS alleles were positively behaving towards SDS-SV, and only Glu-A1c (Null allele) was behaving negatively. There were seven NILs for LMW-GS, five of which represented different alleles of Glu-A3 locus. LMW NILs for allele Glu-A3c were showing significantly negative effect from HD2329 and that of Glu-A3a was showing no effect, and the rest of the LMW NILs (Glu-A3e, Glu-A3d, Glu-D5a and Glu-B3ks) were showing positive effect over recurrent parent for SDS-SV test. Two separate NILs with Glu-A3d showed strong positive effect over recurrent parent HD2329 but effect of NIL Glu-A3d-1 was lower than Glu-A3d-2 (Table 2). This could be due to difference in the linked gliadin polypeptides as Glu-A3d-1 has multiple x-gliadin bands which have negative effect of dough strength. All the twelve gliadin NILs showed either negative or no significant effect on the SDS-SV of recipient variety HD2329. The albumin NILs showed no significant effect on SDS-SV. Farinograph Physical Dough Properties Similar to the effect on SDS-SV both the triticin NILs, Tri-A1a and Tri-D1a, showed highly significant positive effect on dough stability but no effect on farinograph dough development time ( Table 2). HMW glutenin subunit NILs with alleles Glu-A1a (1), Glu-B1b (7 ? 8), Glu-B1b* (7* ? 8), Glu-B1i (17 ? 18) and Glu-D1d (5 ? 10) showed significant positive effects on Farinograph dough stability over HD2329, while Glu-A1c (null allele) showed significant negative effect. LMW-GS NILs Glu-A3c showed significant negative effect on Farinograph dough stability, while Glu-A3a showed no effect rest all the LMW NILs (Glu-A3e, Glu-A3d, Glu-D5a and Glu-B3ks) showed positive effect over recurrent parent for Farinograph dough stability test. Farinograph dough development time showed no significant difference over HD2329 except for allele Glu-D5a which has a low dough development time. All the gliadin NILs, except c-Gli-me and x-Gli-B1b showed significant negative effect, while albumin NILs either showed significantly negative (Alb-mf) or no significant effect (Alb-ma, Alb-mb and -Alb-mg) on dough stability ( Table 2). All the gliadin alleles showed no significant effect except x-Gli-B1c, x-Gli-A1g and c-Gli-md which behaved negatively for dough development time. All the albumins also showed low dough development time. Bread Loaf Volume As expected from the data on SDS-SV and Farinograph physical dough properties, triticin alleles Tri-A1a and Tri-D1a showed highly significant positive impact on the loaf volume of wheat variety HD2329 that was consistent in 2 years of testing. The effect of triticin alleles Tri-A1a and Tri-D1a coming from a traditional high-quality wheat variety K68 was as high as the effect of well-known HMW glutenin subunits 5 ? 10. The bread loaf volume was 620 cc for Glu-D1d, whereas it was 630 cc and 610 cc, for Tri-D1a and Tri-A1a, respectively as compared to 550 cc for HD2329. (Table 2; Fig. 7). HMW-GS (NILs Glu-A1a, Glu-B1b, Glu-B1b*, Glu-B1i and Glu-D1d) showed significant positive effect, while Glu-A1c showed significant (Table 2). Among the LMW-GS NILs, only Glu-A3d-1, Glu-A3d-2 and Glu-D5a showed significant positive effect on loaf volume, and the remaining LMW-GS NILs (Glu-A3c, Glu-A3e, Glu-A3a and Glu-B3ks) showed negative or no effect on bread loaf volume over HD2329. All the gliadin NILs showed significant negative effect on loaf volume except x-Gli-B1d and x-Gli-B1a which had no significant effect ( Table 2). Albumin NILs had no significant effect on bread loaf volume as compared to the recurrent parent HD2329 (Table 2). Chapati Quality Score The bulk of Indian wheat is consumed in the form of chapati, and it is realized that over the years during and post green revolution era, the chapati quality of Indian wheat varieties has declined. Thus, some of the older varieties grown in the central India, so called MP wheat, still fetch premium price in the market [42]. Similar to their effect on SDS-SV, dough physical properties and bread loaf volume, triticin alleles Tri-A1a and Tri-D1a showed significant positive impact on chapati quality score also, which was consistent in 2 years of testing. We evaluated the chapati-making quality of HMW-GS and LMW-GS NILs and found that all the HMW-GS and LMW-GS NILs generally showed positive effects on the chapati quality of HD2329, except for alleles HMW-GS Glu-A1c and LMW-GS Glu-A3a, Glu-A3e, Glu-A3c and Glu-D5a which showed no significant effect. None of the gliadin and albumin alleles showed significant effect on chapati quality score. Validation of the Effect of Triticin Alleles in Segregating Bi-Parental Populations For further validation of the effect of triticin alleles TriA1a and Tri-D1a, two segregating populations were developed by crossing the respective triticin NILs with recipient variety HD2329. Homozygous lines with two segregating alleles of the Tri-A1 and Tri-D1 genes were selected by SDS PAGE. The triticin patterns of each Tri-A1a, Tri-A1b, Tri-D1a and Tri-D1b homozygous line are shown in Fig. 8. Tri-A1a and Tri-D1a were obtained from K68, the donor variety of triticin NILs, whereas Tri-D1b and Tri-A1b were from recurrent parent HD2329. Twenty eight such homozygous F 3 segregating lines were multiplied in the net house, and bulk F 4 seeds were harvested for quality analysis by SDS-SV and a small-scale dough Extensigraph. Effect of Tri-D1a and Tri-A1a alleles was significantly positive on SDS-SV and dough Extensiograph force over HD2329. The overall effect of all the four triticin alleles was significant on SDS sedimentation value (P \ 0.05) and Extensiograph force but extensibility was not affected significantly (Table 3). Tri-D1a and Tri-D1b alleles gave consistently higher Extensigraph force value than HD2329 but the effect of Tri-D1a was higher than Tri-D1b. Discussion A wheat cultivar can produce good quality bread even with moderate protein content, if the protein quality is good. In fact, in many breeding programmes, consciously or not, some HMW-GS alleles, in particular Glu-D1d (subunits 5 ? 10), were frequently used for increasing end-use quality [38]. Earlier studies have provided evidence for strong association between the presence of specific HMW-GS alleles and bread-making quality [17,20]. Further studies have shown that allelic variations in both HMW-GS and LMW-GS are important in determining the breadmaking quality of wheat flour [7]. In our study, no significant difference was found in the protein content of the thirty-one NILs with different seed storage protein alleles, so the effect on wheat quality was primarily due to protein quality i.e. amino acid sequence variation of alleles. A number of studies have been done for evaluating the effects of different HMW, LMW glutenin subunits and gliadin alleles on bread-making quality of wheat but contribution of wheat triticin has not yet been investigated. Triticin is a minor seed storage protein which accounts for only about 5 % of the total endosperm protein in wheat. It is leguminlike protein and has a lysine-rich repetitive domain in its hyper variable region which offers new opportunities to genetic engineers for increasing lysine content of wheat [36]. Our study found two alleles of triticin showing significant positive effect on bread-making quality parameters. SDS sedimentation volumes were 46 and 44 ml in Tri-D1a and Tri-A1a, respectively, which are comparable to the effect of HMW-GS Glu-D1d with volume of 45 ml. Glu-D1d has already proved to be a good contributor towards bread-making quality. Triticin allele's effect was comparable to Glu-D1d consistently in the 4 years trials. The effect was more pronounced on dough strength where Tri-D1a and Tri-A1a NILs showed Farinograph dough stability time of 17.5 and 16 min, respectively, compared to 16 min for Glu-D1d NIL and 11 min for the recipient parent HD2329. Similar positive effect was seen on bread loaf volume which was over 610 cc for the triticin NILs as compared to 550 cc for HD2329. Alleles of HMW-GS and LMW-GS showed expected effects as described in the earlier studies. All the HMW-GS alleles, except the null allele Glu-A1c showed positive effect on SDS-SV and Farinograph dough stability. Earlier Values in the same column followed by different superscript letter are significantly different at cutoff P value of 5 % Fig. 8 SDS-PAGE patterns of homozygous F 3 lines selected from segregating bi-parental populations obtained by crossing recipient parent HD2329 with triticin NIL Tri-A1a (b-g) and Tri-D1a (i-n). a, h Recipient parent HD2329, b-d lines with triticin allele Tri-A1a (marked with arrow), e-g lines with triticin allele Tri-A1b (identical to HD2329), i-k lines with triticin allele Tri-D1a (marked with arrow), l-n lines with triticin allele Tri-D1b (identical to HD2329) LMW glutenin subunits have also been shown to significantly impact the dough strength in bread wheat [6]. In our study, seven NILs with different LMW glutenin subunits were analysed, and it was found that in comparison to HD2329 allele (Glu-A3b) other Glu-A3 alleles showed negative or no significant effect on bread-making quality, except allele Glu-A3d which had a significant positive effect. Earlier Gupta et al. also found association of gliadins and linked Glu-A3b allele with dough resistance and extensibility in bread wheat [6]. Glu-A3a was found to be superior to Glu-A3e and Glu-A3b was superior to Glu-A3c [5,7,8,17]. In our study, Glu-A3a, Glu-A3c and Glu-A3e negatively affected the quality, whereas Glu-A3d, Glu-B3ks and Glu-D5a showed positive effect on the dough properties. Effect of gliadins in determining bread-making quality was variable. Some alleles of c-gliadins were found to have no significant effect on dough properties, while others were found to be negatively correlated with loaf volume, but none of the Gliadin NILs showed positive effect. Studies on the effect of albumin on wheat quality are limited. Here, we found that four NILs with different alleles of albumin have no significant effect on wheat quality. While effect of glutenin subunits have been reported in several earlier studied, this is the first report on effect of triticin on bread-and chapatimaking quality of wheat which can be utilized for the improvement of wheat end-use quality. Conclusions and Prospects Significant differences in dough rheological properties and end-use quality for bread and chapati making were observed among NILs with different seed protein alleles in a common genetic background of wheat variety HD2329. While results with the major classes of seed storage proteins namely glutenin and gliadin were confirmatory in nature, a positive effect of wheat storage globulin triticin on the bread-and chapati-making qualities of wheat is demonstrated here for the first time. Also, it is for the first time that the effect of allelic differences at five different classes of seed protein loci have has been analysed in common genetic background. In future, we can study interaction effects of these genes in the common background of HD2329 by making intercrosses and selecting different assortments of alleles. We also need to develop more NILs for a comprehensive coverage of seed storage protein alleles. The information generated here will be very useful in developing high yielding wheat varieties with improved end-use quality.

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2019-04-02T13:13:57.207Z

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Evaluation of genetic diversity for yield and quality parameters of different potato (Solanum tuberosum L.) germplasm An investigation was carried out at Vegetable Research Centre, G.B. Pant University of Agriculture and Technology, Pantnagar during spring-summer season 2011 and 2012 to study the genetic diversity using Mahalanobis’s D – technique among thirty five potato (Solanum tuberosum L.) germplasm for important yield attributing and quality traits. The D values were calculated and thirty five potato genotypes were grouped into nine clusters for growth characters and ten clusters for quality traits respectively. All the genotypes included in the present investigation, were indigenous, but their grouping in different clusters, suggested that genotypes did not follow the geographic distribution. The cluster I contained the maximum number of genotypes with respect to both yield attributing and quality traits. The inter cluster distance in most of the cases were higher than the intra-cluster distance indicating wider genetic diversity among the genotypes of different groups. Average tuber weight of potato plant contributed maximum (31.76%), followed by number of tuber per plant (27.56%), internodal length (14.45%) and plant dry matter content (13.61%) for growth characters. For quality characters, ascorbic acid content (24.70%), protein content of tuber (20.84%) and TSS of tuber (20.00%) contributed effectively towards genetic divergence. So, these traits will offer a good scope for improvement of yield and quality through rational selection of parental genotypes for future potato breeding. The findings indicated that use of parents selected from the same cross or from a cross involving a common parent should be avoided in hybridization. The results broadly showed there was no parallelism between geographical and genetic divergence. INTRODUCTION Besides its significance to human food security, potato (Solanum tuberosum) is also a crop with fascinating genetic traits and cultural history (Swaminathan, 1999). Exploitation of variability displayed by different Solanum germplasm for breeding the cultivated potato (S. tuberosum) requires phenotypic and genotypic characterization of germplasm resources (Barone et al., 2010). Selection and hybridization are the two basic methods for improving crop plants. Selections concentrate favorable genes in cultivars for better performance. The success of selection and hybridization programme depends upon the extent of variability, heritability and association among yield contributing characters and quality traits and their effects upon yield and quality (Rizvy et al., 2007). Identification of diverse but desirable parents remained a difficult task for plant breeders. In the past, generally, ecological or geographic diversity has been considered as an index of genetic diversity. However, this is an inferential criterion, and may not be so effective in , 2231-5209 (Online) All Rights Reserved © Applied and Natural Science Foundation www.ansfoundation.org quantification and differentiation between populations. Therefore, genetic divergence analysis is highly essential to estimate the extent of diversity existed among selected genotypes (Mandal, 2003). Genetic diversity is used for discriminating divergent populations, which are reinstated by more scientific and advanced biometrical techniques viz., multivariate analysis based on Mahalanobis D 2 -statistic (Mahalanobis, 1936). The success of potato breeding programs depends on identification of the amount and distribution of genetic diversity in the gene pool, to identify the gaps in germplasm collections and to develop effective conservation and management strategies (Esfahani et al., 2009). Selection as well as hybridization programme from locally available germplasm result in minor progress in development of varieties because of low variability in the germplasm available. Diverse genetic materials are, therefore required to meeting the ever-increasing demands of plant improvement. Information on genetic diversity in available germplasm collection is therefore, of paramount importance. Therefore, success in development programme of potato can be achieved through utilization of the broad genetic base of cultivated, as well as wild relatives of crop plants available in different parts of the world in this crop species (Ragassa et al., 2007). Keeping view towards the above facts and to characterize potato germplasm, the present study was under taken with the objective to study genetic diversity among potato genotypes for important yield attributing and quality traits of S. tuberosum. MATERIALS AND METHODS The present investigation was carried out at Vegetable Research Centre, G.B. Pant University of Agriculture and Technology, Pantnagar during spring-summer season 2011 and 2012. Pantnagar is situated at 29.5° latitude and 79.3°E longitude and at an altitude of 243.84 meters above the mean sea level in sub-mountainous region of Shivalik hills, known as Tarai. The climate of this place is humid and sub-tropical and frost can be expected from last week of December to end of the January. The experimental materials comprised of thirty five genotypes of potato including twelve commercially released varieties from Central Potato Research Institute (CPRI), Shimla, Himachal Pradesh and one commercially released variety from G. B. Pant University of Agriculture and Technology, Pantnagar and twenty two breeding lines under trial at Central Potato Research Institution, Shimla, Himachal Pradesh ( Table 1). The tubers were planted (60 cm × 20 cm) in a 5.4 m 2 plots in the month of October. The fertilizers were applied @ 160: 100: 120 (NPK kg/ha) in the form of Urea, single super phosphate and muriate of potash, respectively. All other cultivation practices were carried out following the standard cultivation procedure applicable for potato cultivation in this region. The experiment was conducted in randomized block design with each treatment replicated three times and the pooled mean values were used for statistical analysis as per methods suggested by Panse and Sukhatme (1989). The observations were recorded manually on whole population basis for various yield attributing and quality traits (as presented in tables 2 to 7). Five plants per plot were selected randomly for each genotype. The genetic divergences in thirty five genotypes were estimated by Mahalonobis "D 2 " statistics (generalized distance). Treating D 2 as the square of generalized distance, all genotypes were grouped into a number of clusters, according to the methods described by Tocher (Rao, 1952).The relative contribution of different characters to the total D 2 between each pair of genotypes was given a score of 1 to 20 (total number of characters) based on the magnitude of the D 2 value due to each character. RESULTS AND DISCUSSION Analysis of genetic diversity and clustering pattern of potato genotypes: Using statistic proposed by Mahalonobis, D 2 values were calculated and thirty five potato genotypes were grouped into nine clusters for growth character (Table 2) and ten clusters for quality characters (Table 3) Ashoka] fell in the cluster-I showing genetic similarities among themselves. Different growth characters like plant height, average tuber weight, number of tubers per plant, plant dry matter content which had higher contribution to total divergence. Whereas, for quality, minor difference were observed among different characters like ascorbic acid, protein content and total soluble solid (TSS) of tuber for individual contribution towards total divergence. Among the nine clusters for growth characters, cluster-II, IV, VI, VIII and XI consisted of single genotypes, namely Kufri Anand, MP/91-132, MS/92 -542, Pant Selection-1 and Kufri Chipsona-2 respectively, which indicate high genotypic differences among themselves. Similarly, for quality attributes, cluster-IX (J/93-3128) and cluster-X (JX/576) having one genotype each, showing high genetic divergence among these genotypes from the others. Razvy et al. (2007) studied the genetic diversity using Mahalanobis's D -technique for tuber yield and its components viz., Plant Height, Number of Leaves/plant, Fresh Weight/plant, Number of Tubers/plant, Number of Eyes/tuber, Average Tuber Weight of Plant and Tuber weight/plant. The 30 potato genotypes were grouped into six clusters. The maximum diversity was contributed by tuber weight/plant (0.1341) followed by average tuber weight/plant (0.0462), plant height (0.0365), fresh weight/ plant (0.0156) and number of leaves/plant (0.0085). The outcomes of the research work conducted by Razvy et al. (2007) are in good agreement with the results of the present experiment. From the results obtained in this investigation, genotypes in highly divergent clusters having many desirable characters could be taken into account for further use. Intra and inter cluster divergence: The intra and inter cluster divergence (average D 2 values) of all clusters has been presented in the Tables 4 and 5. Intra-cluster average D 2 values ranged from 0.00 to 131.72 for growth characters and for quality characters, it ranged from 0.00 to 9.84. In quality characters, cluster-VIII showed maximum intra D 2 values with two genotypes and clusters-IX, X showed minimum intra D 2 value each with one genotype. The inter-cluster average D 2 values for growth characters was maximum (478.65) between cluster-V and VII and 29.97 between clusters IX and X, respectively. The minimum inter-cluster values for quality characters was 5.05 between cluster homogenous nature between and within groups, respectively (Mandal, 2003). Study of clustering pattern indicated that genotypes related by pedigree fell in either same cluster or in cluster with low inter cluster distances. High inter-cluster distances were the main cause of heterogeneity in composition of clusters. Cluster mean for characters: A perusal of these cluster means for different characters indicated considerable difference between the clusters for all the characters (Tables 6 and 7). For quality characters highest mean values recorded for tuber dry matter content (23.04%) and specific gravity (1.20) in cluster-VIII. Maximum cluster mean value for protein found in cluster-III (2.43%) and for ascorbic acid it was cluster V (22.77 mg/100 g of tuber) but cluster-IX shows highest mean values for total soluble solid content of tuber (6.94%). For growth characters, cluster-II had the highest cluster mean values for number of stem arises from each tuber (4.34) but cluster -IX had maximum value for number of leaves per shoot (10.56), plant height (45.35 cm) at 40 days after planting, plant dry matter content (15.25%) and internodal length (5.48cm). Pandey and Gupta (1995) also observed comparatively higher cluster mean values under various clusters for characters like dry weight, protein content, TSS, internodal length while working with 16 varieties of potatoes. So, it may be seen here that the findings of the present investigation have corroborated well the results of the research work of Pandey and Gupta (1995). Contribution of different characters towards divergence : The per cent contribution for growth characters towards genetic divergence ranged from 0.00 % to 31.76% (Table 8) and for quality characters 15.46% to 24.70% (Table 9). Average tuber weight contributed maximum (31.76%), followed by number of tuber per Number of leaves per shoot 0.00 3. Number of internodes per shoot 9.24 7. Number of stolons per plant 1.01 9. Number of tubers per plant 27.56 10. Tuber yield (t/ha) 1.51 The greater diversity in the present materials was due to these four yield attributing characters and three quality characters, which will offer a good scope for improvement of yield as well as quality through rational selection of parents genotypes for potato breeding. Conclusion Study of clustering pattern keeping in view the pedigree of genotypes indicated that genotypes related by pedigree fell in either same cluster or in cluster with low inter cluster distances. Kufri Jawahar and MS/92-1090 had one parent Kufri Jyoti to be common and were distributed in cluster-IV and cluster-III. Twelve Indian released varieties (viz., Kufri Badshsh, Kufri Anand, Kufri Ashoka, Kufri Bahar, Kufri Puskar, Kufri Chipsona-2, Kufri Jawahar, Kufri Jyoti, Kufri Pukhraj and Kufri Sutlej, Kufri Lalima and Kufri Arun) were distributed in different clusters indicating considerable genetic diversity in material. The findings indicated that use of parents selected from the same cross or from a cross involving a common parent should be avoided in hybridization. High inter-cluster distances were the main cause of heterogeneity in composition of clusters. The highest genetic distance for quality was observed between cluster-IX (J/93-3128) and cluster-X (JX/576) and for growth characters it was found between cluster -V and VII. Average tuber weight, number of tuber/ plant, internodal length and plant dry matter content for yield attributing characters and ascorbic acid content, protein content and TSS of tuber for quality characters, showed maximum contribution towards total divergence among the genotypes. So, these traits will offer a good scope for improvement of yield as well as quality through rational selection of parents genotypes for future potato breeding involving the germplasm investigated in the present research work. The results broadly showed there was no parallelism between geographical and genetic divergence.

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Phytochemical Profiling and Evaluation of Pharmacological Activities of Hypericum scabrum L. Phytochemical investigations of ethyl acetate-soluble part of the aerial part of Hypericum scabrum L. delivered eight pure phenolic compounds 1–8. The pure compounds were identified through physico-chemical, NMR (1D, 2D) and mass spectrometric studies as: 3-8′′-bisapigenin (1), quercetin (2), quercetin-3-O-α-l-arabinofuranoside (3), quercetin-3-O-α-l-rhamnoside (4), quercetin-3-O-β-d-glucopyranoside (5), quercetin-3-O-β-d-galactopyranoside (6), (−)-epicatechin (7), (+)-catechin (8). Total polyphenolic compounds and total flavonoids contents were determined in the extract as 0.107 mg∙mg−1 and 0.023 mg∙mg−1 of the dried extract, respectively. Antioxidant activity using DPPH free radical scavenging assay delivered very strong activity for compounds 2 and 5, 6 and crude extract 10. Protein tyrosine phosphatase 1B (PTP-1B) inhibition experiment of isolated compounds and crude extracts resulted in significant inhibition activity for samples 2, 7a, 8a, 11 and 12 with IC50 values ranging from 1.57 to 2.91 µM. Antimicrobial activity of the pure compounds and extracts produced average results against Staphylococcus aureus, Escherichia coli and Candida albicans strains. From our literature survey, it appears that all pure compounds except 2 were isolated and reported for the first time in H. scabrum. Introduction Medicinal plants have a long history of use in traditional systems of medicines, and are considered the primary sources of important medicines [1]. Flavonoids belong to a group of natural substances with variable phenolic structures and are found in fruit, vegetables, grains, bark, roots, stems, flowers, tea, and wine [2,3]. These natural products were known for their beneficial effects for health as crude plant material or plant extracts long before flavonoids were isolated in pure forms as effective compounds for various pharmacological activities such as antioxidant, antidiabetic, antimicrobial etc. [4,5]. Crude extracts of fruits, herbs, vegetables, cereals, nuts, and other plant materials rich in phenolic compounds are increasingly of interest in the food industry [6]. They are the group of compounds which received considerable attention from the researchers as depicted from the scientific literature. Mostly, they are present in plants as glycosides but can also be isolated in free aglycon form [7,8]. The genus Hypericum of the Hypericaceae consists of over 450 species, with worldwide distribution in warm temperate, subtropical and mountainous tropical regions [9], and a number of Hypericum species are widely used in folk and modern medicine. Hypericum species are medicinal plants known as healing herbs due to their various medicinal properties for the last two hundred years. Hypericin, an aromatic polycyclic anthrone, extracted from Hypericum perforatum L., has been shown to have potent, broad spectrum antimicrobial activity. This compound significantly inhibited the replication of several viruses, including HIV, influenza A, cytomegalovirus (CMV), Herpes simplex 1 and 2 (HSV-1 and HSV-2), and Epstein-Barr virus (EBV. H. perforatum L. oils have also shown notable biological activities including antiviral, wound healing, antioxidant, antimicrobial, antifungal, anxiolytic and anticonvulsant activities [9]. The essential oil compositions and antimicrobial activities of Hypericum have been recently reviewed [9]. Twenty-six components were characterized in the H. scabrum oil, accounting for 95.6% of the oil, which was dominated by α-pinene (44.8%). Other major components in H. scabrum oil were spathulenol (7.1%), verbenone (6.0%), trans-verbenol (3.9%), and γ-muurolene (3.5%). Thus, the H. scabrum oil from Tajikistan is qualitatively similar to essential oils from previous studies in that α-pinene was the major component, but does show some differences such as the absence of thymol, carvacrol, myrcene, or limonene [9]. The essential oil of the plants has been shown to possess anti-microbial and antioxidant activities and thus can be used in cosmetics, food and pharmaceutical industries [10]. H. scabrum aqueous extract showed remarkable anti-hypoxic and antidepressant effects thus, lend pharmacological justification to the use of the plant extract by traditional medicine practitioners. Hypericum scabrum L. growing in Tajikistan had not been systemically investigated yet. Thus, the systematical study on its chemical constituents was undertaken since such an effort is an important route to discover bioactive compounds. The aim of this study was investigation of the chemical composition of the ethyl acetate fraction from the aboveground part of Hypericum scabrum. Further, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity, protein tyrosine phosphatase 1B (PTP-1B) inhibition assay and antimicrobial activity for pure compounds and also for crude extracts were determined. From our literature survey it appears that all the pure compounds except 2 were isolated and reported for the first time in H. scabrum. Total Polyphenolic Compounds Determination of total polyphenolic compounds were performed employing the Folin-Ciocalteau assay using gallic acid as the standard. Total polyphenolic compounds were calculated as equivalent of gallic acid. Seven point calibration curve was obtained after plotting the absorbance of the working standards against their respective concentrations using the Microsoft Excel sheet. The curve was passed through zero and this delivered a curve with R 2 value as 0.9956. The amount of total polyphenolic compounds were calculated as 0.107 mg·mg −1 of the dried extract using the regression equation obtained from the calibration curve. Total Flavonoids contents Aluminium-flavonoids complex formation assay was used for the quantification of contents of total flavonoids in the Hypericum scabrum extract. Seven points calibration curve was obtained with an R 2 value as 0.9971 passing the curve through zero. Quantity of total flavonoids (x) was determined as 0.023 mg·mg −1 of the dried extract using the straight line equation (y = 9.157x). The 1 H-NMR spectrum of 5 exhibited signals from the H-6 and H-8 position of the flavone molecule at δ 6.20 (1H, d, J = 1.9 Hz, H-6) and 6.41 (1H, d, J = 1.9 Hz, H-8), respectively. One broad singlet at δ 7.58 (1H, br. s), two doublets (J = 8.6 Hz) at δ 6.85 and 7.59 ppm indicated the presence of H-2′, H-5′ and H-6′, respectively. The proton of hydroxyl group typical for 5-OH at δ 12.66 (1H, s) and seven broad singlet signals resonance at δ 10.88, 9.76, 9.25, 5.32, 5.10, 4.98, 4.30 ppm indicated the presence of hydroxyl groups in molecule. In addition, 1 H-NMR signal at δ 5.48 was assigned to anomeric proton of sugar moiety. The sugar moiety was deduced to be attached at position C-3, on the basis of HMBC experiment cross-peak between H-1′′ (δ 5.48) and C-3 (δ 133.30). The anomeric configuration of glucose in 5 was concluded to be β-form from value of the coupling constant of H-1′′ (d, J = 7.5 Hz). In 13 C-NMR spectrum of 5, six carbon signals of glucose moiety were observed along with signals of quercetin. The molecular ion observed at m/z 463.5 in the ESI-MS negative mode. The above results support the structure of 5 as quercetin-3-O-β-D-glucopyranoside. Compound 6 was obtained as a yellow amorphous powder, m. p. 245-246 °C. Comparison of the 1 H-and 13 C-NMR data for 5 and 6 indicated a close relationship for the two structures, although 5, 6 were measured in DMSO-d6. The major difference between 5, 6 were the value of Rf (0.48 for 5 and 0.45 mm for 6), the proton multiplicity of sugar moiety and carbon chemical shift values of sugar moiety. Aglycon part of 1 H-NMR spectrum of compound 6 displayed the typical signals for quercetin. The carbon signals were elucidated to be galactose unit. The position of the sugar moiety in quercetin was established from HMBC cross-peak between H-1′′ (δ 5.38) and C-3 (δ 133.45). Thus, the structure of compound 6 was determined to be quercetin-3-O-β-D-galactopyranoside. The compounds 7, 8 were obtained as a pale red powder. It gave one spot on TLC chromatogram (CHCl3/MeOH/H2O, 65:35:5). The compounds 7, 8 on the basis 1 H-and 13 C-NMR were identified as a mixture of epicatechin (75%) and catechin (25%). The quantity of compounds 7 and 8 in sample were determined on the basis of integral intensities of the signals in 1 H-NMR spectrum. Melting point of the mixture of standard epicatechin and catechin with the same ratio delivered the same result as of our purified compound. Pharmacological Activities Antidiabetic, antimicrobial and antioxidant activities of the six pure compounds (1-6) and seven crude extracts (7a, 8a, 9a, 10-13) were determined. Pharmacological activities of the two pure compounds (7) and (8) could not be performed due to their low amounts which is not sufficient for these in vitro assays. Table 1. Seventy percent ethanol extract (sample 12) showed highest antidiabetic activity (PTP-1B inhibition) with an IC50 1.57 µM among the crude extracts and even more than the PTP-1B inhibitor. This significant activity in the initial extract with low activity in the fractionated extracts proved the synergistic relationship among the compounds in the 70% ethanol extract, which weakened after the separation of the synergistically bound analytes. In Vitro Antimicrobial Screening The pure compounds 1-6 and crude extracts 7a, 8a, 9a, 10-13 were evaluated through in vitro analysis for their antimicrobial activities and the results are tabulated in Table 1. Compounds were tested for antimicrobial activity against Staphylococcus aureus ATCC 6538 (Gram positive bacteria), Escherichia coli ATCC 11229 (Gram negative bacteria) and Candida albicans ATCC 10231 (Fungi) strains. All the pure compounds except the 3 and 4 were active against SA delivering highest zones of inhibition 12 for compound 2. Zones of inhibition of compounds 1, 5 and 6 were 5.5, 11 and 11, respectively, against SA. Compounds 3, 4, and 6 showed activity against EC with inhibition zones as: 5.5, 8 and 6.5, respectively. Among the six pure compounds, only compounds 2 and 4 delivered mild results for the inhibition of CA with zones of inhibition as 6.5 and 5.5, respectively. 8a, 9a, 11-13 did not show activity against any microbial strain while the crude extract 7a showed activity with zones of inhibitions 7, 10 and 7 against SA, EC and CA, respectively. Sample 10 was only active against the EC and showed highest activity with zones of inhibitions as 12 among the crude extracts. The obtained results suggest that the tested compounds showed average and mild activity in comparison to the reference drugs Ampicillin and Amphotericin B. Antioxidant Activity The free radical scavenging activity of the fractions was measured in vitro by 2,2-diphenyl-2picrylhydrazyl (DPPH) assay. A lower IC50 value implied a better antioxidant activity of the tested samples. Quercetin (2) and quercetin glycoside (5) were the most active, with an IC50 value of 24.88 μM and 24.78 μM, respectively, which were even better than that of Vitamin C (IC50 30.32 µM). Other glycosides also showed significant antiradical activities. The crude extracts 10 showed potent antiradical activities with IC50 value of 1.19 μg·mL −1 and 8a, 9a, 11-13 showed relatively average antiradical activities with IC50 values 13.86, 24.62, 6.18, 10.81 and 16.29, respectively. The results of the antioxidant activity determined by the DPPH assay procedure using the standard Vitamin C, in pure compounds and crude extract with IC50 values are presented in Table 1. Results showed that ethyl acetate extract (sample 10) delivered highest antioxidant activity with IC50 value of 1.19 μg·mL −1 followed by n-butanol extract (sample 11), which is completely in agreement with the previous literature [7,8]. 1-6 and 7a, 8a, 9a, 10 Our investigations clarified the use of crude extract as well as the flavonoids from H. scabrum for the development of formulations based on enzyme inhibition PTP-1B and antioxidants. Further confirmation of anti-diabetic and antioxidant activities of the extract and pure flavonoids from H. scabrum needs more research efforts, which may be applied in the food, agriculture and medicinal industry as a source of anti-diabetic and anti-oxidative agents. The underlying antimicrobial and antioxidant mechanisms of the flavonoids and crude extract, their contents in plant materials as well as their preparation on a large scale also need to be studied further. General Experimental Procedures The NMR spectra were recorded on a Varian MR-400 (400 MHz for 1 H and 100 MHz for 13 C) and Varian VNMRS-600 (600 MHz for 1 H and 150 MHz for 13 C) spectrometers. Mass spectra were measured in a 2690-ZQ 4000 Water-Alliance LC-MS spectrometer (Applied Biosystems/MDS Sciex Concord, ON, Canada). Melting points were determined using a BUCHI Melting Point B-540 apparatus (Sigma-Aldrich, Darmstadt, Germany). Sephadex LH-20 gel (Amersham Pharmacia Biotech, Stockholm, Sweden) and Silica gel (100-300 mesh, Qingdao Haiyang Chemical Factory, Qingdao, China) were used for column chromatography. The fractions were monitored by TLC, and spots were visualized by heating Silica gel plates sprayed with 5% H2SO4 in EtOH. Preparation of Sample One gram of dried 70% ethanol extract of Hypericum scabrum was reconstituted in 20 mL of 70% methanol and this extract was used for the determination of total polyphenolic compounds and total flavonoids contents. Total Polyphenolic Compounds Polyphenolic compounds were determined by Folin-Ciocalteau method [4] using gallic acid as the reference standard in the concentration range 0.02 mg·mL −1 to 0.2 mg·mL −1 in water. One milliliter standards/extract/blank (water) and 5 mL of diluted FC reagent (1:10 FC reagent to water) were mixed in a test tube. To each test tube 4 mL of Na2CO3 solution (7.5%) was added after 8 minutes and mixed. The test tubes were covered and allowed to react for 2 h at room temperature protecting them from strong light. Absorbances of these test solutions were determined at 740 nm against the prepared blank using UV-visible spectrophotometer. Determination of Total Flavonoids Amount of total flavonoids were estimated using the procedure adopted by Chang et al. 2002 [11]. Quercetin was used standard and six working standard solutions were prepared in the concentration range: 0.01 mg mL −1 to 0.1 mg mL −1 in methanol, for constructing the calibration curve. Then, 0.5 mL of plant extract/standard solution/Blank (methanol) was mixed with 1.5 mL of methanol in a test tube. To the test tube, 0.1 mL of 10% aluminum chloride, 0.1 mL of 1 M potassium acetate and 2.8 mL of distilled water were added and mixed thoroughly after each addition. The solutions were stored for 30 min. at room temperature. The absorbances of the reaction mixtures were measured at 415 nm using the UV-visible spectrophotometer correcting the absorbance with the prepared blank solution. Extraction, Isolation and Purification Procedures The air-dried and powdered aerial part (2.65 kg) was extracted with a mixture of petroleum ether and hexane (1:1) several times to remove oil and fatty acids. The defatted plant material was then extracted with 70% ethanol (3 × 5 L) three times for 48 h at room temperature. The extract was concentrated under vacuum and dried to give the residue (475 g). The dried residue was suspended in water and successively partitioned in hexane, chloroform, ethyl acetate and n-butanol to afford the hexane fraction (45 g), CHCl3 fraction (58 g), EtOAc fraction (67 g), n-butanol fraction (45 g) and H2O fraction (105 g). Part of the ethyl acetate fraction (25 g) was subjected to silica gel column chromatography (100 to 200 mesh, 900 g), starting the elution with petroleum ether/EtOAc (1:1). The polarity was increased gradually to 100% EtOAc and ended with EtOAc/MeOH (4:1) affording 112 (1 to 112) fractions of 150 mL. The fractions were analyzed by silica gel thin layer chromatography (TLC) using the mobiles phases (CHCl3/MeOH/H2O, 65:35:5 and 73:24:4). Similar fractions were combined as: 13 to 48 (A), 50 to 58 (B), 59 to 68 (C), 69 to 80 (D) and 85 to 107 (E) delivering five main fractions. Mass Spectrometry Mass spectrometric analyses of the pure compounds (1 to 8) were performed on a linear ion trap mass spectrometer (4000 Q TRAP) from AB Sciex. Mobile phase consisted of H2O and MeOH with a ratio of 20:80. MS was operated in negative ionization mode with ESI voltage −4500 V and the scanning was performed in the mass range m/z values from 100 to 1000. Two microliters of sample was injected and the capillary temperature was 450 °C. 60.13 (C-6′′). The 1 H-NMR and 13 C-NMR spectral data of compound 6 were consistent with the reported literature [16]. Protein Tyrosine Phosphatase 1B (PTP-1B) Inhibition Assay The individual compounds 1 to 6 and crude extracts 7a, 8a, 9a, 10 to 13 were tested for the measurement of PTP-1B activity using pNPP (p-nitrophenyl phosphate disodium salt) as substrate. Compounds were pre-incubated with the enzyme at room temperature for 5 min. Then, 178 µL of buffer solution (20 mM HEPES, 150 mM NaCl, 1 mM EDTA) was added in 96-well plates. One microliter of PTP-1B protein solution (0.115 mg·mL −1 ) was added to the buffer solution. After that, 1 µL of test and positive control sample were added. Thereafter, 20 µL of the substrate pNPP (35 mM) was added and mixed for 10 min. The plate was incubated in the dark for 30 min and the reaction was terminated by adding 10 µL of 3 M NaOH. The absorbance was then determined at 405 nm wavelength. The system does not contain the enzyme solution in a blank, using a micro plate reader Spectra Max MD5 (USA Molecular Devices) absorption was measured at 405 nm. Inhibition (%) = [(OD405 − OD405 blank)/OD405 blank] × 100. IC50 was calculated from the percentage inhibition values. Antimicrobial Activity Antimicrobial activity of compounds 1 to 6 and crude extracts 7a, 8a, 9a; 10 to 13 were measured using the agar well diffusion method. Bacterial and fungal pathogens such as S. aureus (ATCC 6538), E. coli (ATCC 11229) and C. albicans (ATCC 10231) were used as indicator strains for this analysis. These microorganisms were aseptically inoculated into appropriate liquid media and incubated at 37 °C. After 16 h, the cells were centrifuged at 6000 rpm for 10 min and then suspended in sterile water. The different cells (1 mL) were added to appropriate agar media (100 mL) prior to plating, and the wells were made using an agar well borer. To these wells, different concentrations of compounds 1 to 6 and the extracts 7a, 8a, 9a; 10 to 13 were added and subsequently incubated at 37 °C for 24 h. Zones of inhibition were estimated by measuring the diameter of the microbial growth inhibition zones. Values were averaged from three independent experiments. DPPH Radical Scavenging Assay Radical scavenging assay was determined by a micro plate spectrophotometric method based on the reduction of DPPH according to the previous report [20]. Various dilutions of pure compounds and crude extracts in the concentration range 0.0625 to 1 mM and 0.0625 to 1 mg·mL −1 , respectively were made. Briefly DPPH solution (100 μL) and pure compound or crude extract in DMSO (100 μL) were added to each well of the micro plate and mixed. The mixture was shaken vigorously and left to stand at 37 °C for 30 min in dark. The absorbance of the solution was then measured at wavelength 515 nm using a micro plate spectrophotometer. Inhibition (%) of free radical (DPPH) was determined as [(Acontrol − Asample)/Acontrol] × 100, where: Acontrol is the absorbance of the control sample containing all reagents except the test sample and Asample is the absorbance of the test sample (pure compound or crude extract). Vitamin C was used as the positive control and tests were carried out in triplicate. The IC50 values were determined for all tested flavonoids, extract and control standard antioxidant. The IC50 value was defined as the concentration (in μM and μg·mL −1 ) of pure compound and extracts that inhibits the formation of radicals by 50%. Conclusions The study confirmed the presence of bioactive compounds like quercetin glycosides, bisapigenin, catechin and epicatechin in the aerial part of H. scabrum in sufficient amounts to be isolated employing routinely applied chromatographic procedures. Seventy percent ethanol extract delivered best results for PTP-1B assay with IC50 1.57 µM among the crude extracts while quercetin showed significant inhibition activity (IC50 2.19 µM) for this enzyme. Quercetin (2) and quercetin glycoside (5) yielded strong DPPH radical scavenging activity among the isolated compounds. Our study suggests the use of aerial part of H. scabrum for antioxidant and antidiabetic formulations based on their proven activities. From our literature survey, it appears that all the pure compounds except 2 were isolated and reported for the first time in H. scabrum.

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2016-05-04T20:20:58.661Z

2015-04-28T00:00:00.000Z

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Antimicrobial and antioxidant flavonoids from the leaves of Oncoba spinosa Forssk. (Salicaceae) Background Naturally occurring flavonoids have been reported to possess various pharmacological properties. The aim of this study was to evaluate the antimicrobial and antioxidant activities of the MeOH extract and flavonoids from the leaves of Oncoba spinosa, a plant used for the treatment of syphilis, wounds and sexual impotence. Methods The plant extract was prepared by maceration in methanol and sequentially fractionated by column chromatography. The structures of isolated compounds were elucidated on the basis of spectral studies and comparison with published data. The MeOH extract and its isolated compounds were evaluated for their antibacterial and antifungal activities by broth microdilution method. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) and trolox equivalent antioxidant capacity (TEAC) assays were used to detect the antioxidant activity. The samples were tested spectrophotometrically for their hemolytic properties against human red blood cells. Results The fractionation of the MeOH extract afforded five known flavonoids including kaempferol (1), quercetin (2), apigenin-7-O-β-D-glucuronopyranoside (3), quercetin 3-O-β-D-galactopyranoside (4) and quercetin 3-O-α-L-rhamnopyranosyl (1 → 6) β-D-glucopyranoside (5). The MeOH extract displayed weak to moderate antimicrobial activities (MIC = 256–2048 μg/ml). Quercetin 3-O-α-L-rhamnopyranosyl (1 → 6) β-D-glucopyranoside (5) and quercetin (2) were respectively the most active compounds against bacteria (MIC = 8–64 μg/ml) and fungi (MIC = 64 – 128 μg/ml). These tested samples also showed high radical-scavenging activities (EC50 = 5.08 – 70.56 μg/ml) and gallic acid equivalent antioxidant capacities (TEAC = 53.76 – 89.86 μg/ml) when compared with vitamin C (EC50 = 4.72 μg/ml). The MeOH extract and compounds 2–5 were non-toxic to human red blood cells indicating their high selectivity to be used as antimicrobial and antioxidant drugs. Conclusion The MeOH extract of O. spinosa as well as compounds 2 – 5 could be a potential source of natural antimicrobial and antioxidant products. Background Infectious diseases are among the main cause of morbidity and mortality worldwide, with HIV, tuberculosis and malaria being the most involved. Despite the progress made in the understanding of microorganisms and their control in industrialized nations, incidents due to drug resistant microorganisms and the emergence of hitherto unknown disease-causing microbes, pose enormous public health concerns [1]. Furthermore, the development of synthetic drugs has slowed down as a result of drug resistance [2]. Consequently, this has created a new renewed interest in the search for new drugs in order to combat resistance. The need for new, effective and affordable drugs to treat microbial diseases in the developing world is one of the major issues facing global health today. Plants have been used as a source of new medicinal compounds throughout history and continue to serve as the basis for many of the pharmaceuticals used today [3]. In recent decades, many studies have been carried out on different plant species to discover compounds of possible interest for medicinal application against oxidative stress, fungal and bacterial infections. Among these studies, several have focused on the biological and phytochemical properties of different species of the family Flacourtiaceae [4][5][6]. Oncoba spinosa Forssk. belonging to the family Flacourtiaceae (Salicaceae sensu lato) is a small tree of about 13 m high which grows under conditions of higher rainfall, of deciduous, secondary and fringing forest from Senegal to West Cameroon, and widely distributed in tropical Africa and Arabia [7]. The plant is traditionally reputed for its medicinal potential particularly in southwest of Nigeria for the treatment of diabetes and cancer [6]. In many African countries, the leaves and roots are used for urethral discharges, infertility [8], epilepsy [9], dysentery and bladder conditions [10]. The α-glucosidase inhibitory, radical scavenging and cytotoxicity activities of the aqueous and chloroform extracts of the leaves of O. spinosa were reported [6]. The methanol extract of the fruits of this plant collected in Yemen demonstrated antimicrobial, anticancer and antioxidant activities [5]. Previous phytochemical studies on the genus Oncoba afforded three tetracyclic triterpenes from the species O. mannii [4]. Phytochemical screening of O. spinosa leaves revealed the presence of anthraquinones, alkaloids, phenols, sterols, tannins, carbohydrates and flavonoids [6]. Experimental Melting points were recorded with a Reichert microscope and are uncorrected. 1 H NMR (500 MHz) and 13 C NMR (125 MHz) were recorded at room temperature in CD 3 OD or (CD 3 ) 2 SO, on a Bruker Avance DRX-500 spectrometer. Chemical shifts (δ) are reported in parts per million (ppm) with the solvent signals as reference relative to TMS (δ = 0) as internal standard, while the coupling constants (J values) are given in Hertz (Hz). COSY, ROESY, TOCSY, HSQC and HMBC experiments were recorded with gradient enhancements using sine shape gradient pulses. The IR spectra were recorded with a Shimadzu FT-IR-8400S spectrophotometer. ESI-MS experiments were performed using a Micromass Q-TOF micro instrument (Manchester, UK) with an electrospray source. Column chromatography was run on Merck silica gel 60 (70-230 mesh) and gel permeation on Sephadex LH-20 while TLC was carried out on silica gel GF 254 pre-coated plates with detection accomplished by spraying with 50% H 2 SO 4 followed by heating at 100°C, or by visualizing with an UV lamp at 254 and 365 nm. Plant material The leaves of O. spinosa Forssk. were collected at Dschang, West Region, Cameroon, in May 2007. Authentication was done at the Cameroon National Herbarium, Yaoundé, where the voucher specimen (No. 21975 HNC) is deposited. Antimicrobial assay Bacterial and fungal strains The studied microorganisms were both reference (from the American Type Culture Collection) and clinical (from Pasteur Institute Paris, France) strains of Enterobacter aerogenes, Escherichia coli, Klebsiella pneumoniae, Candida albicans, and Cryptococcus neoformans. Also, included were two clinical isolates of Candida parapsilosis and Staphylococcus aureus collected from Pasteur Centre (Yaoundé-Cameroon). The bacterial and fungal species were grown at 37°C and maintained on nutrient agar (NA, Conda, Madrid, Spain) and Sabouraud Dextrose Agar (SDA, Conda) slants respectively. Preparation of microbial inoculum The inocula of yeasts and bacteria were prepared from overnight cultures by picking numerous colonies and suspending them in sterile saline (NaCl) solution (0.90%). Absorbance was read at 530 nm for yeasts or at 600 nm for bacteria and adjusted with the saline solution to match that of a 0.50 McFarland standard solution. From the prepared microbial solutions, other dilutions with saline solution were prepared to give a final concentration of 10 6 yeast cells/ml and 10 6 CFU/ml for bacteria [14,20]. Antimicrobial assay The antimicrobial activity was investigated by determining the minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs) and minimum fungicidal concentrations (MFCs). MICs were determined by broth micro dilution [12,21]. Stock solutions of the tested samples were prepared in 10% v/v aqueous dimethylsulfoxide (DMSO) solution (Fisher chemicals, Strasbourg, France) at concentration of 4096 μg/ml. This was two-fold serially diluted in Mueller-Hinton Broth (MHB) for bacteria and Sabouraud Dextrose Broth (SDB) for fungi to obtain a concentration range of 2048 to 0.25 μg/ml. For every experiment, a sterility check (10% aqueous DMSO and medium), negative control (10% aqueous DMSO, medium and inoculum) and positive control (10% aqueous DMSO, medium, inoculum and water-soluble antibiotics) were included. One hundred microliters of each concentration was introduced into a well (96-wells microplate) containing 90 μl of SDB or MHB and 10 μl of inoculum was added to obtain a final concentration range of 4096 to 0.125 μg/ ml. The plates were covered with a sterile lid, and incubated on the shaker at 37°C for 24 h (bacteria) and 48 h (yeasts). MICs were assessed visually after the corresponding incubation period and were taken as the lowest sample concentration at which there was no growth or virtually no growth. The assay was repeated thrice. For the minimum microbicidal concentration (MMC) determination, 10 μl aliquots from each well that showed no growth of microorganism were plated on Mueller-Hinton Agar or Sabouraud Dextrose Agar and incubated at 37°C for 24 h (bacteria) and 48 h (yeasts). The lowest concentration that yielded no growth after the sub-culturing was taken as the MBCs or MFCs. Chloramphenicol (Sigma-Aldrich, Steinheim, Germany) for bacteria and nystatin (Sigma-Aldrich, Steinheim, Germany) for yeasts were used as positive controls. Antioxidant assay DPPH free radical scavenging assay The free radical scavenging activity of the MeOH extract as well as some of its isolated compounds was evaluated according to described methods [22,23] with slight modifications. Briefly, the test samples, prior dissolved in DMSO (SIGMA) beforehand, were mixed with a 20 mg/l 2,2-diphenyl-1-picryl-hydrazyl (DPPH) methanol solution, to give final concentrations of 10, 50, 100, 500 and 1000 μg/ml. After 30 min at room temperature, the absorbance values were measured at 517 nm and converted into percentage of antioxidant activity. L-ascorbic acid was used as a standard control. The percentage of decolouration of DPPH (%) was calculated as follows: The percentage of decolouration of DPPH (%) was plotted against the test sample. Also, the percentage of decolouration of DPPH was converted in probits. The probit values were plotted against the logarithmic values of concentrations of the test samples and a linear regression curve was established in order to calculate the EC 50 (μg/ml), which is the amount of sample necessary to decrease by 50% the absorbance of DPPH [11]. All the analyses were carried out in triplicate. Hemolytic assay Whole blood (10 ml) from a healthy man was collected into a conical tube containing heparin as an anticoagulant (blood group O was used). Authorization for the collection of blood was obtained from the Medical and Ethical Committee (in Yaoundé-Cameroon). The written informed consent for participation in the study was obtained from a parent of 39 years old. Erythrocytes were harvested by centrifugation for 10 min at 1,000 × g and room temperature and washed three times in PBS solution. The top layer (plasma) and the next, milky layer (buffy coat with a layer of platelets on top of it) were then carefully aspirated and discarded. The cell pellet was resuspended in 10 ml of PBS solution and mixed by gentle aspiration with a Pasteur pipette. This cell suspension was used immediately. For the normal human red blood cells, which are in suspension, the cytotoxicity was evaluated as previously described [26]. MeOH extract (at concentrations ranging from 64 to 2048 μg/ml) and compounds 2-5 (32 to 512 μg/ml), were incubated with an equal volume of 1% human red blood cells in phosphate buffered saline (10 mM PBS, pH 7.4) at 37°C for 1 h. Ampicillin and chloramphenicol were tested simultaneously. Non-hemolytic and 100% hemolytic controls were the buffer alone and the buffer containing 1% Triton X-100, respectively. Cell lysis was monitored by measuring the release of hemoglobin at 595 nm with a spectrophotometer (Thermo Scientific, USA). Percent hemolysis was calculated as follows: A595 of sample treated with compound-A595 of sample treated with buffer ð Þ ½ A595 of sample treated with Triton  -100 -A595 of sample treated with buffer ð Þ ½  100 Statistical analysis Statistical analysis was carried out using Statistical Package for Social Science (SPSS, version 12.0). The experimental results were expressed as the mean ± Standard Deviation (SD). Group comparisons were performed using One Way ANOVA followed by Waller-Duncan Post Hoc test. A p value of 0.05 was considered statistically significant. Antimicrobial activity The MeOH extract and four of its isolated compounds (2)(3)(4)(5) were examined in vitro against bacterial and fungal species and the results are depicted in Table 1. Kaempferol (1), obtained in small amount, was not tested. The MeOH extract and compounds 2, 3, 4, 5 showed selective activities; their inhibitory effects being noted respectively on 7/7 (100%), 7/7 (100%), 4/7 (57.14%), 7/7 (100%) and 4/7 (57.14%) of the studied microorganisms. Klebsiella pneumoniae ATCC11296 and Enterobacter aerogenes ATCC13048 were the most sensitive bacteria while the most sensitive fungi were Candida parapsilosis and Cryptococcus neoformans IP 90526. The MeOH extract showed only fungistatic activity against yeast strains while the killing effects of many tested samples could be expected on the sensitive strains at the MMC values not more than twofold their corresponding MICs [27]. The MeOH extract, compounds 2 and 4 were found to be active against all the microbial strains. Compound 5 was more active than 4 and the later than 2, against all the bacterial strains. The reverse observations were noted with the fungal strains with compound 2 more active than 4, and compound 5 being inactive. The three compounds have the same aglycon moiety. Therefore, the sugar moieties at position 3 in 4 and 5 should be responsible for the difference in the observed activity. The lowest MIC value for these tried compounds (8 μg/ml) was recorded with compound 5 on K. pneumoniae ATCC11296. This compound displayed the largest antibacterial activity. The antibacterial and antifungal activities of the tried samples were in some cases equal or more important than those of two reference drugs chloramphenicol (MIC = 16 -64 μg/ml) and nystatin (MIC = 128 -256 μg/ml), highlighting their good antimicrobial potency. Taking into account the medical importance of the tested microorganisms, this result can be considered as promising in the perspective of new antimicrobial drugs development. The present study showed antimicrobial activity of flavonoids (phenolic compounds) and MeOH extract from O. spinosa leaves against the microorganisms including bacterial and fungal species. Compounds 1-5 were previously obtained from other sources, but they are isolated here for the first time from the genus Oncoba. In addition, this is the first time that secondary metabolites are isolated from O. spinosa. Flavonoids and their glycosides have attracted considerable interest because of a large variety of biological activities, such as antioxidant [28], antiplasmodial [29], cytotoxic [30], anti-inflammatory [31], antidiabetic [32] and antimicrobial [19,33]. However, no study has been reported on the antimicrobial activity of the compounds 2-5 and MeOH extract from the leaves of O. spinosa against these types of pathogenic strains. The mechanism of the active compounds (2)(3)(4)(5) is still to be studied; nevertheless, their activity is probably due to their ability to complex with extracellular and soluble proteins and to complex with bacterial cell walls. More lipophilic flavonoids may also disrupt microbial membranes [34]. In addition to the flavonoid compounds, alkaloids, tannins, sterols, and anthraquinones were previously detected in the 70% aqueous ethanol, hexane and chloroform extracts from O. spinosa [6]. Phenolic compounds such as flavonoids are known to be potential antioxidant due to their ability to scavenge free radicals and active oxygen species such as singlet oxygen, superoxide anion radical and hydroxyl radicals [35,36]. Therefore, the presence of such compounds could be responsible for the antioxidant activity found in the MeOH extract. To the best of our knowledge, this is the first systematic screening for the Hemolytic activity Human red blood cells provide a handy tool for toxicity studies of compounds, because they are readily available, their membrane properties are well known, and their lysis is easy to monitor by measuring the release of hemoglobin [26]. To investigate the potential use of MeOH extract and compounds 2-5, the cellular toxicity also has to be determined. In this study, none of the tested samples showed hemolytic activities against human red blood cells at concentrations up to 512 μg/ml and 2048 μg/ml for isolated compounds and MeOH extract respectively (results not shown). This finding highlights the fact that the observed biological efficacy is not due to cytotoxicity. Conclusion The phytochemical study of the MeOH extract of O. spinosa leaves afforded five known flavonoids including kaempferol (1), quercetin (2), apigenin-7-O-β-D-glucuronopyranoside (3), quercetin 3-O-β-D-galactopyranoside (4) and quercetin 3-O-α-L-rhamnopyranosyl (1 → 6) β-D-glucopyranoside (5). The MeOH extract and compounds 2-5 possess significant antimicrobial and antioxidant activities with no toxicity to human red blood cells. They may be used as phytomedicines at low cost and easily affordable by the target population with caution of clinical studies currently going on in our Laboratory. Figure 3 Gallic acid equivalent antioxidant capacity (TEAC; μg/ml) of tested samples. Bars represent the mean ± SD of three independent experiments carried out in triplicate. Letters a-e indicate significant differences between samples according to one way ANOVA and Waller Duncan test; p < 0.05.

v3-fos

2019-03-30T13:04:02.655Z

2015-02-19T00:00:00.000Z

85572807

s2

Evaluation of some soil test methods in acid soils for available phosphorus for soybean of Imphal East District, Manipur (India) Evaluation of nutrient status in soil is important for nutritional, environmental and economical aspects. Phosphorus is a very important nutrient for leguminous crops. In order to evaluate the phosphorus availability, six soil test methods were carried out for predicting response of soybean to phosphorus application on twenty surface soils (0 15 cm) of Imphal East District, Manipur were studied. The suitability of these extractants was in the descending order: Bray P1 > Mehlich P1 > Bray P2 > Troug P > Olsen P > Morgan P. Bray’s P1 extractable phosphorus showed the highest and positive correlations with phosphorus content (control), phosphorus uptake (control), Bray’s percent yield and uptake. Therefore, this extractant may be used as an index of available phosphorus for soybean (JS-335) grown on acid soils of Imphal East District, Manipur, the critical level being 13 ppm (mg kg). The critical limit of phosphorus concentration in plant at 40 days of planting was 0.22%. INTRODUCTION Phosphorus deficiency in plants has been reported from various parts of India. The responses of oilseeds to applied phosphorus, particularly soybean had been reported by many workers. Soil testing allows one to access a soil's current nutrient status and decide on appropriate fertilizer rates to maximize crop production. But no information is available on the status of available phosphorus in these soils. The knowledge of relationship between soil test methods and soil phosphorus fractions help in selecting a suitable soil test method for a particular soil to grow the crop profitable. The most appropriate soil test method for a soil would be one which extracts predominantly that fraction of phosphorus playing the major role towards plant uptake. Therefore, the present investigation was planned to select the most promising extractant which may predict the availability of Mehlich (1978) phosphorus to soybean (JS-335) grown in acid soils. MATERIALS AND METHODS Twenty soils samples (0 -15 cm) were collected from various cultivated fields of Imphal East District, Manipur. The physical and chemical characteristics of these soils were determined by standard methods (Jackson, 1973) and reported in Table 1. Four kilograms of soil was filled in earthen pots and phosphorus was applied at 0, 60 and 90 kg P 2 O 5 ha -1 through single superphosphate. The treatments were replicated thrice in a completely randomized design. A basal dose of 20:60 kg NK ha -1 was applied in the form of urea, and muriate of potash in each pot. Soybean (var.JS-335) seeds were sown and thinned to six plants after ten days of sowing. The pots were irrigated with distilled water as and when required. The crop was harvested 40 days after germination. The plant samples were washed in water and dried in oven at 65°C for 48 h and the dry matter yield was recorded. The samples were then powdered and requisite quantities of the same were digested in nitricperchloric acid mixture. Phosphorus was determined by using vanadomolbdophosphoric acid reagent. To test the suitability, six soil test methods were used ( Table 2). The soil samples were shaken for two minutes with soil to solution ratio of 1:10. Extractable phosphorus was determined spectrophotometrically. Extractable phosphorus The available P obtained with different chemical extractants revealed that the varying amounts of P extracted from different soils depended on the nature of extractant (Table 2). Based on the mean values of extractable P, the extractants were arranged in the following decreasing order: BrayP 2 > Truog P > BrayP 1 > Olsen P > Mehlich P 1 > Morgan P. This was in conformity with the findings reported by Jaggi et al. (1990) and Ravindra and Ananthanarayana (1999). The higher solubility in Bray P 2 may be due to its relatively higher strength of acidity and complexing of Al 3+ and Fe 3+ ions with Fions and consequent release of P adsorbed by these trivalent ions (Ballard and Fiskell, 1974). The lowest quantity of P was extracted by Morgan reagent. This might be due to the presence of weekly buffered salt solution such as acetic acid-sodium acetate solution. Similar finding was also reported by Hesse (1971). Correlation between different chemical extractants The data on the simple correlation coefficients among the different methods of phosphorus extractants (Table 3) revealed that the extractants were closely interrelated. Such a close relationship between the different extractants suggested that these extractants were able to extract more or less the same forms of phosphorus indicating the existence of dynamic equilibrium among different forms of phosphorus but relatively to different degrees. Significant correlation between extractable phosphorus by all the procedures indicates that they extract similar pool of phosphorus in the soils but with varying degree. Similarly, Bhattacharya et al. (1990) and Jaggi (1991) reported that the transformation and availability of P in the soils was dependent on its various forms. Correlations of soil P with yield and uptake The simple correlation coefficients of coefficients between soil test results and yield were presented in Table 4. The data revealed that different soils significantly affected the dry matter production of soybean and also P uptake by soybean. The relative yield ranged from 46.70 to 95.32%, P content (control) 0.16 to 0.45%, and average P uptake from 6.81 to 24.67 mg pot -1 . Out of the six extractants, four were found to be significantly correlated with Bray's percent yield and five were also significantly correlated with P uptake by the crop. Among the extractants used for this investigation, Bray P 1 showed higher degree of co-efficient of correlation with Bray's percent yield as well as phosphorus uptake by the soybean, with 'r' values of 0.670** and 0.709**, respectively than the other extractants, that is, Bray P 1 > Mehlich P 1 > Bray P 2 > Troug P > Bray P 1 > Morgan P. So, Bray P 1 was the most suitable test for determining available soil phosphorus in the studied soil as the degree of co-efficient of correlation between the quantities of P extracted by this extractant and yield parameters were of higher order. Base on the simple correlation, it can be suggested that for acid soils of Imphal East District, Manipur, Bray-P 1 extractable phosphorus is found to be the suitable test for evaluation of available phosphorus in the soils for growing soybean plants. Critical level of phosphorus It was observed that the critical level of phosphorus in the soils for growing of soybean plants varied with the methods of phosphorus extraction. According to graphical procedure of Cate and Nelson (1965), the critical level of soils ranged from 13 to 20.2 ppm phosphorus depending upon the methods of phosphorus extraction. A high degree of correlation between Bray P 1 reagent extractable P and Bray's percent yield against Bray P 1 reagent extractable P was found to be 13 ppm (Figure 1) as the critical limit of available P in these soils for demarcating the phosphorus responsive soil from the non-responsive ones. Similar observations were also reported by Tandon (1987), Gupta and Vyas (1993), Mullen et al. (2009) andLaBarge (2013). The result revealed that the critical level of phosphorus concentration in soybean plant was found to be 0.22% as shown in scatter diagram (Figure 2) partitioning the dimensional percentage yield versus phosphorus content in 40 days old soybean plants scattered into two groups. Similar observations were also reported by Ali et al. (2006) and Mallarino (2010).

v3-fos

2019-03-28T13:42:42.877Z

2015-01-01T00:00:00.000Z

54761877

s2

Agrobacterium-mediated Genetic Transformation in Cucumber (var. Shital) as Influenced by Explant, Inoculation Time and Co-cultivation Period To study genetic transformation for abiotic stress resistance in cucumber (var. Shital), leaf, nodal and internodal calli were subjected to Agrobacterium tumefaciens mediated transformation using LBA4404 strain containing CIPK sense gene. Transformation ability was examined by histochemical assay of GUS reporter gene in survived calli. Conspicuous GUS positive (blue colour) region were detected in callus tissue. There were 3 factors in this investigation. Factor A consisted of three types of explants viz. leaf, nodal and internodal callus, factor B consisted of two durations of inoculation time viz. 3 and 5 min and factor C consisted of two co-cultivation periods viz. 24 and 48 hours. The highest GUS positive transgenic callus obtained from leaf explants (3.30) and internodal explants gave the lowest number (1.79) of GUS positive transformants. Inoculation time is an important factor in transformation experiment mediated by A. tumefaciens. Transformation ability was increased with increase of inoculation time. Percentage of survived callus was higher (53.68 %) when the calli were immersed for 5 min in bacterial suspension. Both number and percentage of GUS positive callus were higher (3.17 and 52.87 %, respectively) when they were kept in bacterial suspension for higher time (5 min) and lower (2.15 and 35.88 %, respectively) when calli were soaked in bacterial suspension for minimum time (3 min). Introduction Cucumber (Cucumis sativus L.) (2n = 14), a member of the family Cucurbitaceae, is one of the oldest vegetable crop supposed to be originate in India, between the Bay of Bengal and the Himalayas (Peirce, 1987) [14]. Cucumis sativus L. is a cucumber species which has commercial importance (Nonnecki, 1989) [13]. The total area and production of cucumber in Bangladesh during 2003 -04 were 13925 ha and 25215 mt, respectively (BBS, 2005) [2]. The production has increased upto 32000 mt during the year 2006-'07 (BBS, 2008) [1]. The data indicates that total production has increased during the last few years with increased demand of cucumber. However, average yields of cucumber during 2002-'03, 2003-'04 and 2004-'05 were 4.45, 4.45 and 4.37 mt/ha, respectively (BBS, 2006) [3] which indicate that the yield has declined slightly. Yield of cucumber is very low in our country compared to leading cucumber producing countries like China (12.24 t/ha), former USSR (7.57 t/ha), Japan (44.23 t/ha), USA (11.06 t/ha), Turkey (16.07 t/ha), Netherlands (192.50 t/ha), Spain (30.00 t/ha) (Nonnecki, 1989) [13]. Abiotic stresses include drought, salinity, extreme temperatures, chemical toxicity and oxidative stress are serious threats to agriculture and the natural states of environment (Wang et al., 2003) [17]. Without these, the population of our country is increasing day by day but the land is decreasing. Therefore, we need to utilize the lands which are not under cultivation at present, such as, coastal zone which have high saline properties. That's why we need millions of healthy cucumber seedlings in a short period of time. In crop improvement, genetic transformation offers the ability to introduce new characters into a plant cultivar without altering its existing traits (Gardner, 1993) [8]. In all transformation experiments, specific reporter gene and one or more selectable marker gene are required to be introduced into the plant cell prior to the integration of gene (s) of interest. In this case, GUS-A (β-glucoronidase) gene and neomycin phosphotransferase II termed as npt II (kanamycin resistant) gene have been used as reporter and selectable marker gene respectively. This reporter gene can be recognized in plant tissue with the help of selectable agents, confirming transformation of the plant tissue (through histochemical GUS assay). So, in this way, one can understand that the plant tissue subjected for transformation has really been transformed or not (Gardner, 1993) [8]. Agrobacterium-mediated Genetic Transformation in Cucumber (var. Shital) as Influenced by Explant, Inoculation Time and Co-cultivation Period The use of genetic transformation may allow the production of abiotic stress resistant plants in a significantly shorter period of time than using conventional breeding, especially if several traits are introduced at the same time. The geographic spread of cucumber production may contribute to food, nutrition security and poverty alleviation in Bangladesh. From the above background information it was revealed that tissue culture and genetic transformation of cucumber depend on several factors. So the present investigation was conducted to see the effect of explant, inoculation time and co-cultivation period on genetic transformation of cucumber Plant material Leaf, nodal and internodal calli of variety Shital were used in present investigation. Genetic transformation material Agrobacterium strain, plasmid and gene Genetically engineered A. tumefaciens strain LBA4404 was used for infection in the pre-cultured explants. The strain is being maintained at the Biotechnology lab. under Bangladesh Agricultural University. This strain contains plasmid pBl121 of 14 kDa (binary vector). This binary vector contains following genes within the right border (RB) and left border (LB) region of the constructi. The uidA gene (Jefferson, 1986) [9] encoding GUS (β-glucuronidase), driven by CaMV promoter and NOS terminator. This reporter gene can be used to assess the efficiency of transformation. ii. The npt II gene encoding neomycin phosphotransferase II (npt II) conferring kanamycin resistance, driven by NOS promoter and NOS terminator. iii. The CIPK sense gene encoding calcineurin B-like protein conferring abiotic stress tolerance. Calcineurin B -like proteins (CIPK) Calcineurin (Cn) is a unique Ca 2+ dependent serine/threonine protein phosphatase (PP2B) of cytosol, which plays an important role in the coupling of Ca 2+ signals to stress responses. Using degenerate primers from the conserved domains and by library screening a full-length cDNA (CIPK, 972 bp) was isolated from pea (accession no: AY883569). Plants respond to adverse environments by initiation a series of signaling processes that often involves diverse protein kinases, including calcineurin B-like protein interacting protein kinases (CIPKs). Putative CIPK genes (O s CIPK01 -O s CIPK30) survived for their transcriptional responses to various abiotic stresses, like drought, salinity, cold, polyethylene glycol and abscisic acid treatment. To prove that some of these stress-responsive CIPK genes are potentially useful for stress-tolerance improvement, three CIPK genes (CIPK 03, CIPK 12, CIPK 15) were over expressed in Japonica rice. Transgenic plants over expressing the transgenes CIPK 03, CIPK 12, CIPK 15 showed significantly improved tolerance to cold, drought and salt stress, respectively. Under cold and drought stresses, CIPK 03, CIPK 12, over expressing transgenic plants accumulated significantly higher content of proline and soluble sugars. and putative proline synthetase and transporter genes had significantly higher expression level in the transgenic plants, against different stresses (Mahajan and Tuteja, 2005) [10]. Methods Treatments: There were 3 factors in this experiment. Factor A consisted of three types of callus, factor B consisted of two inoculation times and factor C consisted of two co-cultivation periods. A. Explants: Leaf, nodal and Internodal callus B. Infection time: 3 and 5 minutes C. Co-cultivation period: 24 and 48 hours Total no. of treatments were 12 (3x2x2). Each treatment consisted of 4 vials and replicated three times. Design: Factorial in Completely Randomized Design (CRD) Media used Media used in the present study were as follows. A. For callus induction For induction and maintenance of callus, MS (Murashige and Skoog, 1962) [11] medium supplemented with different concentrations and combinations of BAP and NAA were used. B. For Agrobacterium culture Two types of culture media, namely, YMB (Yeast Extract Mannitol Broth) medium and LB (Luria Broth) medium were used with kanamycin as antibiotic to grow the strain of genetically engineered Agrobacterium tumefaciens. YMB medium was used for Agrobacterium maintenance and LB medium was used as Agrobacterium working culture medium for transformation work. C. For co-cultivation MS media without growth hormones were used for co-cultivation. D. For washing explants after co-cultivation Cefotaxime (200 mg/l) was used for washing the explants after co-cultivation. E. For Post-cultivation and regeneration MS media supplemented with 2 mg/l BAP, 1 mg/l NAA and 100 mg/l cefotaxime were used for this purpose. High selection medium: MS media supplemented with 2 mg/l BAP, 1 mg/l NAA, 30 mg/l kanamycin and 100 mg/l cefotaxime were used. Preparation of Culture Media Preparation of MS medium The MS media used in this investigation were fortified with different concentrations and combinations of required auxin and cytokinin. They were added in the medium before the adjustment of P H of the solution. Preparation of Agrobacterium culture medium YMB medium was used for the maintenance of Agrobacterium strain LBA4404. The composition (Begam, 2007) [4] of the medium given below- The P H was adjusted to 7.0-7.2 before adding agar at 1.5%. After autoclaving the medium was cooled to 50-55 0 C and kanamycin was added at a rate of 0.05 mg/l and separated in petridishes. Preparation of LB (Luria Broth) medium To prepare one liter (1000 ml) of LB medium, the following steps were followed-15.5 g of LB (Luria Broth) powder was taken into a 2-liter beaker on a magnetic stirrer. i. 400-500 ml of distilled water was poured in the beaker to dissolve the powder ii. After dissolution the medium was transferred to a 1 liter measuring cylinder or volumetric flask and volume was made up to the mark with distilled water. iii. Then the p H of the medium was adjusted to 7.0-7.2 with 0.1 N NaOH iv. The medium was transferred back to stirred beaker to allow full mixing. v. Batched (25-50 ml) of medium was transferred to clean 250 ml conical flasks and plugged with non-absorbent cotton wool. The tops were covered with aluminum foil. Preparation of GUS assay solution The GUS straining solution is composed of the following chemicals. For the preparation of 10 ml GUS straining solution, the following steps were followed- All necessary glasswares were autoclaved.  The 8.89 mg X-gluc was weighted.  Few drops of DMSO (Dimethyl Sulphoxide) were taken in a beaker and X-gluc was added.  Gently shaken until all the X-gluc was dissolve.  200 µl of chloramphenicol was added into the beaker.  10% titron X was prepared. Then 100 µl Titron X from this solution was added to the X-gluc solution.  2 ml methanol was added to the solution and gently mixed and P H was adjusted to 7.15 by adding P H -10 buffer solution. Sterilization of culture media The glasswares with medium were sterilized under 1.09 kg/cm 2 pressure at 121 0 C for 25 min. Sterilization of glasswares and instruments Beakers, test tubes, conical flasks, pipettes, metallic instruments like forceps, scalpels, and inoculation loop, micropipette tips, eppendorf tubes, needles, spatulas were wrapped with aluminium foil, vials were capped with plastic cap and then sterilized in an autoclave at a temperature of 121 0 C for 30 minutes at 1.16 kg/cm 2 pressure. Culture techniques i. Explant culture Explants (Leaf, node and internodal calli) were produced in present experiment from the shoots of cucumber seeds (variety Shital). Explants were separately placed horizontally on each vial and gently pressed into the surface of the sterilized culture medium supplemented with various concentrations and combinations of BAP (0, 1 and 2 mg/l) and NAA (1, 2 and 3 mg/l). The culture vials containing explants were placed under dark in growth room with controlled temperature (25 ± 1 0 C). The vials were checked daily to note the response and the development of contamination if any. ii. Agrobacterium culture For maintenance the strain, one single colony from previously maintained Agrobacterium stocks was streaked onto freshly prepared petridish containing YMB medium having kanamycin. The petridish was sealed with parafilm and kept at room temperature for at least 48 hours. This was then kept at 4 0 C to check over growth. Such culture of Agrobacterium strain was thus ready to use for liquid culture. The cultures were subcultured regularly at each week in freshly prepared media to maintain the stock. For infection single colony of A. tumefaciens was picked and inoculated in a conical flask containing liquid LB medium with 50 mg/l kanamycin. The culture was allowed to grow at 28 0 C to get optimum growth of Agrobacterium for infection and co-cultivation of explants (calli). Agrobacterium-mediated Genetic Transformation in Cucumber (var. Shital) as Influenced by Explant, Inoculation Time and Co-cultivation Period iii. Infection The Agrobacterium grown in liquid LB medium was used for infection. Prior to this, optical density (OD) of the bacterial suspension was determined at 600 nm (OD 600 = 0.60) with the help of a spectrophotometer. Following the determination of density, to pre-culture explants (calli) were dipped into bacterial suspension for 3 and 5 min, respectively, before transferring them to co-cultivation medium. iv. Co-cultivation Following infection, the explants were co-cultured on co-cultivation medium. Prior to transfer of all explants (callus) to co-cultivation medium they were blotted with sterile tissue papers for a short period to remove excess bacterial suspension. All the explants were maintained in co-cultivation medium for 24 and 48 hours, respectively. Co-cultured explants were placed under fluorescent illumination with 16/8 hours light/dark cycle at (25±2 0 C). The intensity of light was maintained at 1800 lux (approximately). The culture vials were checked daily to observe any contamination and the behaviors of the explants. v. Washing and post-cultivation After co-cultivation for required periods, the infected explants were washed twice with sterile ddH 2 O and once with sterile ddH 2 O containing 200 mg/l cefotaxime. Then the explants were transferred onto post-cultivation medium containing 100 mg/l cefotaxime. vi. Transfer to selection medium Following one week of post-cultivation, the explants were transferred onto low selection MS medium supplemented with 2 mg/l BAP + 1 mg/l NAA + 20 mg/l kanamycin + 100 mg/l cefotaxime and also onto high selection MS medium fortified with 2 mg/l BAP + 1 mg/l NAA + 30 mg/l kanamycin + 100 mg/l cefotaxime. vii. GUS (β-Glucuronidase) histochemical assay From each batch of calli following each transformation experiment, randomly selected survived calli were examined for GUS histochemical assay. For this test survived calli were immersed in X-gluc (5-bromo-4-chloro-3-indoly-1glucuronide) solution and were incubated at 37 0 C for overnight. A characteristic blue color would be the expression of GUS (β-Glucuronidase) gene in the plant tissue. Proper control for GUS histochemical assay was done with the explants having no Agrobacterium infection. After X-gluc treatment explants were transferred to 70% alcohol for degreening. Following degreening explants were observed under stereomicroscope (Begam, 2007) [4]. viii. Transfer of the selected materials to regeneration medium After ten days, the survived calli were transferred to regeneration medium consisting of MS medium supplemented with 1 mg/l NAA + 2 mg/l BAP + 20 mg/l kanamycine + 100 mg/l cefotaxime for regeneration. Data Recording To investigate the effects of different treatments and responses of different varieties to callus induction subsequent inoculation and regeneration, data were collected from the different parameters as given below. Effect of explant (callus) Influence of explants was found significant in all the parameters studied (Table 1). Among the explants leaf callus showed the best performance over nodal and internodal callus. The highest number (12.75) leaf callus was survived in co-cultivation medium followed by nodal callus (12.10) and the lowest number (10.95) was found in internodal callus. Regarding percentage of survived callus, leaf explants also showed the best performance (53.11 %) and the lowest survival percentage was found in internodal explants (45.60 %). In an investigation of Agrobacterium mediated genetic transformation of potato Begum (2005) [5] found that both number and percentage of survived calli per pertridish were higher (4.55 and 25.01 %, respectively) in leaf calli than internodal calli (2.33 and 11.16 %, respectively). The highest GUS positive transgenic callus was obtained from leaf explants (3.30) and the internodal explants gave the lowest number (1.79) of GUS positive transformants. In case of percentage of GUS positive callus leaf explants gave the highest percentage (55.04 %). Nishibayashi et al. (1996) [12] reported that very young leaves of more than 50 percent plantlets when treated with x-gluc displayed strong GUS expression. Rajagopalan and Perl-Treves (2005) [15] obtained the highest (75 %) GUS positive callus from cotyledon explants in cucumber. From above findings it was revealed that transformation ability may differ from explant to explant. Means in a column followed by uncommon letter (s) varied significantly at 5% level of significance Effect of inoculation time Inoculation time is an important factor in transformation experiment mediated by A. tumefaciens. The Agrobacterium mediated transformation system is historically the first successful plant transformation system, marking the breakthrough in plant genetic engineering in 1983. The breakthrough in gene manipulation in plants came by characterizing and exploiting plasmids carried by the bacterial plant pathogens. These provide natural gene transfer, gene expression and selection systems. In recent times, A. tumefaciens used as nature's most effective plant genetic engineer (Chawla, 2002) [6]. In present investigation, three types of callus were immeresed for 3 and 5 min, respectively in Agrobacterium suspension to see the effect of inoculation time regarding transformation ability. Following immersion the calli were blotted and placed on co-cultivation medium. From Table 2, it was revealed that both inoculation times had highly significant influence on different parameters studied. Transformation ability was increased with increase of inoculation time. Percentage of survived calli were higher (53.68 %) when the calli were immersed for 5 min in bacterial suspension. Both number and percentage of GUS positive calli were higher (3.17 and 52.87 %, respectively) when callus were kept in bacterial suspension for higher time (5 min) and were lower (2.15 and 35.88 %, respectively) when they were soaked in bacterial suspension for minimum time (3 min). Rajagopalan and Perl-Treves (2005) [15] reported that they found maximum GUS expression (93 %) when calli were immersed either for 60 min or 120 min in bacterial suspension. They also mentioned that although GUS expression events increased with inoculation time but survival rate and regeneration capacity were dramatically reduced. So they selected 10 min as recommended inoculation time. From these findings it was understood that inoculation time play a vital role in Agrobacterium mediated gene delivery system. Effect of co-cultivation period Duration of co-cultivation also an important factor in Agrobacterium-mediated plant genetic transformation. Two co-cultivation periods had highly significant effects in all the parameters studied ( Table 2). The longer co-cultivation period (48 hrs) showed better performance than shorter one. The higher number and percentage of callus (12.45 and 51.87 %, respectively) were survived when they were kept in co-cultivation medium for two days (48 hrs). Rajagopalan and Perl-Treves (2005) [15] observed the highest survival rate (72 %) in cotylendonary explants of cucumber when those were co-cultivated for 24 hrs. But survival rate declined (69 %) with increase of co-cultivation period upto 48 hrs. They also observed the lowest survival rate (41 %) in contrast of the highest co-cultivation period (120 hrs). In present investigation there was scope to see the effect of survival rate as well as GUS response of variety Shital with more co-cultivation time considered in present investigation. Nishibayashi (1996) [12] successfully obtained transgenic cucumber plants after co-cultivated the calli for 5 days. The highest percentage (48.36 %) GUS +ve calli were obtained when co-cultivation time was 48 hrs. Rajagopalan and Perl-Treves (2005) [15] obtained the highest GUS foci (88 %) from co-cultivation period of 120 hrs. Fang and Grumet (1990) [7] reported that three day co-cultivation period was the best for successful transformation in muskmelon. In an investigation of Agrobacterium -mediated transformation of potato Begam (2007) [4] observed that GUS expression increased with the increase of co-cultivation period. She reported that calli co-cultivated for 5 days gave higher (58.53 %) response to the GUS assay. Both the present and Begam's investigation were conducted in the same laboratory. From the above findings it was revealed that survival percentage of callus influenced by co-cultivation period. From last two decades, A. tumefacines mediated plant genetic transformation has become well established in numerous laboratories and at present the most preferred method for cucumber transformation. The advantages of this method includes high transformation ability, minimal rearrangement of the transgene and a relatively high percentage of the transgenic plants that harbour a single copy of the transgene (Roy et al. 2000) [16]. The future aim of cucumber breeding may be use this transformation technique to develop value-added transgenic cucumber varieties by transforming single or many transgenes into commercially important cucumber varieties of Bangladesh or any part of the world. Figure 1 showed transformed and non-transformed leaf calli in eppendorf tubes. Figure 2 and 3 showed blue patches that indicated GUS activity and Conclusions An efficient protocol for genetic transformation in cucumber was developed which showed transfer of CIPK sense gene in variety Shital and integration of two marker genes (GUS and npt II). The highest and the lowest GUS positive transgenic calli were obtained from leaf and internodal explants, respectively. Transformation ability was increased with increase of inoculation time (5 mins.). Duration of co-cultivation also an important factor in Agrobacterium mediated plant genetic transformation. The higher co-cultivation period (48 hrs.) showed better performance than lower one. For the development of abiotic stress tollerant cucumber varieties in Bangladesh this transformation protocol could be utilized successfully. Further transgenecity confirmation test like PCR, southern blotting, sequencing etc. to be needed to confirm transformation of putative transformed callus.

v3-fos

2019-03-22T16:15:05.659Z

2015-01-01T00:00:00.000Z

84996427

s2

Relation between O3-Inhibition of Photosynthesis and Ethylene in Paddy Rice Grown under Different CO2 Concentrations Abstract This study was conducted to evaluate the role of ethylene in acute ozone (O3: 0, 0.1, and 0.3 cm3 m–3; O0, O0.1 and O0.3, respectively)-induced photosynthetic inhibition of paddy rice leaves grown under different atmospheric carbon dioxide concentrations (CO2: 400 and 800 cm3 m–3; C400 and C800, respectively). Ethephon and silver thiosulfate complex (STS) were applied one day before exposure to O3. Gas exchange, chlorophyll fluorescence, and ascorbic acid were measured immediately before (BE), immediately after (AE-0), and 1 d and 3 d after (AE-1, AE-3) the start of the exposure to O3. In the plants exposed to O3, visible leaf symptoms on the adaxial leaf surface appeared at AE-3. O3 decreased photosynthesis-related parameters, total ascorbic acid content, and redox state of ascorbic acid (RDS), and C800 ameliorated O3-induced damage. STS ameliorated the O3-induced visible leaf symptoms and O3-inhibition of photosynthesis but ethephon worsened slightly or did not affect them. Additionally, we evaluated the effects of O3 and CO2 on ethylene production in rice leaves. Although elevated CO2 did not affect ethylene production, exposure to O3 greatly increased ethylene production at AE-0 and rapidly reduced it at AE-1. These results indicate that ethylene is an important component of signal transduction for the extension of O3 injury in paddy rice. Recently, plant hormones such as jasmonic acid (JA), salicylic acid (SA), and ethylene have been recognized as important signals in O 3 stress (Rao and Davis, 2001;Kangasjärvi et al., 2005). In paddy rice, Imai (2012b, 2013b) reported that methyl jasmonate (MeJA) and SA application ameliorated the O 3 -inhibiton of P N . In addition, jasmonates (JA and MeJA) and SA contents in rice leaves are increased by O 3 Imai, 2012b, 2013c). However, the amelioration of O 3inhibition by SA application and the increase of endogenous SA in rice leaves (Kobayakawa and Imai, 2012b) are less than in other plants such as Arabidopsis (Sharme et al., 1996), tobacco (Yalpani et al., 1994) and soybean (Zhao et al., 2010). Moreover, elevated CO 2 induces SA production and suppresses JA production in leaves such as tomato, soybean, and ginger (DeLucia et al., 2012). In paddy rice, Kobayakawa and Imai (2013c) found that endogenous JA and MeJA contents in leaves were decreased slightly by elevated CO 2 (800 cm 3 m -3 ). Therefore, the roles of plant hormones on O 3 -inhibition might differ among plant species, and might change under future CO 2 concentrations. Ethylene is the simplest olefin synthesized from methionine through intermediates S-adenosyl-methionine (SAM) and 1-aminocyclopropane-1-carboxylic acid (ACC). In higher plants, ethylene production increases during leaf abscission, flower senescence, and fruit ripening. In addition, ethylene biosynthesis increases under stress conditions such as drought, flooding, chilling and mechanical wounding (Taiz and Zeiger, 2010). The O 3exposure increases ethylene production in leaves of Arabidopsis (Rao et al., 2002), pea (Mehlhorn et al., 1991) and tobacco (Mehlhorn et al., 1991;Ogawa et al., 2005). Likewise, Rao et al. (2002) reported that ethyleneoverproducer mutants (eto1, eto3) were more sensitive to O 3 than wild type. Overmyer et al. (2000) showed that ethylene-insensitive mutant (ein2) was more insensitive to O 3 than wild type, and O 3 -sensitive mutant (rcd1) produced ethylene more than wild type, and was therefore more sensitive to O 3 . Furthermore, the O 3 -induced visible leaf symptoms are ameliorated by ethylene biosynthesis inhibitor, aminoethoxyvinylglycine (AVG), application in pinto bean and tobacco (Mehlhorn et al., 1991). However, there were few studies on the relation between O 3inhibition of photosynthesis and ethylene in paddy rice, although O 3 exposure increased ethylene production in rice leaves (Ohki et al., 1999). The objective of current study is to clarify the role of ethylene on O 3 -inhibition in rice leaves under different CO 2 concentrations. The level or function of ethylene can be manipulated in different ways. In this study, we used the following two methods to determine possible associations of ethylene in the O 3 -inhibition of photosynthesis under different CO 2 concentrations (Exp. 1); application of ethylene-generating agent (ethephon) or ethylene-action inhibitor (STS). The former is intended to increase the level of ethylene and the latter is to suppress ethylene functions even in the presence of it. We also examined the endogenous ethylene level under different CO 2 concentrations (Exp. 2). Our hypothesis is that elevated ethylene promotes and deficient ethylene suppresses the O 3 -inhibition in rice leaves. However, this situation may change under elevated CO 2 . In Exp. 1, immediately after full expansion of the eighth leaves (ca. one month from sowing), a solution of 0.1% ethephon (Nissan Ethrel 10; Nissan Chemical Industries Ltd., Tokyo, Japan) or 2 mM STS (K-20C; Chrysal Japan Ltd., Oosaka, Japan) was applied to the plants. Ethephon was sprayed to the shoots using a mister, and STS was applied to the soil for absorption from the plant roots. One day after ethephon or STS application (Exp. 1), or immediately after full expansion of the eighth leaves (Exp. 2), rice plants were exposed to 0 (< 0.002), 0.1, and 0.3 cm 3 m -3 O 3 (expressed respectively as O 0 , O 0.1 , and O 0.3 ) during 5-hr local daytime (0800 -1300) using three chambers under growth CO 2 concentrations. Ozone was supplied using a high-voltage O 3 generator with dry air (ED-OG-R6; Ecodesign Inc., Ogawa, Saitama, Japan), and CO 2 was supplied from cylinders containing liquid CO 2 . These gases were injected into air that had been filtered through activated charcoal layers. The O 3 and CO 2 concentrations were measured every 1 min and computer-controlled using an ultraviolet absorption-type O 3 analyzer (EG-2001F; Ebara Jitsugyo Co. Ltd., Tokyo, Japan) and infrared CO 2 analyzer (ZRH; Fuji Electric Systems Co. Ltd., Tokyo, Japan), respectively. The average concentrations of O 3 in the O 0.1 and O 0.3 treatments were 0.106 and 0.307 cm 3 m -3 , respectively, and those of CO 2 in the C 400 and C 800 treatments were 396 and 792 cm 3 m -3 , respectively. These were within 3 % of target values. Gas exchange measurements In Exp. 1, in situ gas-exchange measurements (1300 -1430, local time) of the attached, eighth leaves at the middle portion were conducted immediately before (BE: 1 -0 hr before), immediately after (AE-0: 0.1 -1.1 hr after), and 1 and 3 d after (AE-1, AE-3) exposure to O 3 at C 400 or C 800 for four replicate plants in each treatment using a portable photosynthesis and transpiration measurements system (LI-6400XT; Li-Cor Inc., Lincoln, NE, USA). Environmental conditions within the LI-COR cuvette during measurements were set at 28ºC leaf temperature, 1.5 kPa VPD, and 1500 µmol m -2 s -1 PPFD (mixed light from red and blue LEDs) under growth CO 2 concentrations. Chlorophyll fluorescence measurements In both Exp. 1 and Exp. 2, chlorophyll fluorescence of the eighth leaves were measured at 28ºC right after (within about 1 min) the gas exchange measurements for four replicate plants using a portable fluorometer (MINI-PAM; Heinz Walz GmbH, Effeltrich, Germany). The chlorophyll fluorescence parameters were obtained by the respective applications of 0.2, 7000, and 1400 µmol m -2 s -1 of measuring light, saturation pulse (0.8 s flash), and actinic light. Before measurements, the leaf was kept in the dark for 10 min because the effect of O 3 remained maximal, although the effects of other non-steady-state factors on chlorophyll fluorescence parameters disappeared during this period (Kobayakawa and Imai, 2013a). Then the minimum (F 0 ) and maximum (F m ) fluorescence were determined, respectively, by irradiating the measuring light and saturation pulses. Thereafter, the steady fluorescence (F ') and maximum fluorescence in the steady state (F m ') were determined under actinic light irradiation. The maximum (F v /F m ) and operating (F q '/F m ') quantum efficiencies of PSII were obtained using the following equations (Baker, 2008;Sonoike, 2009). Ascorbic acid measurements In Exp. 1, four eighth leaves for each treatment were sampled and used for the measurements of ascorbic acid (reduced form, AA; oxidized form, DHA) contents right after (within about 5 min) the chlorophyll fluorescence measurements. Immediately after measurements of the fresh weight (FW) and leaf area, leaves were frozen in liquid N 2 and ground with a mortar and pestle by adding metaphosphoric acid to obtain leaf extracts. Then they were determined using the hydrazine method (Fujita and Yamada, 2002) with a spectrophotometer (Ubest-30; Jasco Corp., Tokyo, Japan), with L(+)-ascorbic acid (Kanto Chemical Co. Inc., Tokyo, Japan) used as a standard reagent. This method uses a color reaction (540 nm) between 2, 4-dinitrophenylhydrazine and sulfuric acid to assay DHA. When sodium 2, 6-dichloroindophenol is added to the sample solution beforehand, the AA is transformed to DHA and total ascorbic acid (AA + DHA) is obtainable. The AA content was calculated by subtraction of DHA from total ascorbic acid. The redox state (RDS) of ascorbic acid was calculated as AA / (AA + DHA). Ethylene production measurements In Exp. 2, four eighth leaves for each treatment were sampled and used for measurements of ethylene production almost simultaneously with chlorophyll fluorescence measurements. Immediately after measurements of the FW, leaves were sealed in a test tube with 4 mL water. They were incubated at 28ºC for 4 hr using an incubator (LTI-700; Tokyo Rikakikai Co. Ltd., Tokyo, Japan). Thereafter, the gas sample in a test tube was injected into a gas chromatograph (GC-2010; Shimadzu Corp., Kyoto, Japan) with a flame ionization detector (FID-2010Plus; Shimadzu Corp., Kyoto, Japan) using a gas-tight syringe (MS-GANX00; Ito Corp., Shizuoka, Japan). Separation of ethylene was conducted using a capillary column (30 m × 0.25 mm × 0.25 µm: Rxi®-1 ms; Restek Corp., Pennsylvania, U.S.) with He as a carrier gas. Temperatures of the injection port and detector were maintained, respectively, at 250ºC and 280ºC. The column temperature was maintained at 170ºC for 5 min. The ethylene retention time was 1.392 min. Statistical analysis All data were subjected to three-way (Exp. 1) or two-way (Exp. 2) analysis of variance (ANOVA) using software (Excel Statistics 2010 for Windows; Social Survey Research Information Co. Ltd., Tokyo, Japan). Because of the lack of chamber replications, the O 3 effect was not separable from the chamber effect. Therefore, the former might be biased. However, the chamber effect did not seem large from our experience with environmental regulation. The significance among treatments in each interval was determined using Tukey's honestly significant difference (HSD) test (P ≤ 0.05). O 3 -induced visible symptoms on adaxial leaf surface (Exp. 1) At AE-3, O 3 -induced visible symptoms appeared in the O 0.1 + C 400 , O 0.3 + C 400 , and O 0.3 + C 800 plants. Elevated CO 2 (C 800 ) and STS application ameliorated but ethephon application slightly worsened the leaf symptoms. Figure 1 shows O 3 -induced visible leaf symptoms in the O 0 + C 400 (control), O 0.1 + C 400 , O 0.1 + C 400 + S, and O 0.1 + C 400 + E plants. The O 3 -induced symptom in the O 0.1 + C 400 and O 0.1 + C 400 + E plants look similar, but injured areas of leaf between these two were different. The symptom appeared only on the edge of leaf in the O 0.1 + C 400 plants, but it spread from the edge to middle portion (around midrib) in the O 0.1 + C 400 + E plants. Effect of ethylene on O 3 -inhibition of photosynthesis under ambient CO 2 (Exp. 1) Photosynthesis-related parameters in the O 0 plants were unaffected by ethephon or STS application during AE-0 to AE-3 under both C 400 and C 800 (Figs. 2 and 3). At AE-0, P N in the O 0.1 + C 400 (▲) and O 0.3 + C 400 (■) plants were 52% and 24%, respectively, of those at BE. Thereafter, the decreases persisted: At AE-3, the former and the latter plants were 47% and 21% of those at BE. The O 3inhibition of P N in the O 0.1 + C 400 and O 0.3 + C 400 plants slightly ameliorated by STS: At AE-0, P N in the O 0.3 + C 400 + S plants (□) was significantly higher than those in the O 0.3 + C 400 plants; at AE-3, this amelioration was clearer. At AE-3, P N values in the O 0.1 + C 400 + S (△) and O 0.3 + C 400 + S plants were, respectively, 81% and 35% of those at BE. However, the O 3 -inhibition of P N in the O 0.1 + C 400 and O 0.3 + C 400 plants was unaffected or slightly worsened (not significantly different) by ethephon. At AE-3, P N values in the O 0.1 + C 400 + E (▲) and O 0.3 + C 400 + E (■) plants were, respectively, 43% and 12% of those at BE (Fig. 2a). At AE-0, g s values in the O 0.1 + C 400 and O 0.3 + C 400 plants were, respectively, 61% and 58% of those at BE. Thereafter, these plants recovered from O 3 -inhibition: At AE-3, g s values in the former and the latter were, respectively, 72% and 82% of those at BE. Ethephon and STS did not affect the O 3 -inhibition of g s in the O 0.1 + C 400 and O 0.3 + C 400 plants at AE-0 (Fig. 2b). Three-way ANOVA for P N and g s exhibited clear effects of O 3 during AE-0 to AE-3 (P ≤ 0.001, Table 1). It also showed interactions between O 3 and ethylene from AE-1 to AE-3 (Table 1). At AE-0, F v /F m in the O 0.1 + C 400 and O 0.3 + C 400 plants were, respectively, 78% and 66% of those at BE. Thereafter, these plants recovered from O 3 -inhibition. At AE-3, F v /F m values in the former and the latter were, respectively, 93% and 72% of those at BE. The O 3 -inhibition of F v /F m in O 0.1 + C 400 and O 0.3 + C 400 plants was unaffected by ethephon or STS at AE-0, but the O 0.3 + C 400 plants were ameliorated by STS at AE-1 and AE-3. The F v /F m values in the O 0.3 + C 400 + S plants at AE-1 and AE-3 were, respectively, 76% and 88% of those at BE (Fig. 2c). At AE-0, F q '/F m ' values in the O 0.1 + C 400 and O 0.3 + C 400 plants were, respectively, 73% and 52% of those at BE. Thereafter, the O 0.1 + C 400 plants recovered from O 3 -inhibition, but the O 0.3 + C 400 plants did not. At AE-3, the O 0.1 + C 400 plants recovered fully, but the O 0.3 + C 400 plants remained at 55% of BE. The O 3 -inhibition of F q '/F m ' in the O 0.3 + C 400 plants was ameliorated slightly by STS. At AE-3, the F q '/F m ' in the O 0.3 + C 400 plants was 75% of that at BE (Fig. 2d). Three-way ANOVA for F v /F m and F q '/F m ' indicated strong effects of O 3 from AE-0 to AE-3 (P ≤ 0.001, Table 1). Effect of ethylene on O 3 -inhibition of photosynthesis under elevated CO 2 (Exp. 1) The O 3 -inhibition of all photosynthesis-related parameters was ameliorated by elevated CO 2 (Figs. 2 and 3). At AE-0, P N values in the O 0.1 + C 800 (▲) and O 0.3 + C 800 (■) plants were, respectively, 85% and 43% of those at BE. intensified by ethephon but was ameliorated by STS. The g s in the O 0.3 + C 800 + S plants at AE-0 was 60% of that at BE. The g s of O 0.3 + C 800 + E plants at AE-3 was 70% of that at BE (Fig. 3b). Three-way ANOVA for P N and g s revealed clear interactions between O 3 and CO 2 during AE-0 to AE-3 (P ≤ 0.001 or 0.05, Table 1 (Fig. 3d). Three-way ANOVA for F v /F m and F q '/F m ' indicated clear interactions between O 3 and CO 2 at AE-0 to AE-3 (P ≤ 0.001 or 0.05, Table 1). Effects of O 3 and ethylene on ascorbic acid contents under different CO 2 concentrations (Exp. 1) The effects of O 3 and ethylene on total ascorbic acid (AA + DHA), AA, and DHA contents expressed per unit of fresh weight (FW) were similar to those expressed per unit of leaf area. Figure 4 shows values obtained on a FW basis. At AE-0, the redox state of ascorbic acid (RDS) in the O 0.1 + C 400 , O 0.3 + C 400 , and O 0.3 + C 800 plants decreased, respectively, to 92%, 88%, and 91% of those at BE. At AE-1, RDS decreased further: RDS in the O 0.1 + C 400 , O 0.3 + C 400 , and O 0.3 + C 800 plants were, respectively, 75%, 62%, and 85% of those at BE. Thereafter, the plants recovered from O 3 -inhibition at AE-3. The O 3 -inhibition of RDS was ameliorated by STS at AE-1 under ambient CO 2 (C 400 ). At AE-1, the O 0.1 + C 400 + S plants (△) recovered fully, but the O 0.3 + C 400 + S plants (□) remained at 79% of BE (Figs. 4c and 4d). Three-way ANOVA for total ascorbic acid contents and RDS showed clear negative effects of O 3 at AE-0 to AE-3 (P ≤ 0.001, Table 1). In addition, because STS ameliorated the negative effect of O 3 on ascorbic acid contents and RDS, three-way ANOVA for those showed clear effects of ethylene during AE-0 to AE-3, except for RDS at AE-3 (Table 1). Effects of O 3 and CO 2 on ethylene production and photosystem II (Exp. 2) The trends of F v /F m and F q '/F m ' resemble those in Exp. 1. At AE-0, F v /F m in the O 0.1 + C 400 , O 0.3 + C 400 , and O 0.3 + C 800 plants decreased, respectively, to 85%, 63%, and 67% of those at BE. The F q '/F m ' in the O 0.1 + C 400 , O 0.1 + C 800 , O 0.3 + C 400 , and O 0.3 + C 800 plants decreased, respectively, to 66%, 75%, 55%, and 56% of those at BE. Thereafter, the F v /F m and F q '/F m ' recovered, more or less, from O 3inhibition: At AE-3, O 0.1 + C 400 and O 0.3 + C 800 plants recovered fully, but the O 0.3 + C 400 plants remained inhibited (86% and 79% of those at BE, respectively) (Figs. 5a and 5b). At AE-0, the ethylene production was increased by O 3 . The production in the O 0.3 + C 400 and O 0.3 + C 800 plants at AE-0 increased drastically (302% and 389%, respectively) compared to those at BE. In the O 0.1 + C 400 and O 0.1 + C 800 plants, it increased slightly (P ≤ 0.10). Thereafter, the increase disappeared from AE-1 to AE-3 (Fig. 5c). Two-way ANOVA for ethylene production indicated a clear effect of O 3 at AE-0 (P ≤ 0.001, Table 2). Discussion Coincident with our previous studies in rice (Imai and Kobori, 2008;Kobayakawa and Imai, 2011a), the photosynthesis-related parameters and ascorbic acid content and its RDS were degraded by O 3 exposure. They were ameliorated by elevated CO 2 (C 800 ) . Generally, the amelioration of O 3 -induced damage of plants by elevated CO 2 is largely attributed to the restriction of CO 2 intake through stomatal closure (Booker and Fiscus, 2005;Imai and Kobori, 2008), although undetermined additional factors might be concerned. In this study, STS application ameliorated the O 3 -induced visible injury (Fig. 1) and O 3 -inhibitions of P N and PSII (F v / F m and F q '/F m '), as in the cases of MeJA and SA applications Imai, 2012b, 2013b). In contrast, ethephon application slightly intensified the O 3 -inhibition of photosynthesis-related parameters. These results demonstrated that ethylene functions as a positive regulator of O 3 injury, in contrast to JA in rice leaves (Kobayakawa and Imai, 2012b), as seen in Arabidopsis (Rao and Davis, 2001). The amelioration of O 3 -induced damage of plants by MeJA application was ascribed to the induction of stomatal closure and increased antioxidant capacity (Kobayakawa and Imai, 2012b). In contrast to MeJA, SA and ethylene-related plant growth regulator (STS and ethephon) application did not affect g s (Figs. 2 and 3; Kobayakawa and Imai, 2013b). Nevertheless, the amelioration of O 3 -inhibition of photosynthesis-related parameters by STS was more evident than that by SA, and was even equal to that by MeJA. With respect to endogenous ethylene, its production in rice leaves increased immediately after exposure to O 3 (AE-0) and decreased rapidly at AE-1 (Fig. 5). These are coincident with the case of Arabidopsis (Rao et al., 2002). Therefore, ethylene is expected to be an important factor in the enlargement of O 3 injury in rice leaves. The inhibition of ethylene action did not affect stomatal function, but slightly increased the total ascorbic acid contents and ameliorated O 3 -induced decrease of total ascorbic acid and RDS (Fig. 4). Therefore, the amelioration of O 3 -inhibition by STS is attributed to the activation of antioxidant capacity, although it does not fully explain it. Although antioxidant ability is an important factor for the amelioration of O 3 injury (Didyk and Blum, 2011), Imai (2011c, 2013b) demonstrated that the increase of ascorbic acid by exogenous AA and SA could not completely prevent O 3 injury in rice leaves. However, STS ameliorated O 3 -induced injury more effectively than ascorbic acid and SA applications. These results suggest that ethylene affects other factors for preventing O 3 injury. Ethylene induces programmed cell death (PCD) and senescence (Trobacher, 2009). In addition, levels of chronic and acute O 3 exposure respectively induce premature senescence and hypersensitive reaction-like PCD (Rao and Davis, 2001). Therefore, it is predicted that O 3 -induced rapid ethylene production and thereby cell death and/or senescence later appeared on the leaf surface as visible leaf symptoms. Nakamura and Saka (1978) demonstrated that kinetin and benzimidazole applications ameliorated O 3 -induced chlorophyll degradation in rice leaves. Because these cytokinins suppress leaf senescence in contrast to ethylene Table 2. Results of statistical analyses of the effects of O 3 and CO 2 on maximum (F v /F m ) and operating (F q '/F m ') quantum efficiencies of photosystem II and ethylene production in rice leaves portrayed in Fig. 4 (Exp. 2). AE-0, AE-1, and AE-3 respectively denote values obtained immediately after, and 1 d and 3 d after gas exposure. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s. -not significant, by two-way ANOVA. (Taiz and Zeiger, 2010), O 3 -induced rapid ethylene production might induce premature leaf senescence. This study showed that ethylene production was unaffected by elevated CO 2 (Fig. 5), but Abeles et al. (1992) reported that elevated CO 2 promoted, inhibited, or had no effect depending on the plant species and tissues. However, the effects of STS on photosynthesis-related parameters were significant under both CO 2 conditions, and the effects of ethephon were noted under elevated CO 2 , as evidenced by significant CO 2 × ethylene or O 3 × CO 2 × ethylene interactions (Table 1, Figs. 2 and 3). These results suggest that elevated CO 2 may reduce the levels or function of ethylene and thereby ameliorate the O 3inhibition of photosynthesis. Kobayakawa and Imai (2013c) found that JA contents in rice leaves were decreased slightly by elevated CO 2 , as reported previously (DeLucia et al., 2012). Likewise, rice plants grown under elevated CO 2 are more susceptible to leaf blast than those grown under ambient CO 2 (Kobayashi et al., 2006). In addition, elevated CO 2 -grown soybean plants exhibited higher sensitivity to acute exposure to O 3 because of the lowered antioxidant capacity under elevated CO 2 (Gillespie et al., 2011). Elevated CO 2 may alter the physiological response in plants including the defense response and sensitivity to various stresses through changes in plant hormone levels. Results of present and previous Imai, 2012b, 2013b, c) studies show that JA and SA function as negative regulators of O 3 -inhibition, and that ethylene functions as a positive regulator of O 3 -inhibition. In addition, jasmonates (JA and MeJA) and ethylene increased immediately (AE-0) and SA increased with a lag time (from AE-1 to AE-3) after exposure to O 3 . Furthermore, the amounts of jasmonates and ethylene increased more than that of SA. Therefore, JA and ethylene may be more important for the determination of O 3 inhibition than SA in rice leaves. This phenomenon might be attributed to high SA contents in rice leaves (8 -30 µg g -1 FW), which is higher than other plants such as Arabidopsis (0.01 -0.1 µg g -1 FW) (Ogawa et al., 2006). According to Yang et al. (2004), rice had two orders of magnitude higher levels of SA than tobacco and Arabidopsis, and was insensitive to exogenous SA. In addition, Kanno et al. (2012) reported that JA and SA contents in rice leaves were increased by the feeding damage of white-back planthopper (Sogatella furcifera), but the increment of SA was less than that of JA because rice plants contain much higher levels of endogenous SA in healthy tissues. Therefore, we conclude that the role of SA in rice is less pronounced than in other plants, and that JA and ethylene are major components of signal transduction causing O 3 injury.

v3-fos

2017-04-15T06:32:39.157Z

2015-10-21T00:00:00.000Z

9193657

s2

Genetic Analysis of East Asian Grape Cultivars Suggests Hybridization with Wild Vitis Koshu is a grape cultivar native to Japan and is one of the country’s most important cultivars for wine making. Koshu and other oriental grape cultivars are widely believed to belong to the European domesticated grape species Vitis vinifera. To verify the domesticated origin of Koshu and four other cultivars widely grown in China and Japan, we genotyped 48 ancestry informative single nucleotide polymorphisms (SNPs) and estimated wild and domesticated ancestry proportions. Our principal components analysis (PCA) based ancestry estimation revealed that Koshu is 70% V. vinifera, and that the remaining 30% of its ancestry is most likely derived from wild East Asian Vitis species. Partial sequencing of chloroplast DNA suggests that Koshu’s maternal line is derived from the Chinese wild species V. davidii or a closely related species. Our results suggest that many traditional East Asian grape cultivars such as Koshu were generated from hybridization events with wild grape species. Introduction Grapes are one of the most important horticultural crops worldwide, and are typically consumed as fresh fruit or used in the production of wine, brandy, juice and raisins. The majority of wine grapes are cultivars of the domesticated grape V. vinifera, which originated in the Caucasus and spread from there to Europe and eventually to grape growing regions worldwide [1]. Cultivars grown for sale as table grapes or juice are often interspecific hybrids of V. vinifera and wild species (e.g. V. labrusca). There are an estimated 60 species in the genus Vitis distributed broadly across the entire northern hemisphere, and many wild grapevines are used to breed new cultivars since they harbor desirable traits like disease resistance and tolerance to abiotic stress [2]. Several grape cultivars from China and Japan are widely grown for commercial purposes. For example, Koshu is one of Japan's most widely planted and popular grape cultivars with approximately 1200 acres under vine in 1997 [3]. It is known for its distinct pale purple skin containing both cyanidin-based and delphinidin-based anthocyanins [4], but not anthocyanin diglucosides [5]. Wine produced from Koshu has aromatic characteristics similar to Sauvignon blanc [6] and has contributed significantly to the growth of the Japanese wine industry. It is widely believed that Koshu is an oriental cultivar of V. vinifera [3] and previous analyses have supported this hypothesis. For example, a phylogeny generated using simple sequence repeats (SSRs) showed that Koshu clusters with well-known V. vinifera cultivars such as Sultanina and Muscat of Alexandria [7]. Similar results were observed from analyses of AFLPs [8] and principal coordinate analysis of SSR data [9]. In contrast, there is also evidence that Koshu may have ancestry from wild Vitis species. For example, Koshu has the SSR allele 4MG1 or n+4 at the VVS2 locus [10], which has only been found in rootstock cultivars, wild species, or cultivars crossed with wild Vitis (Boursiquot JM, personal communication). Similarly, Koshu and other East Asian cultivars possess unique SSR alleles that are not found in European V. vinifera cultivars [9,10]. To verify the ancestry of popular East Asian cultivars on a genome-wide scale, we genotyped a panel of 48 ancestry informative markers (AIMs) in the Japanese cultivars Koshu and Koshusanjaku, and the Chinese cultivars Longan, Huotianhong, Baijixin. The AIMs are a subset of SNPs from the Vitis9kSNP array [11] that clearly differentiate wild Vitis species from V. vinifera. Our ancestry estimates are based on a method developed by Sawler et al. [12], who showed that genotyping fewer than 50 SNPs leads to accurate estimates of the genomic contributions of V. vinifera and North American wild Vitis in interspecific hybrid grape cultivars from the USDA grape germplasm collection. Here we apply the same method to quantify the potential genomic contribution of wild East-Asian Vitis to popular East Asian cultivars. In addition, the maternal ancestry of these cultivars was investigated by partially sequencing their chloroplast DNA and comparing them to published sequences. Marker selection and genotyping Forty-eight AIMs were selected as those with the highest loadings along the first principal component (PC1) from PCA performed on 6114 SNPs genotyped in 1031 samples labeled as V. vinifera and 786 labeled as wild Vitis grapevine accessions from the USDA grape germplasm collection [12]. For each SNP, pairs of PCR primers were designed based on 100 bp flanking each side of the SNP. The amplified fragments were purified with a Nucleospin Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany) and sequenced directly, or after gel electrophoresis and extraction from the gel when necessary. The genotyped SNPs and their primer sequences according to the 8x Pinot Noir reference genome [13] are shown in S1 Table. Genetic material The ten DNA samples genotyped for the AIMs in this study are Koshu, Pinot Noir, Koshu-sanjaku, Longan, Huotianhong, Baijixin, V. amurensis, V. coignetiae, V. ficifolia var. ganebu, and V. shiragai. The full genotype data are shown in S2 Table. Among them V. amurensis, V. coignetiae, V. ficifolia var. ganebu, and V. shiragai were extracted using standard methods from young leaves donated by Prof. Horiuchi and Dr. Mochioka of Osaka Prefecture University. All other DNA was extracted from young leaves of vines in an experimental vineyard in Higashi-Hiroshima. For the analysis of chloroplast DNA, partial chloroplast sequences were obtained from the following six cultivars: Koshu, Koshu-sanjaku, Longan, Huotianhong, Baijixin, and Chardonnay. and analysis, decision to publish, or preparation of the manuscript. The specific role of this author is articulated in the 'author contributions' section. Competing Interests: The authors have the following interests: Co-author Jason Sawler is employed by Anandia Labs. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. Principal components analysis and ancestry estimation PCA of the 48 AIMs was first performed using 33 V. vinifera cultivars and 33 accessions of East-Asian wild Vitis species. These two groups of accessions are considered ancestral populations for the purposes of this study. Except for four East Asian wild Vitis samples obtained for this study, the genotypes for these ancestral groups were obtained from previously published data from the Vitis9KSNP genotyping microarray [14,15]. Genotype data from the 48 AIMs were obtained for this study by genotyping four East Asian wild species (V. amurensis, V. coignetiae, V. ficifolia var. ganebu, and V. shiragai), five oriental cultivars (Koshu, Koshusanajku, Longan, Huotianhong, Baijixin) and one V. vinifera (Pinot noir). After establishing the PC axes using the 33 V. vinifera and 33 wild accessions described above as ancestral populations, the remaining samples were subsequently projected onto the first two PCs. The proportion of V. vinifera ancestry in the samples genotyped as part of this study was then calculated as follows: '% V. vinifera = b/(a+b)', where a and b are the chord distances along the first principal component from the centroids of the V. vinifera cultivars and wild species in PC space, respectively, according to [12]. Partial sequencing of chloroplast DNA Following the method of Zecca et al. [16] with minor modification, four spacer regions of chloroplast DNA (trnH-psbA, trnK-rps16, trnF-ndhJ, and rpl32-trnL) were amplified and sequenced. The PCR and sequencing primers are shown in S3 Table. The sequences obtained from this have been submitted to DDBJ [LC054004-LC054031]. The reported sequences of wild Vitis species and those of V. vinifera subsp. sylvestris (HQ656029-HQ656577) were downloaded from GenBank and compared with the sequence obtained in this study. Phylogenetic analysis of chloroplast sequence The wild species for which sequence data were unobtainable from one or more of the four chloroplast genomic regions were omitted from the analysis. The downloaded sequences were trimmed to the same region of the sequence obtained in this study after alignment with Clus-talW. The total resulting sequence length was 2081 bp including gaps. For each accession, the four regions were joined and a dendogram was constructed using the Neighbor-Joining (NJ) method in MEGA6 [17]. Distances were calculated with Kimura's 2 parameter model and substitutions included transitions and transversions. Gaps and missing data were treated as pairwise deletions. Bootstrap confidence values were calculated from 100 bootstrap replications. From this distance matrix, principal coordinate analysis (PCoA) was also carried out using GenAlEx [18] with standardization. PCA based ancestry estimation PCA of the two ancestral populations, wild East Asian species and V. vinifera, resulted in a clear separation of these two groups along PC1. Samples from the four East Asian wild species (V. amurensis, V. coignetiae, V. ficifolia var. ganebu, and V. shiragai) genotyped as part of this study all clustered with the East Asian wild ancestral population genotyped in [14]. The Pinot Noir sample genotyped as part of this study also clustered as expected with the 33 V. vinifera cultivars genotyped in [15] and had an estimated V. vinifera ancestry proportion of 1.0. The V. vinifera ancestry proportions for the five East Asian cultivars genotyped as part of this study had values ranging from 0.715 to 1 (Fig 1). Analysis of chloroplast diversity We find that the partial chloroplast sequence of Chardonnay is identical to the reported sequence of V. vinifera cv. Maxxa (NC_007957.1) except for one site at position 205, as reported by Tabidze et al [19]. Differences were observed between Koshu and Chardonnay at 8 sites (Table 1). All other oriental cultivars share an identical chloroplast sequence with Chardonnay (V. vinifera). A PCoA plot based on a chloroplast genetic distance matrix enables the relationships among the accessions' chloroplast sequences to be visualized in two dimensions (Fig 2). Koshu derives its chloroplast DNA from an Asian wild species, as it clusters most closely with V. davidii (S1 Fig). Discussion The ancestry of some of the most common East Asian grape cultivars is often claimed to be derived exclusively from the domesticated grape, V. vinifera, which originated in the Caucasus region of Central Asia. Studies investigating these claims have so far been limited in marker number and/or sample size. Here we use a set of genome-wide ancestry informative SNPs and chloroplast DNA sequences to investigate the potential contribution of wild grape species to the genomes of present-day East Asian grape cultivars. Accessions of wild Asian grape species (V. amurensis, V. coignetiae, V. ficifolia var. ganebu, and V. shiragai) genotyped for the present study clustered closely in PC space with wild Asian species genotyped in a previous study using the Vitis9KSNP array [14] (Fig 1). In addition, the V. vinifera accession we genotyped here as a control (Pinot Noir) also clustered with the V. vinifera accessions genotyped previously [15]. Thus, we are confident that the genotype data obtained from the AIMs we identified are concordant with genotype data generated from the Vitis9KSNP array and that the use of merged data for the purposes of ancestry assignments is justified. Our analysis of 48 AIMs in the Japanese grapes Koshu and Koshu-sanjaku suggests that 20-30% of their genomes are derived from wild Vitis species (Fig 1). Thus, we conclude that Koshu and Koshu-sanjaku are not cultivars of V. vinifera, in contradiction to previous work [3,[7][8][9]. It is likely that hybridization with wild Vitis, intentional or by chance, occurred at some point in the history of these commercially successful Japanese wine grapes. We find that the chloroplast genome of Koshu appears most similar to V. davidii and not V. vinifera (Fig 2 and S1 Fig), indicating that at least one of its East Asian ancestors was a female plant. Although we can safely conclude that Koshu is an interspecific hybrid, we cannot conclusively identify the type or number of wild species in its lineage as these species may be extinct, unidentified, or simply missing from the present analysis. It is also worth noting that, based on our analysis of partial chloroplast DNA sequences, accessions from a single Vitis species did not always cluster together in genetic space: it was often the case that accessions from a single species were more genetically distant than accessions from different species (Fig 2 and S1 Fig). This may be due to curation error, or it may the case that there is incomplete lineage sorting: the partial chloroplast sequences studied here may segregate within and among Vitis species. The clear separation of North American and Asian wild Vitis species, together with the close relationship between V. vinifera and its wild ancestor V. vinifera subsp. sylvestris, provide support that the genetic distances based on chloroplast DNA sequences at least partially capture the evolutionary relationships among the samples studied here (Fig 2 and S1 Fig). A more comprehensive analysis of chloroplast DNA sequences is required to refine the relationship between Koshu and its chloroplast ancestor. The errors associated with the PCA-derived ancestry estimates presented here provide support to our conclusions and refine the possibilities of how wild introgression took place in the history of commercial East Asian grapes. Using simulated offspring from F1 and F2 hybrids between V. vinifera and wild Vitis species, Sawler et al. [12] used the same PCA-based approach to accurately estimate the simulated offsprings' V. vinifera ancestry with 95% confidence intervals of approximately ± 0.04. Assuming similar variation in estimates in the present study, Koshu (71.5% V. vinifera) could therefore be F2 plants derived from an F1 hybrid parent (V. vinifera x East Asian wild species) and a V. vinifera parent. We cannot rule out the possibility, however, that this cultivar has a more complex pedigree including wild Asian Vitis and V. vinifera. Koshu-sanjaku (81.8% V. vinifera) has a higher estimated proportion of V. vinifera ancestry than Koshu and, according to the estimated error, is unlikely to be an F2 hybrid as described above. Thus, Koshu-sanjaku is more likely to be the result of a complex pedigree with V. vinifera and East Asian wild Vitis relatives. Longan (91.7% V. vinifera) and Huotianhong (90.4% V. vinifera) both cluster close to the V. vinifera population in Fig 1. They may be genetically distinct V. vinifera cultivars or possess a very small proportion of wild Vitis ancestry. Finally, we determined that the Chinese cultivar Baijixin (100% V. vinifera) is in fact most likely a V. vinifera cultivar. With sufficiently dense genotype data from a comprehensive sample of V. vinifera and oriental cultivars, it may be possible in the future to identify the V. vinifera cultivars that contributed to the ancestry of commercial oriental cultivars. Taking into account the results of the SNP based ancestry estimation and observed genetic relatedness to wild Vitis based on chloroplast sequencing, the simplest hypothetical origin of Koshu is that it originated from a cross between an F1 hybrid (V. davidii or related x V. vinifera) and a second V. vinifera cultivar. V. davidii is a synonym of V. armata, and has a wide geographic distribution in China [20]. Cultivars of V. davidii are grown in China, and are used for both wine making as well as breeding stock. The branches of V. davidii possess many spines, and it is sometimes referred to as "spiny Vitis". The young shoots of Koshu have small spines that may be a result of V. davidii ancestry. In addition, V. davidii is highly resistant to disease and is tolerant to wet climates [20]. These traits may have contributed to the success of Koshu given the wet and hot summers in Japan. However, nearly all anthocyanins of V. davidii are delphinidin-based and diglucosides [21], which differ from those produced by Koshu. As previously discussed, V. davidii is only one of many possible ancestors of Koshu, and further work examining more samples and markers will be required to confirm or refute the conjectures outlined here. Studies of European grape cultivars have demonstrated introgression from local wild vines as humans brought grapes into Europe [15]. While our data do not enable us to determine the timing of the hybridization events that led to the East Asian cultivars studied here, there was likely ample opportunity for wild introgression during the journey of the domesticated grape along the Silk Road trade route to Japan. Further studies are required to refine the timing and precise nature of the hybridization events that led to the grapes currently grown commercially in East Asia. Supporting Information S1 Fig. NJ tree based on partial sequence of chloroplast DNA. Labels on the branches are bootstrap confidence values. The letters following the species names refer to the IDs used in [16]. (TIF) S1 Table. Primer sequence for the 48 ancestry informative SNPs genotyped. The SNP ID is composed of the chromosome, followed after the colon by the position according to the 8x Pinot Noir (PN40024) genome sequence [13]. (XLSX) S2 Table. SNPs of 10 grape accessions for 48 AIMs. The SNP ID is composed of the chromosome, followed after the colon by the position according to the 8x Pinot Noir (PN40024) genome sequence [13]. When only a single allele appears, it indicates that the sample is homozygous for that allele. (XLSX) S3 Table. Primers for partial sequencing of chloroplast DNA. (XLSX) role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

v3-fos

2019-08-18T19:27:25.403Z

2015-01-01T00:00:00.000Z

222239747

s2

Effect of irrigation and nitrogen fertilization on agronomic traits of sweet corn1 Brazil is highlighted as a major corn producer, with a great potential for also producing sweet corn (Zea mays var. saccharata Sturt) (Ferreira et al. 2011). Sweet corn is quite popular in temperate countries, such as the United States, Canada and European nations, but not in Brazil (Borin et al. 2010). The lack of improved varieties and knowledge about cultivation techniques under ABSTRACT RESUMO INTRODUCTION Brazil is highlighted as a major corn producer, with a great potential for also producing sweet corn (Zea mays var. saccharata Sturt) (Ferreira et al. 2011). Sweet corn is quite popular in temperate countries, such as the United States, Canada and European nations, but not in Brazil (Borin et al. 2010). The lack of improved varieties and knowledge about cultivation techniques under ABSTRACT RESUMO tropical conditions contribute to the low interest on sweet corn in Brazil. The sweet corn crop cycle lasts from 90 to 100 days (Teixeira et al. 2009), allowing its production throughout the year (Zárate et al. 2009). It can be grown under a monocrop or intercrop system, and it is an alternative for small and medium farmers (Rocha et al. 2011). Almost all its production is destined for human consumption, either processed or in natura (Araújo et al. 1999, Pereira et al. 2009). 1. Manuscript received in Jan./2015 and accepted for publication in Aug./2015Aug./ (http://dx.doi.org/10.1590Aug./ /1983 Irrigation and nitrogen fertilization are management practices that have positive results for the corn crop. This study aimed at evaluating the effect of nitrogen fertilization and irrigation on agronomic traits of sweet corn. Two experiments were carried out in two crop seasons (winter/spring and summer/autumn), in a split-plot design, with the main plots consisting of four irrigation levels (50 %, 75 %, 100 % and 125 % of the crop evapotranspiration -ETc) and subplots consisting of four nitrogen doses (0 kg ha -1 , 100 kg ha -1 , 200 kg ha -1 and 300 kg ha -1 ), applied at the V3 and V8 stages, via urea, in a randomized blocks design experiment, with four replications. Leaf nitrogen content, root depth, plant height, stem diameter, ear yield and water use efficiency were evaluated. In the winter/spring season, nitrogen fertilization did not affect yield, while in the summer/autumn season the dose that maximized yield was 300 kg ha -1 . Sweet corn showed better results when irrigated with replacements of 50 % and 125 % of ETc, respectively in the summer/autumn and winter/ spring seasons. KEY-WORDS: Zea mays var. Saccharata Sturt; drip irrigation; specialty corn; water use efficiency. The instability of water regimes may restrict the development of corn crops, but the proper use of irrigation and nitrogen fertilization may improve yield and minimize risks during the production process (Borin et al. 2010). A great amount of nitrogen is absorbed by sweet corn, and its availability affects yield (Okumura et al. 2011). This fact induces producers to use fertilizers in larger quantities, expecting to increase yield. However, Farinelli & Lemos (2010) argue that the increase of nitrogen fertilizer doses reduces its efficiency and, as a result, the economic and environmental damages are increased. The increase of efficiency can be achieved by identifying the doses that maximize the fertilization effect. This study aimed at evaluating the effect of irrigation and nitrogen fertilization on the agronomic traits of sweet corn grown in two crop seasons, in the northeast of Mato Grosso do Sul State, Brazil. MATERIAL AND METHODS The experiment was conducted at the Universidade Federal de Mato Grosso do Sul (UFMS), in Chapadão do Sul (18º46'24"S, 52º37'25"W and 820 m of altitude), Mato Grosso do Sul State, Brazil. The climate is classified as humid tropical. Air temperature, relative humidity and rainfall averages, during the experimental period, are shown in Figure 1. The soil of the experimental area was classified as clayish red-yellow Dystrophic Latosol (Oxisol), with density of 1.21 g cm -3 , field capacity water content of 0.2632 dm 3 dm -3 and plant permanent wilting point of 0.1887 dm 3 dm -3 . The soil chemical properties were evaluated before each harvest in laboratory (Table 1). Two experiments with sweet corn were conducted in two distinct seasons: winter/spring (August 17th to November 24th, 2012) and summer/ autumn (March 3rd to May 31st, 2013). The experiments were arranged in a split-plot randomized blocks design, with four replications, where irrigation regimes were the plots (50 %, 75 %, 100 % and 125 % of the crop evapotranspiration -ETc) and nitrogen doses (0 kg ha -1 , 100 kg ha -1 , 200 kg ha -1 and 300 kg ha -1 ) the subplots. Experimental units consisted of 2.5 m long (0.5 m of border) and 4.8 m wide (0.8 m of border) The soil preparation consisted of plowing and harrowing. Soil acidity was corrected according to Sousa & Lobato (2004) and fertilization at sowing was carried out based on soil chemical properties (Sousa & Lobato 2004) (Table 1). Fertilization at sowing consisted of 60 kg ha -1 of P 2 O 5 , 60 kg ha -1 of K 2 O and 30 kg ha -1 of N for the first season (winter/ spring), and 120 kg ha -1 of P 2 O 5 , 60 kg ha -1 of K 2 O and 30 kg ha -1 of N for the second crop (summer/autumn). The nitrogen, phosphorus and potassium sources were respectively urea, single superphosphate and potassium chloride. The sweet corn was sown on August 17th, 2012 (winter/spring), and February 03rd, 2013 (summer/autumn), spaced 80 cm between rows, with density of 75,000 seeds ha -1 . The hybrid used was the Tropical Plus ® (Syngenta), which has high yield potential, 90-110 days cycle, light yellow grain color, thin pericarp, sweet flavor and resistance to major diseases. The sidedress nitrogen fertilization was divided and applied in the V3 and V8 phenological stages (Magalhães & Durães 2006). The urea was applied in the plant rows, next to the drip tapes, which were turned on after fertilization to minimize volatilization losses. At V3, potassium fertilization was also applied at 80 kg ha -1 of K 2 O. A drip irrigation system was used, with the following equation applied to calculate the actual irrigation required to treat 100 % of the ETc: where: AIR LOC = actual irrigation required in localized irrigation systems (mm); ET 0 = reference evapotranspiration (mm day -1 ); K C = crop coefficient (dimensionless); K S = soil moisture coefficient (dimensionless); K L = location coefficient (dimensionless); P E = effective rainfall in the period (mm). The Penman-Monteith methodology was used to calculate the reference evapotranspiration (ET 0 ). Crop coefficients (K C ) were 0.7 for the stage I (1st to 20th day after planting) and 1.1 for the stage III (51st day to harvest) (Bernardo et al. 2008). The K C daily values for the stage II (21st to 50th day) were obtained using the linear weighting between the values from the stages I and III. Soil moisture coefficients (K S ) and location (K L ) were established according to Bernardo et al. (2008). The aboveground phenotypic evaluations were performed when the plants were at full male flowering. Ten plants were randomly sampled within the useful area of each plot. The evaluations were: a) leaf nitrogen content: the central third from ten opposite leaves below the ear were collected (Carmo et al. 2012) and evaluated according to Silva (2009); b) plant height: length (cm) from the ground level to the highest leaf insertion point, using a tape measure (Carmo et al. 2012); c) stem diameter: diameter of the second internode (largest diameter), measured by a caliper (Carmo et al. 2012). The corn ears were harvested at the R3 phenological phase (November 24th, 2012, for the winter/spring, and May 31st, 2013, for the summer/ autumn), in the early morning hours (Kwiatkowski & Clemente 2007). The yield comprised all corn ears from the useful area of each experimental unit, which were subsequently weighed (kg plot -1 ), with values extrapolated to t ha -1 (Carmo et al. 2012). After harvest, longitudinal trenches were opened following the plant lines up to the last roots of the sweet corn, plus the excavation of 20 cm to confirm the absence of roots. Roots were measured (cm) from the ground surface to the last root exposed, with a tape measure. The water use efficiency was determined by the ratio between sweet corn yield and amount of water used in each treatment. Data were submitted to regression analyzes, testing the linear and quadratic models. The models were chosen based on the significance of the regression coefficients (t test, at 5 %), coefficient of determination (R 2 ) and on the biological phenomenon. The program Sigmaplot v11.0 was used to perform the statistical analyses. RESULTS AND DISCUSSION Effective rainfall was higher for sweet corn in the winter/spring season (Table 2), due to the fact that rainfall was concentrated in periods when the crop presented higher values for crop (K C ) and location (K L ) coefficients ( Figure 1). The sweet corn grown in the winter/spring season (Table 2) showed higher water consumption because of two main reasons: a) greater water demand caused by higher daytime temperatures and lower air humidity (Figure 1), which coincided with the period when the sweet corn reached higher crop coefficient values; b) a longer crop life cycle during this season (99 days, whereas in the summer/autumn it was 90 days). Both crop cycles were within expectation: 90 to 100 days (Tan et al. 2009). In both crop seasons irrigation regimes had a negative linear effect on leaf nitrogen content (Figure 2), probably due to the fact that sweet corn changes the carbon allocation to non-nitrogenous compounds, such as cellulose and lignin, rather than to proteins and amino acids. Chun et al. (2005) stated that the increase in carbon allocation from shoot to root formation, aiming at increasing surface area, results in a reduced nitrogen content in corn leaves. Franco et al. (2008) evaluated leaf nitrogen content of Urochloa decumbens, using two water levels (20 % and 60 % of the soil maximum water retention capacity), and observed an increase of 10.3 % in nitrogen content in the lower water level treatment, when compared to the higher one. The nitrogen content in the sweet corn cultivated in the summer/autumn season was not affected by the increase in nitrogen doses. Two possible reasons for this are: a) data variability resulted in a low value for the coefficient of determination, consequently affecting the adjustment of the regression equation; b) a proximity to the optimum point of leaf nitrogen content was reached for sweet corn in that season, hindering the response to the application of higher nitrogen doses. Nascimento et al. (2012) report that the adequate nitrogen content for corn crop ranges from 2.7 % to 3.5 %. Regardless of crop season, the irrigation regimes had a negative linear effect on the depth of sweet corn roots (Figure 2). In treatments with lower water depths, probably plants deepened their roots, in order to search for water at deeper soil layers. Schlichting et al. (2015) also observed this trend in the growth of corn roots as a defense to water stress conditions. The nitrogen doses had no effect on the depth of sweet corn roots (Figure 2). Soares et al. (2009) also found no difference for root depth in six corn cultivars, applying two nitrogen doses (zero and 6 mmol L -1 of soil) in a Red Latosol. According to these authors, high nitrate concentrations can even reduce root growth, since this element inhibits the auxin flow to the roots. The irrigation regimes had a quadratic effect on plant height in the winter/spring season (Figure 2). According to the regression equation, the water depth that maximized plant height was 111.3 % of ETc, with plants reaching 1.77 m. In the summer/autumn season, this effect was linear negative. Regardless of crop season, the nitrogen doses had no effect on plant height (Figure 2), as also observed by Valderrama et al. (2011). According to these authors, the crop response depends on the cropping history of the area, on weather conditions and nitrogen fertilization. The cultivar and plant density also influence this effect. On the other hand, some studies have shown positive response in plant height with nitrogen fertilization (Silva et al. 2006, Khazaei et al. 2010, Pereira Júnior et al. 2012. The lack of response in this study may have occurred because the nitrogen availability in the soil was already at optimum levels for the crop. This hypothesis is supported by the lack of response on leaf nitrogen content (Figure 2). The irrigation regimes had a quadratic effect on stem diameter, in the summer/autumn season (Figure 3). According to the regression equation, the water depth that maximized the stem diameter was 83.0 % of the ETc, resulting in a stem of 19.89 mm. According to Calonego et al. (2011), the increase in plant height reduces the stem diameter, partly agreeing with the results in this study, as observed in the summer/autumn season (Figures 2 and 3). Regardless of crop season, the nitrogen fertilization had no effect on stem diameter (Figure 3). The irrigation regimes had a positive linear effect on ear yield, in the winter/spring season (Figure 3), showing that there was water restriction in treatments with lower water depths. This result suggests an irrigation regime of 125 % of the ETc for the winter/spring season. In the summer/autumn season, the irrigation regimes had a negative linear effect on ear yield ( Figure 3). Therefore, an irrigation regime of 50 % of the ETc is suggested in this season, in order to ensure higher yield and lower water and electricity loss. This response was not expected, but may be explained by the fact that rainfall in this crop season was concentrated at the beginning of the cycle. Therefore, treatments that received lower water depths had no water restrictions, and treatments with larger water depths had moisture contents near to field capacity, preceding rainfall, resulting in higher water percolation and possibly nutrient lixiviation. Another possible explanation is based on the root system performance (Figure 2). The increase in irrigation reduced the roots' depth, and consequently the soil volume explored by them, possibly decreasing the nutrients input by the sweet corn. This hypothesis has support on the lower leaf nitrogen content with increasing irrigation (Figure 2). The same effect was also observed in the winter/spring season, although in a milder way, as observed in the regression coefficients ( Figure 2). According to the equations in Figure 2, the root depths in treatments irrigated with 125 % of the ETc were 60 cm in the summer/autumn season and 95 cm in the winter/spring. Heinemann et al. (2009), evaluating common corn in different locations in the Goiás State, Brazil, concluded that water deficit stress is not the main impediment to the development of corn crops in normal seasons. The actual concern occurs in soils that hinder root development by physical, chemical or biological factors. The nitrogen doses had a positive linear effect on ear yield, in the summer/autumn season (Figure 3). According to Okumura et al. (2011), the increase of nitrogen doses increases the composition of amino acid, protein, chlorophyll and other essential enzymes that stimulate the sweet corn growth and development. The nitrogen response was related to the irrigation regimes, since the nitrogen fertilization increased yield in treatments with lower water depths. Winter/spring Summer/autumn SD = 1.9E + 1 SD = 1.3E + 1** + 1.7E -1*ID -1.0E -3*ID 2 R 2 = 0.2983 p = 0.0230 EY = 1.1E + 1** + 5.1E -2**ID R 2 = 0.6334 p = 0.0010 EY = 2.4E+1** -8.3E -2**ID + 9.5E -5*ID ND R 2 = 0.8192 p < 0.0001 WUE = 4.0E + 0** + 9.4E -3*ND -4.6E -5*ID ND -2.1E -5*ND 2 R 2 = 0.4993 p < 0.0196 WUE = 7.6E + 0** -3.0E -4*ID 2 + 2.9E -7*ID 2 ND R 2 = 0.9240 p < 0.0001 regime of 50 % of the ETc and nitrogen fertilization of 300 kg ha -1 . This value exceeds the highest yield (19.5 Mg ha -1 ) found by Carmo et al. (2012), applying 150 kg ha -1 of nitrogen in a summer crop in Palmeiras, Goiás State, Brazil. In the winter/spring season, nitrogen doses did not increase yield (Figure 3), agreeing with Aguiar et al. (2012), which applied nitrogen doses from 0 to 144 kg ha -1 to sweet corn grown in Gurupi, Tocantins State, Brazil. The effect of nitrogen doses was lower than expected for some parameters, probably due to inadequate potassium levels or to the interaction between absorption and use of these two macronutrients (Costa et al. 2008). The reduction of potassium contents in the soil (Table 1) between the two crop seasons, and the fact that the potassium supplementation was not proportional to the nitrogen doses, in order to balance the interaction between these two elements, reinforces this hypothesis. This interaction is related to the activity of the nitrate reductase enzyme, which acts in the inorganic nitrogen incorporation (Silva et al. 2011). Researches applying sidedress fertilization in corn at the V4 and V6 stages reached positive results (Repke et al. 2013, Rotili et al. 2014. Therefore, the methodology used in the present research, applying nitrogen at the V3 and V8 stages, suggests that the sidedress fertilization occurred too early or too late, influencing the effect of the nitrogen doses in the evaluated parameters. Thus, new researches should be developed to study this hypothesis. The water use efficiency decreased as the water depth increased (Figure 3). This was already expected, since these factors are inversely proportional. In the summer/autumn season, the reduction in water use efficiency was higher also due to the reduced ear yield. In the winter/spring season, nitrogen fertilization had a quadratic effect on water use efficiency. According to the regression equation, the nitrogen dose that maximized this parameter was 168.4 kg ha 1 (Figure 3). Nitrogen doses in the summer/autumn season had a positive linear effect on water use efficiency. 2. In the winter/spring crop season nitrogen fertilization did not affect yield, while in the summer/autumn the nitrogen dose that maximized the sweet corn yield was 300 kg ha -1 .

v3-fos

2018-04-03T01:01:25.892Z

2015-04-21T00:00:00.000Z

27763147

s2

Interaction between Meloidogyne incognita and Rhizoctonia solani on green beans The interaction between Meloidogyne incognita (race 2) and Rhizoctonia solani (AG 4) in a root rot disease complex of green beans (Phaseolus vulgaris) was examined in a greenhouse pot experiment. Three week-old seedlings (cv. Contender) were inoculated with the nematode and/or the fungus in different combinations and sequences. Two months after last nematode inoculation, the test was terminated and data were recorded. The synchronized inoculation by both pathogens (N + F) increased the index of Rhizoctonia root rot and the number of root galls; and suppressed plant growth, compared to controls. However, the severity of root rot and suppression of plant growth were greater and more evident when inoculation by the nematode preceded the fungus (N → F) by two weeks. Nematode reproduction (eggs/g root) was adversely affected by the presence of the fungus except by the synchronized inoculation. When inoculation by nematode preceded the fungus, plant growth was severely suppressed and roots were highly damaged and rotted leading to a decrease of root galls and eggs. Introduction Green beans (Phaseolus vulgaris L.) is a very important summer vegetable crop in Saudi Arabia, and grown in the open fields and greenhouses mainly for its green pods. The crop is frequently attacked by Meloidogyne javanica (Treub), Chitwood and Meloidogyne incognita (Kofoid & White) Chitwood (Al-Hazmi, 1985;Al-Hazmi et al., 1995). Green beans are also very susceptible to Rhizoctonia solani Kuhn (Hall, 1991). In a field survey of fungal pathogens associated with green beans in the central region of Saudi Arabia, 17 species of pathogenic fungi were recorded (Al-Osaimi, 2005). Among these fungi, R. solani was found to be the second most common species and the most severe on green beans. The fungus R. solani was found frequently associated with the root-knot nematodes. Interaction between these two pathogens in our field soils might play a very damaging role in our green bean fields. Since the first recorded case of nematode-fungus interaction in 1892 (Atkinson, 1892), interest in such interactions and their damages to many economic crops has been attracting many scientists. Interactions between the root-knot nematodes (Meloidogyne species) and the root rot fungus R. solani have been studied and documented in several host crops including green beans. Several reviews on the subject have been published (Powell, 1971;Back et al., 2002;Shahzad and Ghaffar, 1992;Mai and Abawi, 1987;Evans and Haydock, 1993). Several reports indicated that Rhizoctonia-root rot was more severe in the presence of root-knot nematodes, including the root rot disease complex caused by R. solani and M. incognita on green beans (France and Abawi, 1994;Mokbel et al., 2007;Abuzar, 2013;Ali and Venugopal, 1992;Batten and Powell, 1971;Chahal and Chhabra, 1984;Shahzad and Ghaffar, 1995;Sharma and Gill, 1979;Anwar and Khan, 2002;Reddy et al., 1979;Bhagwati et al., 2007). Most of these reports indicate a synergistic interaction between these two important pathogens. Rhizoctonia-root rot generally affects seedlings, but fungus can also infect mature plants and induce root rot leading to plant wilt and finally death of infected plants. The objective of this present study was to evaluate the interaction of M. incognita (race-2) and a local isolate of R. solani in a root rot disease complex of green beans (cv. Contender) under the greenhouse conditions. The nematode inoculum consisted of eggs which were extracted in 0.05% sodium hypochlorite (Hussy and Barker, 1973) from a pure greenhouse culture of M. incognita (race-2) on tomato plants. The egg suspension was immediately washed several times with sterilized distilled water, and then, adjusted to contain 1200 eggs/ml of the suspension. The nematode inoculum used was 12,000 eggs/seedling. A pure culture, on Potato Dextrose Agar (PDA), of R. solani originally isolated from green bean plants from local fields, was obtained from the Mycology Unit (Dr. Saleh El-Hussaini), laboratory of fungal plant diseases, Department of Plant Protection, King Saud University, Riyadh, Saudi Arabia. The fungus was, then, maintained on PDA in petri plates (at 25-27°C) for a week. For inoculum preparation, 250 ml conical flasks, each containing about 10 g of barley grains soaked overnight in sterilized distilled water, were used. The media in flasks were autoclaved for 30 min in two consecutive days. After the flasks were cooled, each one was inoculated with a small block (5 mm diam.) taken from the periphery of the 7-day-old cultures on PDA. The flasks were, then, incubated at 27 ± 2°C for two weeks. During incubation, the flasks were shaken twice a day to ensure the proper growth of fungal mycelium on the barley seeds. The fungal-colonized barley seeds were used as inoculum at the rate of 15 g/seedling. The green bean (Phaseolus vulgaris L.) cultivar used in this study was ''Contender'' which is known to be susceptible to both M. incognita (race-2) and the fungus R. solani. Uniform 3-week-old seedlings were transplanted singly into sterilized plastic pots (14 cm diam.) containing a steam-sterilized mixture (1500 g soil) of equal parts of sand, soil, and peat moss. The seedlings were fertilized with Hogland's solution and left in the greenhouse for two weeks before treatments. At inoculations with nematode and/or fungus, each seedling was inoculated with 12,000 eggs and/or 15 g of the fungus inoculum on barley grains depending on the designated treatment (Table 2). Non-inoculated seedlings served as control. The nematode egg inoculum, suspended in 10 ml of water, was equally distributed through three small holes made in the soil around the seedling stem and deep enough to contact the roots. Inoculation with the fungus was made by distributing and mixing the fungal inoculum thoroughly with the soil surface of the designated pots. Each treatment was replicated five times, and treatments were arranged on a bench in the greenhouse (25-27°C) in a completely randomized design. All seedlings were irrigated and fertilized with Hogland's solution as needed. Sixty days after inoculation, plants were uprooted, washed under tap water and growth parameters were recorded. Nematode infection was determined by the number of root galls and host growth, whereas the nematode reproduction was determined by the number of eggs on roots. Gall index (Taylor and Sasser, 1978) and reproduction factor (RF) of the nematode (Oostenbrink, 1966) were calculated. Root rot of each root system was determined according to four categories of root system necrosis: 0 = none; 1 = less than 25%; 2 = 26-50%; 3 = 51-75%; 4 = 76 = 100% (Aoyagi et al., 1998). Disease severity of root rot was also calculated according to the formula by Aoyagi et al. (1998). Values are means of five replicates. Means, in each column, followed by the same letter (s) are not significantly, different at P 6 0.05. * Sequences: N + F = simultaneous inoculation, N fi F = nematode applied 2 weeks before fungus, F fi N = fungus applied 2 weeks before nematode. ** On a scale of 0-4, where 0 = healthy; 1 = 1-25%; 2 = 26-50%; 3 = 51-75%, and 4 = 76-100% root area is infected (Aoyagi et al., 1998). *** % disease severity ¼ P Disease index  No: plants in each category of the index Higher value of the index  No: of all inoculated plants  100 (Aoyagi et al., 1998). Data were statistically analyzed (SAS, 2013), and treatment means were separated by protected Fisher's least significant difference (LSD). Results Our results showed that root rot indices and disease severity (%) increased (P 6 0.05) in plants inoculated with both pathogens (N + F) compared to the plants inoculated with the fungus alone (F) ( Table 1). However, the greatest root rot disease was observed when the nematode preceded the fungal inoculation by two weeks (N fi F). This last increase was significantly higher than that of the simultaneous inoculation (N + F). The reciprocal treatment (F fi N) did not increase the root rot disease compared to treatment of fungal alone (F) ( Table 1). The total fresh weights of plants inoculated with either or both pathogens were reduced (P 6 0.05) compared to the non-inoculated plants (Table 2). This suppression of plant growth was more (P 6 0.05) severe (À78.0%) when the nematode preceded the fungal inoculation by two weeks (N fi F). In fact, plants in this treatment (N fi F) were severely damaged and their very small roots were severely rotted and, subsequently, had lesser galls and egg production ( Table 2). Number of root galls in the other two treatments (N + F, and F fi N) were higher than the nematode alone (N) treatment ( Table 2). Reproduction of M. incognita was adversely affected (P 6 0.05) when the fungus preceded the nematode inoculation (F fi N) (Table 3). In the reciprocal treatment (N fi F), the roots were severely damaged and had fewer eggs. The concomitant inoculation with both pathogens (N + F) showed similar nematode reproduction to that caused by the nematode alone (N) ( Table 3). Discussion Our results showed that the index of root rot caused by R. solani has increased in the presence of the root-knot nematode M. incognita. However, the severity of the root rot disease was more (P 6 0.05) pronounced when the inoculation by the nematode preceded the fungus by two weeks (N fi F). This increase of root rot indicates that the infection by both pathogens, whether simultaneously or the nematode first, would result in a synergistic interaction leading to a greater plant damage (1 + 1 > 2). This was supported by the reciprocal treatments when the fungus preceded the nematode (F fi N) where the severity of the root rot was not significant compared to the infection by the fungus alone. The present results support previous similar reports of synergistic effects of R. solani and M. incognita, on different crops, such as mung beans (Shahzad and Ghaffar, 1995), cardamom (Ali and Venugopal, 1992), sunflower plants (Mokbel et al., 2007), cotton (Carter, 1981), okra (Bhagawati et al., 2007;Safiuddin and Shahab, 2012;Safiuddin et al., 2014), tomato (Chahal and Chhabra, 1984), chili (Abuzar, 2013), potato (Sharma and Gill, 1979), eggplant (El-Nagdi and Abd-El-Khair, 2008), grapes (Walker, 1994) and on other vegetables and field crops (Shahzad and Ghaffar, 1995). The increase of root rot severity in the presence of M. incognita may be due to the fact that the infection by this endo-parasitic nematode (M. incognita), whether simultaneously (N + F) or prior to the fungal infection (N fi F), causes physiological and anatomical great changes in the root tissues leading to predisposing the plants to increased fungal infection (Carter, 1981;Powell, 1968;Porter and Powell, 1967;Batten and Powell, 1971). Giant cells induced by Meloidogyne spp. often make suitable sites for the pathogenic and non-pathogenic soil-borne fungi (Mai and Abawi, 1987;Mayol and Bergeson, 1970). All these changes caused by the nematode may explain the great increase of root rot and damage in the plants infected first by the nematode (N fi F) compared to the plants in the other two treatments of fungus alone (F) or fungus first (F fi N). Similar findings were reported by other researchers (Reddy et al., 1979;Ghaffar, 1992, 1995;Ali and Venugopal, 1992). Root galls increased in both the synchronized treatment (N fi F) and when the fungal preceded the nematode inoculation (F fi N). This might be attributed to the increase of the penetration rate by J2 into the roots (Tu and Cheng, 1971). However, the plants inoculated with the nematode first (N fi F) were much greatly damaged and their roots were severely rotted. Therefore, these roots became unsuitable for juvenile penetration and nematode development, and have much less galls and eggs. Growth of plants inoculated with the nematode and/or the fungus was suppressed (32-78%), supporting similar previous findings (Reddy et al., 1979). However, the greatest suppression of growth (78%) was observed when the nematode preceded the fungal inoculation. Undoubtedly, the pathogenic physiological Values are means of five replicates. Means followed by the same latter are not significantly different at P 6 0.05. * Sequences: N + F = simultaneous inoculation, N fi F = nematode applied 2 weeks before fungus, F fi N = fungus applied 2 weeks before nematode. ** Plants in this treatment were severely damaged. Values are means of five replicates. Means, in each column, followed by the same letter (s) are not significantly, different at P 6 0.05. * Sequences: N + F = simultaneous inoculation, N fi F = nematode applied 2 weeks before fungus, F fi N = fungus applied 2 weeks before nematode. ** RF = Pf/Pi. alterations caused by the nematode and/or the fungus are the main factor for this growth suppression. It has been also reported that green beans infected with M. incognita contain much less quantities of chlorophyll, carbohydrates and nitrogenous compounds; and show low capacity to absorb water and nutrients from soil, resulting in a growth and yield decrease (Melakeberhan et al., 1985;Wilcox and Loria, 1986). The reproduction of M. incognita (eggs and RF) was suppressed by the presence of the fungus R. solani, especially in the two sequential treatments (F fi N, and N fi F). This finding agrees with previous similar reports on the adverse effects of several soil-borne fungi on the reproduction of Meloidogyne spp. on several crops (Mokbel et al., 2007;Al-Hazmi, 1985;France and Abawi, 1994;Griffin and Thyr, 1988;Sharma and Gill, 1979;Powell, 1971;Mousa and Hague, 1988). The great damage on roots caused by R. solani may have affected the nematode feeding process within the root tissues and, subsequently, adversely affected the nematode reproduction. Furthermore, the toxic metabolites produced by many pathogenic fungi may have deteriorated the giant cells which are necessarily for the nematode feeding and, then, reproduction, as well as the juvenile movement (Fattah and Wbester, 1989;Mokbel et al., 2007). These effective damages on nematode biology may explain the decrease of a number of eggs produced by the nematode. Development of a successful strategy to manage this nematode/fungus disease complex should depend primarily on an applicable integrated disease management including suitable different methods to suppress the population of both pathogens in our field soils.

v3-fos

2019-04-12T13:55:11.557Z

2015-08-11T00:00:00.000Z

109081317

s2

Hydrogeochemical Characteristics and Quality Assessment of Groundwater in University of Science and Technology Port Harcourt The quality and suitability of boreholes water quality in the Rivers State University of Science and Technology were assessed for potability and irrigation purposes by analyzing the water for physico-chemical parameters, microbial contents and irrigation indices using standard methods. The results obtained were compared with permissible limits for drinking water provided by World Health Organization and Standard Organization of Nigeria The results showed pH ranged from 4.09 6.77 at 26.4 30.3°C, Turbidity <0.01 NTU in all the samples, Electrical Conductivity 20 407 μS/cm, Salinity <0.01 0.20‰,TDS 12 274 mg/l, Chloride <1.0 12.3 mg/l, Sulphate <1.0 15.5 mg/l, Phosphate <0.05 1.9 mg/l, Nitrate 0.30 6.20 mg/l, Total Alkalinity (as CaCO3) 2 8 mg/l, Total Hardness <0.1-34.6 mg/l, Calcium <0.08 9.2 mg/l, Magnesium <0.05 2.8 mg/l, Sodium <0.01 44.62 mg/l and Potassium <0.01 11.88 mg/l. The results of microbial analyses showed Total Heterotrophic Bacteria population ranged from Nil 3000 cfu/ml, Total Coliform Bacteria 0 210 MPN/100ml while Faecal Coliform Bacteria were not present in all the samples. The Groundwater within the University is fresh, soft and has low pH. The water in some parts had high microbial count and therefore not suitable for drinking. The ground water in the area should be regularly monitored and treated to avoid serious pollution problems. The irrigation indices showed the water is suitable for irrigation and other purposes. Introduction The Niger Delta region is one of the largest wetlands in the world. Some rural inhabitants take what they can from the creeks, ponds and rivers. The Federal Ministry of Water Resources says efforts over the past century to develop National water resources have not yielded much. [1] quoted Central Bank of Nigeria statement that "the population of Nigerians with access to potable water rose from 30% in 1999 to 65% in 2005". Water is an essential requirement for human and industrial developments. It is also used directly and indirectly by many people for several purposes. Water in general plays a critical part in the maintenance of plant and animal life. Owing to the presence of water in cells and body fluids, such as blood, human beings are approximately 60 -75% water [2]. The major sources of water include springs, ponds, streams, rivers, oceans, rain and ground water. The variety of water sources brings in water with different degrees of impurities. The presence of impurities therefore reduces the use to which the water may be deployed. In this study on Rivers State University of Science and Technology (RSUST), it is limited to bore holes. For potable water, it must be safe for human consumption while the one for irrigation and some industrial processes may not be as pure as that for human consumption. So water must therefore be analyzed to determine its acceptability for the intended purpose. The levels of parameters obtained may be the cause for rejection or acceptance of the sample. Sources of water for various uses include atmospheric (i.e. rainwater), surface water (i.e. streams, rivers, ponds, lakes and dams) and groundwater (i.e. springs, wells and mono pumps / boreholes). As a result of prevailing conditions connected with some of these sub-water sources; most industrial, governmental and private sectors in urban areas resort to boreholes as water source for their potable and various needs [3]. Sources of groundwater contamination or pollution include leachate from landfill / refuse dumpsite, industrial liquid effluent, domestic waste, agricultural waste, salt water intrusion, oil pollution and geological formations [4]. Many wells (i.e. groundwater sources) in the United States of America have been closed because of contamination by various toxic substances [5]. In the Niger Delta, water is mostly abstracted from the aquiferous Benin Formation [6]. In urban and industrial areas; hydrochemistry, geochemistry and processes at solid / liquid interfaces are among the important issues in environmental risk assessment studies in water resources policies [7]. The chemical composition of groundwater is dependent on the geology and the geochemical processes within the aquifer [8], [9]. Thus, water quality analyses are focal and imperative in groundwater investigation by monitoring both the water level (where possible) and trends of the water quality parameters that are influenced by the geological formations and the anthropogenic activities in a given area [10]. The contamination of shallow groundwater sources leading to incidents of water borne diseases like abdominal disorders, typhoid fever, dysentery and urinary track infectious has been reported in some communities [11], [12]. The main objective is to determine, establish and document the current status of boreholes water quality on the University campus Port Harcourt, with population well over 1 million lies within latitudes 4 º 43´ 07 " and 4 º 54´ 32 " N and longitudes 6 º 56´ 04 " and 7 º 03´ 20 " E with a mean annual rainfall of over 2000mm and mean annual temperature of 29 º C [13]. The study area Rivers State University of Science and Technology ( Fig. 1.1 [14]. The domination of loose sands in the Benin Formation makes the ground in Port Harcourt porous and permeable to wastes on the soil surface. This is because during the rainy season, rainwater will cause leachates from the wastes to percolate downwards and pollute the groundwater over time. Source: Urban & Regional Planning Dept. RSUST, P.H. The ground water level, measured from newly drilled boreholes show that water levels are close to the surface, commonly between 3.05 -9.14metres. For example, the water level at East -West (Nkpolu, Rumuigbo) is 3.05metres but about 6.10metres at Eneka, etc. [15]. Sample Collection Samples for physico-chemical analysis were collected in 1.5-litre plastic bottles while those for microbiology were collected in sterile McCartney bottle. All the samples were preserved in iced cool box and transported to the laboratory for analyses. Table 1 shows the sampling stations and GPS Co-ordinates taken with a hand held GPS by Garmin. Field Measurements Some in situ measurements were taken in the field for pH, Temperature, Conductivity, Salinity and Total Dissolved Solids using Extech DO700 meter calibrated with buffer pH 4.0, 7.0 and 10.0 as well as 1413 µS/cm conductivity solution. Laboratory Analyses Except otherwise stated, the laboratory methodologies used were from Standard Methods for the Examination of Water and Wastewater by American Public Health Association [16] and American Society for Testing & Material [17]. Total Alkalinity was determined by titration with 0.02N H 2 SO 4 -using methyl orange indicator [16]. Chloride was determined titrimetrically by the Argentometric method in the presence of potassium chromate as the indicator. Phosphate was determined by the Ammonium Molybdate (NH 4 ) 2 MoO 4 ) method. Nitrate was determined using the Brucine Method [16]. Sulphate was determined by Turbidimetric method [16]. 100mls of water sample was taken. 2mls of buffer solution was added along with a pinch of Eriochrome black T indicator and titrated with 0.0IM EDTA until a blue colour was observed. Sodium and Aluminium were determined by direct aspiration into Flame Atomic Emission Photometer. Microbiology The ten-fold serial dilution was used to obtain appropriate dilutions of the samples. Aliquots of the required dilutions were plated in duplicates onto the surface of dried sterile nutrient agar plates for total heterotrophic bacteria. In the case of total/faecal coliform bacteria, the most probable number (MPN) technique was employed for estimation of their numbers in water. Appropriate volumes of undiluted water samples were inoculated into test tubes of MacConkey broth containing Durham tubes. All inoculated media were incubated at 37°C for 24 hours except for faecal coliform bacteria which was incubated at 44°C [16]. Results The range, mean and standard deviation values of the physico-chemical as well as microbiological properties of groundwater samples within the Rivers State University of Science and Technology for July and September 2013 are shown in Table 2. Also contained in the Table 2 are Standard Organization of Nigeria (SON) limits for drinking water and World Health Organization (WHO) standards [18], [19]. Physico-Chemical Parameters pH The ground water pH ranged from 4.09 -6.77 with a mean of 4.93±0.58. In July all the samples (100%) were in the range 4.30-5.40 while in September 12.5% of the samples were within 6.73-6.77 range whereas 87.5% of the samples had pH ranging from 4.09-6.39. The lowest pH 4.30 came from ISS2 in July while in September pH 4.09 was obtained at the Main Gate Motor Park (Fig. 2). Temperature and Turbidity Groundwater Temperature ranged from 26.4 -30.3°C with a mean of 28.3±1.2°C. Turbidity was <0.05 NTU in all the samples. Conductivity, Salinity and TDS Conductivity values ranged from 20 -407 µS/cm with a mean of 103±102 µS/cm. Salinity values ranged from <0.01 -0.20‰ with a mean of 0.03±0.06‰. Similarly, TDS values varied from 12 -274 mg/l with a mean of 67±67 mg/l (Fig. 3). The correlation existing among the parameters measured are shown in Table 3 Chloride, Sulphate, Phosphate and Nitrate Chloride concentrations ranged from <1.0 to 12.3 mg/l with a mean of 2.9±2.6 mg/l. Similarly, Sulphate concentrations varied from <1.0 -15.5 mg/l with a mean of 2.4±3.0 mg/l. Phosphate concentrations ranged from <0.05 -1.9 mg/l with a mean of 0.05±0.29 mg/l while the nitrate concentrations varied from 0.30 -6.20 mg/l with a mean of 2.65±1.32 mg/l. Total Alkalinity and Hardness Total Alkalinity values (as CaCO 3 ) were from 2 -8 mg/l with a mean of 3.8±1.8 mg/l whereas the Hardness values varied from <0.1-34.6 mg/l with a mean of 8.0±7.8 mg/l. Calcium and Magnesium The Calcium concentrations ranged from <0.08 -9.2 mg/l with a mean of 1.7±1.9 mg/l whereas Magnesium values varied from <0.05 -2.8 mg/l with a mean of 0.9±0.8 mg/l. Sodium and Potassium Sodium concentrations ranged from <0.01 -44.62 mg/l with a mean of 11.21±13.2 mg/l whereas Potassium concentrations ranged from <0.01 -11.88 mg/l with a mean of 1.50±2.64 mg/l. Table 4 contains the July and September means of the parameters required in the characterization of RSUST groundwater. A plot of these hydro-geochemical data on the Piper diagram [20] is shown in Fig. 4. Graphical presentation showing order of occurrence of the major anions (HCO 3 -, Cl -, SO 4 2and CO 3 2-) and the corresponding major cations (Na, Ca, K and Mg) are shown in the Pie chart (Fig. 5). Microbiological Analysis The results of the microbial analyses showed Total heterotrophic bacteria (THB) population ranging from Nil -3000 cfu/ml with mean of 109±570 cfu/ml; while total coliform bacteria (TCB) values varied from Nil -210 MPN/100ml with a mean of 8.6±43.6 MPN/100ml. Faecal coliform bacteria (FCB) were not present in all the samples. The highest THB (3000 cfu/ml) and TCB (210 MPN/100ml) occurred at the Institute of Education (Figs. 6 and 7). Physico-Chemical Parameters pH The groundwater pH, a measure of the hydrogen ion concentration depicted variation between acidic (pH 4.09) and near neutral (pH 6.77). This implies that the groundwater in the area is acidic. The low pH values (4.0-4.9) indicates that the aquifer may be associated with waters containing free acids derived from oxidizing Sulphide minerals of the parent bedrock materials. Those that have moderately low pH values (5.0-5.9) might be associated with small amount of mineral acids from Sulphide sources or with organic acids from decaying vegetation. The moderate pH values (6.39-6.77) which are slightly acidic in reaction occurred around ICT, Medical Lab. Sc. and Council Unit. This could be attributed to aquifer having high bicarbonate content [21]. It is observed in Fig 2 that Prolonged intake of acidic water may predispose one to the dangers of acidosis, which according to Health Experts may lead to cancer or cardiovascular damage including the constriction of blood vessels and reduction in Oxygen supply even at mild levels [25]. It could also cause leaching of valuable minerals such as Calcium, Magnesium, Sodium and Calcium from the body. Closely related to the pH is the alkalinity, which is a measure of the buffering capacity of the system. The recorded values were low (2 -8 mg/l as CaCO 3 ) and they are mostly due to bicarbonate contents. Water hardness is another quality parameter that determines the use of water for drinking / domestic and industrial purposes. Simply put, the hardness is the capacity of water to lather on the application of soap and these increases with the softness of the water. The hardness level of <0.1 -34.6 mg/l as CaCO 3 is within the 0 -60 mg/l as CaCO 3 classification of soft water. These hardness values are within the national and international limits. However, continued intake of soft water has been linked to cardiovascular diseases incidents [26], [27]. The TDS, which is a measure of all the dissolved substances in the water, was from 12 to 274 mg/l. These values are within WHO (600 mg/l) and SON (500 mg/l) limits for drinking water. TDS correlated positively with conductivity, salinity, chloride, sulfate and nitrate. The levels of these parameters in the groundwater were within the stipulated standards (Table 1). All the conductivity values are within acceptable limit (1000 µS/cm, SON and 1200 µS/cm WHO). The TDS values ranged from 12 mg/l to 274 mg/l with mean of 67±67 mg/l. These values are within both [18] and [19] potable water limits (Fig. 3). The values obtained in the study for Conductivity, Salinity and TDS indicate that the ground water is fresh [21]. Temperature and Turbidity The groundwater temperatures (26.6-30.3˚C) in July and September showed no significant difference indicating similarity in chemical behavior in the water characteristics within the study area. Turbidity values in both periods were <0.01 NTU indicating clear water devoid of suspended solids caused by clay, silt and other substances that enter boreholes from the aquifer or from the soil surface. Chloride, Sulphate, Phosphate and Nitrate The low Chloride concentrations (<1.0 -10.3 mg/l) indicate that the aquifer recharge is high due to high rainfalls, it is not overdrawn and there are no contact with water of marine origin or leaching from the upper soil layers. RSUST is situated close to the mangrove swamp and overdrawn aquifer may give rise to saltwater intrusion. Sulphate contents are attributable to the sedimentary basin of the Niger Delta region. The low Sulphate levels (<1.0 -15.5 mg/l) could be related to the removal of Sulphate by Sulphur bacteria in the sub-surface water [28]. Phosphate concentrations are mostly less than 0.05 mg/l; with the exception of water at Health Services station which had 1.90 mg/l. This is indicative of absence of Phosphoruscontaining mineral apatite in the area. There is no potable water standard for Phosphorus by SON and WHO. Nitrate levels in July (0.30 -4.30 mg/l) are lower than those of September (1.0 -6.20 mg/l). These Nitrate concentrations are within both [18] and [19] limits of 50 mg/l. The higher levels in September could be due to leaching by percolating water that reached the groundwater fast because nitrate compounds are highly soluble [28]. Microbial Analysis Total heterotrophic bacteria (THB) population in some boreholes exceeded WHO limit (100 cfu/ml) [18]. The boreholes (15.4%) that exceeded stipulated WHO limit for THB were ISS2, UWA Day Care, Council Unit and Institute of Education. This implies that 15.4% of the boreholes studied had unacceptable THB values. Total coliform bacteria (TCB) in the borehole at Institute of Education exceeded SON limit of 0-2 MPN/100ml [19]. The non-detection of faecal coliform bacteria in all the samples indicates no pollution with faecal matters. The presence of microbes could be attributed to myriads of activities of microorganisms in the subsurface, shallow depth and water pressures not being high enough to deter microbial activity as many bacteria can survive under high osmotic pressures [29]. Also indigenous bacterial activity and active micro-flora exist in deep formations due to contamination by surface water seepage to an aquifer unprotected by relatively fine textured soil [30]. Droppings from birds into open water tanks also contributed to microbial contamination of the water. This could be the case of the Institute of Education borehole water which is exposed, located near the mangrove swamp forest and may be vulnerable to migration of contaminants from the creek water if the subsoil is coarse-textured. Water Characterization The groundwater in Rivers State University of Science and Technology has been classified based on the hydrogeochemical characteristics obtained in the Piper's diagram (Fig. 4). Suitability for Irrigation Sodium gets to the aquifer from rainwater in coastal areas and / or dissolution of rock as rainwater percolates and the groundwater flows through the aquifer. As a result of effects of sodium on soil and plants; it is considered one major factor that governs the use of groundwater in irrigation [31], [32]. The suitability of groundwater for agricultural purposes (such as irrigation) is based on its Sodium Adsorption Ratio (SAR). The SAR was calculated using the formula (Richards, 1954): Where, Ca 2+ , Mg 2+ and Na + are in mili-eqivalent per litre (meq/l) concentration of the metals in the groundwater. Also, the soluble sodium percent (SSP) is another parameter used to indicate water that is suitable for irrigation. It was calculated from the formula: Where, Ca 2+ , Mg 2+ and Na + are concentrations in meq/l. SSP values less or equal to 50 indicates good quality water while values greater than 50 are contrary and unsuitable for irrigation. The SAR and SSP values obtained for RSUST water samples are in Table 5. Conclusions and Recommendations Based on the findings of this study the tap water within RSUST is fresh and soft with low to moderate dissolved solids. All the borehole water quality on Campus are not potable due to low pH; in addition, borehole water at Council Unit, ISS, UWA and Institute of Education had high microbial count and therefore not suitable for drinking. The use of the water in its present state for aqua-culture might be detrimental to fishes. The water is suitable for irrigation and other purposes except drinking. There is need to urgently commence treatment of water supplied to the University community and create awareness to educate people on the need to boil and/or filter the water prior to consumption.

v3-fos

2018-12-06T04:43:21.923Z

2015-12-01T00:00:00.000Z

55165847

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An empirical model approach for assessing soil organic carbon stock changes following biomass crop establishment in Britain Land-use change (LUC) is a major in fl uence on soil organic carbon (SOC) stocks and the global carbon cycle. LUC from conventional agricultural to biomass crops has increased in Britain but there is limited understanding of the effects on SOC stocks. Results from paired plot studies investigating site-speci fi c effects document both increasing and decreasing SOC stocks over time. Such variation demonstrates the sensitivity of SOC to many factors including environmental conditions. Using a chronosequence of 93 biomass crop sites in England and Wales, mainly of 1 e 14 y age, empirical models were developed of SOC trajectory following LUC from arable and grassland to short rotation coppice (SRC) willow and Miscanthus production. SOC stocks were calculated for each site using a fi xed sampling depth of 30 cm and changes were estimated by comparing with typical pre-conversion SOC stocks. Most LUCs had no demonstrable net effect on SOC stocks. An estimated net SOC loss of 45.2 ± 24.1 tonnes per hectare ( ± 95% con fi dence intervals) occurred after 14 y following LUC from grassland to SRC willow. Soil texture and climate data for each site were included in multivariable models to assess the in fl uence of different environmental conditions on SOC trajectory. In most cases the addition of explanatory variables improved the model fi t. These models may provide some preliminary estimates of more region-speci fi c changes in SOC following LUC. However, the model fi t did not improve suf fi ciently as to provide a basis for adopting a more tar- geted LUC strategy for lignocellulosic biomass crop production. Introduction Soils globally represent the most significant long term organic carbon store in terrestrial ecosystems, containing 4.5 times as much carbon (C) as all living biomass [1] and 3.1 times as much as the atmosphere [2]. Soil organic carbon (SOC) storage results from a dynamic equilibrium between C continuously entering the soil through organic matter inputs and leaving through decomposition and mineralisation, dissolved organic carbon leaching and erosion. Land-use change (LUC) from natural to agro-ecosystems has a major impact on this balance and is the second largest source of anthropogenic greenhouse gas (GHG) emissions after fossil fuel combustion [3]. This vulnerability to human impact is recognised in Articles 3.3 and 3.4 of the Kyoto Protocol with signatory states required to report SOC stock changes resulting from LUC in their annual GHG inventories. Consequently, efforts are being made to identify land-uses that increase SOC storage and utilise the C sink capacity offered globally through agricultural and degraded soils [4,5]. LUC from conventional agriculture to purpose-grown lignocellulosic biomass crop production has become increasingly common in Europe [6]. It has been argued that using land as a source for bioenergy crops has the potential to offset anthropogenic CO 2 emissions through soil C sequestration as well as fossil fuel substitution [4,7]. Purpose-grown biomass crops have been promoted as a source of lignocellulosic feedstock for the production of heat and electricity as well as for the future production of liquid biofuels [8]. It has been suggested that lignocellulosic biomass crops are a more sustainable resource than using food crop-based biofuels [9e11]. Studies indicate that lignocellulosic biomass crops require fewer inputs and can grow on marginal land [7,12,13] but concerns remain over competing land-use where purpose-grown biomass crops will replace food production. Miscanthus x giganteus and short-rotation coppice Salix spp. (SRC willow) are the most prevalent lignocellulosic biomass crops in the UK and currently cover estimated areas of 79e135 km 2 and 22e55 km 2 respectively [6,14,15]. However, this is expected to increase, with 9,300e36,300 km 2 of land being identified as available for lignocellulosic biomass crop production in the UK [16]. Although life-cycle assessments indicate Miscanthus and SRC willow have significant potential for GHG mitigation through fossil fuel substitution [7], an absence of data relating to the effects of LUC on SOC and biogenic GHG emissions remains a barrier to their promotion through policy formulation [17]. The effects of LUC on SOC stocks are difficult to assess and long term monitoring of SOC stocks through repeated assessment of soil inventories is time-consuming and complex, often showing insignificant changes in SOC or inconsistent temporal and spatial trends [18e21]. The potential to measure changes in SOC over time is limited with detectability dependent on the number of samples taken as well as the rate of change [22,23]. Attempts have been made to develop simple and cost-effective practical indicators of SOC stock changes that would avoid repeated sampling [24,25]. However, such measurements have not been widely tested and require validation for a range of soil and land-use types. Due to the many problems associated with long term measurements, spacefor-time substitution methods are preferred to infer the effects of LUC over time. Results of paired plot studies investigating effects of land conversion to lignocellulosic biomass crops on SOC stocks often report short term gains in SOC following the conversion of arable land to Miscanthus in temperate Europe [26e28] while losses and gains have both been inferred for LUC from arable crops to SRC willow [29]. Studies typically infer no significant change in SOC following the conversion of grassland to Miscanthus [26,30,31], and a loss of SOC following LUC from grassland to SRC willow [29,32]. However, the trajectory and magnitude of change differs between studies, reflecting the general sensitivity of SOC to site-specific factors such as climate, soil texture, crop management, previous land-use and SOC stocks [33]. A large number of study sites representing LUC under a range of conditions would be required to ascertain the overall net effect of LUC on a landscape scale. The carbon response function (CRF) concept was developed as a simple statistical tool to describe the relative SOC change rate after LUC as a function of time [34]. With this approach, SOC stock changes (DSOC%) are inferred using reference sites and regression models are fitted to the dataset with the best-fit model, or 'general carbon response function' (CRF gen ), identified to provide an overall measure of change across multiple sites [35]. To investigate the influence of environmental parameters on SOC change rate and to improve the model fit, additional variables are used in a multivariable model designated 'specific carbon response function' (CRF spec ) for the purpose of more region-specific estimates [35,36]. These empirical models are more transparent and less complex than process-based simulation models although they require large datasets to provide reliable estimates of temporal trends in SOC following LUC. CRF models have been developed to estimate the effects of major LUCs in temperate Europe [36,37]. For these historic LUCs large retrospective datasets were available from which paired sites that were adjacently situated could be selected to ensure similar pedological conditions. However, in circumstances where suitable reference sites were unavailable and rather than limiting the number of study sites, average pre-conversion SOC stocks obtained from soil surveys have been employed to provide a baseline measurement with which to estimate relative changes in SOC [37]. This method has also been employed in the present study to assess the impact of LUC for lignocellulosic biomass crop production, since this is a recently emerging LUC in Britain and we were subsequently constrained by a lack of retrospective datasets and suitable reference sites. Here two approaches have been combined to assess SOC trajectory following biomass crop establishment: (i) free-intercept models were used to determine the post-conversion trajectory of SOC for a selection of sites that can be assumed to follow a similar trajectory and; (ii) forced-intercept CRFs were developed to estimate net changes in SOC from a hypothetical baseline and to assess the effects of environmental parameters on SOC changes. The main purpose is to assist in targeting future research efforts and to provide preliminary evidence for policy makers. Site selection A list of 150 commercial SRC willow and 121 Miscanthus plantations was compiled in England and Wales, from which 45 SRC willow and 48 Miscanthus plantations were selected for soil sampling. To limit variance arising from site-specific factors the following were excluded from the list: (i) sites with anomalously high SOC content (>8% SOC) or wetland soil, (ii) crops established on reclaimed land, and (iii) land where organic fertiliser (sewage sludge or manure) had been applied in the five years prior to sampling. Of those remaining, 93 sites were selected to obtain as far as possible a broad, even range of age and an equal representation of SRC willow and Miscanthus plantations established on arable and permanent grassland. Due to the relatively recent emergence of these crops as a biomass resource in Europe, all plantations were between 1 and 14 y old at the time of sampling, apart from one plantation, a 22-y old SRC willow crop. The number of plantations established on former grassland sites was limited, owing to declining policy support. All available conversions from permanent grassland were sampled and supplemented by sites comprising setaside fields that had been under grassland management for at least five years prior. Sites from each crop type were generally located in the same broad geographical area ( Fig. 1) with similar climatic characteristics and soil texture to ensure similar site trajectory (Table 1). Site climate was categorised using mean annual precipitation (MAP) and mean annual temperature (MAT), based on 1981e2010 observations, obtained for the Met Office weather station closest to each study site. Soil texture at 26% of the sites was 'light' (<15% clay), 70% of sites had 'medium' texture (15e30% clay) and 4% were 'heavy' textured (>30% clay). All sites fall within a range of 10e38% clay content. The distribution of sites was affected by historic planting efforts, with a concentration towards the northeast and south-west of England (Fig. 1). To reduce bias only one field was sampled on a given farm, even if another stand age was present. Soil sampling Soil sampling at the 93 study sites was undertaken between March and November 2011. Each field was divided into a grid of 100 intersections of which 25 were randomly selected for sampling. Soil cores (30 mm diam.) were taken to 30 cm depth and divided into two layers (0e15 and 15e30 cm). Where roots or large stones were present, the sample was taken from within 10 cm of the grid intersection. Samples were combined by depth and stored at 4 C for a maximum of 2 weeks before processing for analysis. Three additional cores of 50 mm diam. were taken to 15 cm depth from randomly selected intersections, using a specialised ring corer kit to measure soil bulk density (BD) (Van Walt, Haslemere, England). Soil analysis The composite samples were used to obtain a site value for SOC for the two depth increments. Soil was sieved (<5.6 mm) and homogenised using the cone and quarter method [38]. A representative sub-sample was then collected and air-dried at room temperature for 7 days, before being crushed with a pestle and mortar, sieved (<2 mm) and milled to a fine powder using a MM200 ball mill (Retsch GmbH, Haan, Germany). 20 mg of sample was analysed for total C and N by dry combustion using a TruSpec elemental analyser (Leco, St. Joseph, MI, USA). SOC was ascertained for each sample as the difference between total C and the mass fraction of inorganic C in dry soil, quantified using an automated acidification module and coulometry (CM 5012 and CM 5130, UIC, Joliet, Illinois). Ratios of clay-(0e2 mm), silt-(2e63 mm) and sand-sized (63e2000 mm) primary particles were determined for the soil mineral fraction using a laser diffractometer (Beckmann Coulter LS230, High Wycombe, England). Samples containing inorganic C > 0.1 g kg À1 were treated prior to analysis as follows. 20 g samples were acidified with 20 ml of 1 mol l À1 sodium acetate (NaOAc), adjusted to pH 5 with glacial acetic acid (CH 3 COOH). Acidified samples were maintained at 70 C overnight in a water bath and then centrifuged. After carbonate removal 10 g of each sample was treated for the removal of organic matter with 20 ml of 9.79 mol l À1 hydrogen peroxide (H 2 O 2 ) for 24 h, maintained at pH 5 with 0.1 mol l À1 NaOAc buffer. Each residue was then rinsed three times with deionised water and oven dried overnight at 80 C [39,40]. Oxidised, carbonate-free residues were dispersed by treating overnight with 25 ml of 0.07 mol l À1 sodium hexametaphosphate (NaPO 3 ) 6 in an ultrasonic bath and sieved (<1 mm) prior to analysis. The >1 mm residue was isolated by vacuum filtration and then oven-dried at 80 C for estimation of volume using an assumed grain density of 2.65 g cm À3 to re-calculate clay, silt and sand particle abundances for the whole <2 mm sample. Statistical modelling Two approaches were employed to assess SOC trajectory following biomass crop establishment. Free-intercept models were used to determine the post-conversion trajectory of SOC by regressing SOC density (t ha À1 ) against time since establishment. This approach demonstrates the general relationship between SOC stocks and age of plantation for a chronosequence of sites. However, in the free-intercept models an intercept is calculated which does not account for a potential land conversion effect and cannot be used to estimate net changes from pre-conversion SOC stocks. For this purpose CRFs were also developed for each LUC based on the approach developed in a number of recent studies [34e37]. These forced-intercept models were produced by regressing relative changes in SOC stocks measured at each site from a preconversion SOC stock (DSOC) against time since establishment. Due to the lack of suitable reference sites available for this study, DSOC values were calculated using pre-conversion SOC stocks derived from soil surveys. Each biomass crop plantation in the chronosequence was categorised into major soil groups of the Soil Survey of England and Wales (SSEW) soil classification system [41] using the National Soil Map for England and Wales [42] (Table 2). Mean SOC stocks for arable and grassland soils and standard deviations were obtained for each corresponding group, as described in Gregory et al. [43] (Table 3). SOC density (t ha À1 ) was calculated using the fixed depth approach (to 30 cm depth) using results from the samples at 0e15 For these calculations it was first necessary to develop a pedotransfer function (PTF) to derive estimates of 15e30 cm BD using the 0e15 cm BD measurements and other measured soil parameters. The best-fit equation predicted BD as a function of SOC [Eq. (3)] (see the online Supporting Information for detail of the PTF derivation). where SOC is soil organic carbon (% mass fraction of dry soil). For both the free-and forced-intercept models, regressions fitted to the data included linear, quadratic, cubic, power and exponential functions. Weighted regressions were used for the forced-intercept CRFs with weights (1/SD 2 ) derived from the standard deviations of the pre-conversion SOC stocks (Table 3). Model selection was based on the corrected Akaike information criterion (AICc) [Eq. (4)]. Overall model robustness was evaluated using the model efficiency index (EF) [44,45] [Eq. (5)]. Root mean square prediction error (RMSPE) [Eq. (6)] was used to measure the overall prediction error. where n is the total number of observations, SSE is the sum of squared errors of prediction and k is the number of parameters plus 1, P i are the predicted values, O i the observed values and O the mean of the observed data. Selected forced-intercept models were designated CRF gen models and used to estimate changing SOC stocks from mean preconversion SOC stocks (±95% confidence intervals). Specific CRFs (CRF spec ) were also created to assess the influence of other explanatory variables on changing SOC stocks (Table 4). Clay, silt and sand density (t ha À1 ) was used instead of relative abundances (%) since these provided a better fit and enabled greater predictive accuracy. Linear and quadratic functions were selected for CRF gen models [Eqs. (7) and (8)] which were enhanced for CRF spec models by entering explanatory variables in a hierarchical manner as direct effects on model coefficients to increase EF and decrease RMSPE [Eqs. 9 and 10]. The order of the variables (e.g. x 1 , x 2 …) indicates their degree of influence with x 1 having the greatest effect. Explanatory variables were added individually and associated coefficients used to indicate either a positive or negative effect on each response function [36]. To take account of any possible effect of sampling season (spring, summer and autumn) on the rate of SOC change, season was assigned categorical values of 1, 2 and 3 respectively, in the order of spring to autumn. Linear CRF gen : DSOC ¼ at Quadratic CRF gen : DSOC ¼ at þ bt 2 (8) Quadratic CRF spec : where t is time after LUC (y), a, and b are constants and x denotes the explanatory variable. All regression analysis, curve fitting and checking of residuals for normal distribution using the Shapiro Wilk test were carried out using Genstat 16 (VSN International, Hemel Hempstead, UK). SSE values were obtained from Genstat 16 and AICc calculated using the method of Motulsky and Christopoulos [46]. Arable to SRC willow Two sets of models were established to describe SOC trajectory following LUC from arable crops to SRC willow: (i) for the initial 14 y period and (ii) including the 22 y old site. Dual analysis was carried out to enable comparison of all LUCs over a similar time frame, but also to explore the longer time frame available here since the 22-y site was not identified as an outlier using the Grubb's test. In both cases, an exponential function provided the best predictive free-intercept model and a linear function provided the best predictive forced-intercept model. The upward trajectory of the freeintercept models suggest a post-conversion increase in SOC stocks, by an estimated 42.2 ± 19.1 t ha À1 from 2 to 14 y and by 78.4 ± 51 t ha À1 from 2 to 22 y (Fig. 2aeb). However, the forcedintercept CRF gen model shows no demonstrable overall change in SOC for this LUC, with an estimate of 19.3 ± 19.8 t ha À1 after 14 y and 30.3 ± 30.3 t ha À1 after 22 y (Fig. 3aeb). These results indicate that, after initial losses, SOC stocks recover during years 2e14 with a greater recovery after 22 y. However, there is no evidence for any overall net effect on SOC relative to pre-conversion SOC stocks after 14 or 22 y. EF was improved for both the 14-y and 22-y CRFs (from 0.05 to 0.45 and from 0.07 to 0.40 respectively) with the addition of explanatory variables (Table 5). Sampling season, clay density and MAT all had an effect on SOC trajectory (Table 6). In both cases a predicted positive effect on the response function occurred from spring to autumn. A negative effect of clay density on the response function indicates greater SOC losses and/or lower SOC accumulation for more clayey soils. A positive effect of MAT indicates greater SOC accumulation in warmer regions. Arable to Miscanthus For LUC from arable crops to Miscanthus a power function provided the best predictive free-intercept model and a quadratic function provided the best predictive forced-intercept model. The downward trajectory of the free-intercept model suggests a postconversion decrease in SOC stocks, by an estimated 23.5 ± 7.8 t ha À1 from 1 to 13 y (Fig. 2c). However, the forcedintercept CRF gen model shows no demonstrable overall change in SOC for this LUC, with an estimate of À1.1 ± 24.6 t ha À1 after 13 y (Fig. 3c). No additional variables improved the model fit. Grass to SRC willow For LUC from grassland to SRC willow an exponential function provided the best predictive free-intercept model and a linear function provided the best predictive forced-intercept model. From years 3e14 the free-intercept model follows a slight downward trend but with no demonstrable overall change in SOC, with a model estimate of À33.5 ± 51.0 t ha À1 (Fig. 2d). However, the forced-intercept CRF gen model indicates an overall net loss of SOC following LUC from grassland to SRC willow, with an estimate of À45.2 ± 24.1 t ha À1 after 14 y (Fig. 3c). EF was improved from 0.09 to 0.26 by the addition of the explanatory variables sand density and MAP (Table 5). Negative effects of sand density and MAP indicate greater SOC losses may occur in sandier and wetter soils. Grass to Miscanthus For LUC from grassland to Miscanthus a power function provided the best predictive free-intercept model and a linear function provided the best predictive forced-intercept model. From years 3e13 the free-intercept model follows a slight upward trend but with no demonstrable overall change in SOC, with a model estimate of 19.0 ± 23.0 t ha À1 (Fig. 2e). Similarly the forced-intercept CRF gen model shows no demonstrable overall change in SOC for this LUC, with an estimate of À7.4 ± 15 t ha À1 after 13 y (Fig. 3e). EF was improved from 0.05 to 0.12 with the addition of the explanatory variables sand density, silt density, MAP and MAT (Table 5). Negative effects of sand and silt density, MAP and MAT indicate potential SOC losses or less accumulation in lighter textured soils and/or in warmer and wetter regions. Discussion The upward trajectory of the free-intercept models indicates a post-conversion increase in SOC stocks following LUC from arable crops to SRC willow. An expected increase in SOC has previously been attributed to reduced tillage, increased C inputs from leaf, woody and root litter production and by increased transfer of assimilates into the external mycelium of mycorrhizal fungi [47e50]. While the 14-y model indicates a declining rate of accumulation, possibly reaching a new equilibrium (Fig. 2a), the 22-y model projects a continued increase, but with a large uncertainty reflected by the broad 95% confidence intervals (Fig. 2b). However, this increase in SOC may have been preceded by an initial loss of SOC stocks following LUC due to the disruption of aggregates caused by soil disturbance, leading to the accelerated decomposition of SOC that has lost physical protection [51]. Since the free-intercept model is unable to account for such a land conversion effect, forced-intercept CRF gen models have been employed to attempt to relate this period of SOC recovery to pre-conversion SOC stocks. These models suggest no overall net increase from pre-conversion levels has occurred after either 14 or 22 y (Fig. 3aeb). Although there is no measurable gain after 22 y, a model estimate of 30.3 ± 30.3 t ha À1 suggests that SOC has recovered to preconversion levels representing a full SOC payback for any initial losses. However, this parameterised model reflects a short term effect and it is unclear whether an increase can be expected beyond this period, or when a new equilibrium may be reached. In contrast, the downward trajectory of the free-intercept model indicates a post-conversion decrease in SOC stocks following LUC from arable crops to Miscanthus. SOC stocks measured at Miscanthus plantations aged 1e2 y are relatively large and this produces a negative exponent used to predict a loss over time. Furthermore, since this free-intercept model is unable to account for any initial losses following LUC, an even greater overall loss from pre-conversion stocks might have been expected. However, the forced-intercept CRF gen model shows no demonstrable overall change in SOC for this LUC. Instead these large SOC stocks for young Miscanthus plantations are more likely to represent increases rather than decreases from pre-conversion levels. Both the exponential and quadratic curves projected by the free-and forced-intercept models respectively appear counter-intuitive since: (i) low-input arable soils have previously been identified as having a large C storage potential [4]; (ii) paired plot studies have previously inferred a significant increase in SOC for LUC from arable crops to Miscanthus [26,28] and; (iii) it is unlikely that SOC would increase in the first few years following LUC and decrease thereafter. Based on previous studies, an overall increase might have been expected here, due to an anticipated reduction in soil disturbance and increased C inputs to the soil from both above-and below-ground [28,52,53]. Reasons why the expected SOC increase was not detected in this research may include patchy Miscanthus crop establishment, which was observed at some sites, although not quantified. It has previously been suggested that poor crop performance may relate to inexperience and inefficient management of a newly emerging crop [54]. It is also possible that the performance of Miscanthus in trials using experimental sites does not adequately reflect that of commercial planting which, due to economic factors, may be more likely to occur on lower grade land. Further research would be required to confirm these effects. No demonstrable changes in SOC stocks are predicted by the free-intercept models for LUC from grassland to either SRC willow or Miscanthus (Fig. 2dee). LUC to SRC willow follows a slight downward trend and LUC to Miscanthus follows a slight upward trend but in both cases there is large uncertainty around model estimates. Fewer study sites were available for biomass crops established on grassland which may contribute to the large Fig. 3. Forced-intercept models with estimated SOC changes (t ha À1 ± 95% confidence intervals) expressed as a function of time following LUC: (a) arable to SRC willow 0e14 y; (b) arable to SRC willow 0e22 y; (c) arable to Miscanthus; (d) grass to SRC willow; (e) grass to Miscanthus. uncertainty reflected by the broad 95% confidence intervals. However, the forced-intercept CRF gen model predicts an overall net decrease in SOC following LUC from grassland to SRC willow. This suggests that the free-intercept model underestimates SOC losses and that, by comparing with typical pre-conversion SOC stocks, uncertainty is lower for the forced-intercept CRF gen model which predicts an overall net loss of 45.2 ± 24.1 t ha À1 after 14 y. Although such a loss appears high for mineral soils, equivalent to 3.2 t C ha À1 y À1 , other studies which have used a paired sites approach to assess LUC in temperate Europe have also reported SOC losses of a similar magnitude. Poeplau et al. [36] estimated a loss for LUC from grassland to cropland of 36 ± 5 (95% CI) % of an initial SOC stock of 115 t C ha À1 after 17 y, which is equivalent to 2e3 t C ha y À1 . They also estimate a loss for forest to cropland of 31 ± 20% of an initial SOC stock of 147 t C ha À1 after 20 y, which is equivalent to 1e4 t C ha y À1 . The loss estimated by the CRF gen may corroborate the results of paired plot studies which have inferred significant losses for this LUC [29,32]. For LUC from grassland to Miscanthus, the forced-intercept CRF gen model projects a slight downward trend also suggesting the free-intercept model may underestimate SOC losses. However, no demonstrable change in SOC is apparent for this LUC from either model approach. Paired plot studies have also reported no significant differences in SOC between Miscanthus and adjacent grassland sites [26,30,31]. EFs of the CRF gen models were low with a range of 0.01e0.09 (Table 5) indicating that 'time since conversion' explains only a small amount of variance in the data. Other explanatory variables were used to enhance the model fit, with soil texture, climate and sampling season all having an effect on SOC trajectory. Sampling season improved the model fit for LUC from arable crops to SRC willow having a positive effect on the response function, suggesting that estimated increases in SOC from sites sampled later in the year may appear artificially high. This may relate to fine root growth, which begins in spring and continues until early autumn [55], or increased litter inputs and decomposition during the course of the year. Although care was taken to remove root material passing the 2-mm sieve, some fine roots may have remained, which may also have influenced the results. Clay density improved the model fit for LUC from arable to SRC willow with a negative effect indicating a lower SOC accumulation Table 6 Explanatory variables used to develop CRF specs . þ indicates a positive and À a negative effect on the response function. Blank cells indicate variables were not included in the CRF for the respective LUC. for more clayey soils. This may reflect a slower rate of change, which would be consistent with trends reported in other studies investigating long term changes in SOC stocks [36] as well as studies that have assessed changes in specific SOC fractions following LUC [24]. Sand and silt density improved the model fit for LUC from grassland to SRC willow and Miscanthus with both variables having a negative effect on the response function. These effects of soil texture can be explained by the higher proportion of mineral and aggregate bound SOC in clayey soils which is more resistant to decomposition than the particulate SOC that is more abundant in sandy soils [56]. If SOC is assumed to follow a 'slow in, fast out' trend then it may be 'slower in' for clayey soils which have a greater C storage capacity in the long term. Climatic factors improved EF with potentially greater SOC losses and/or less accumulation in warmer and wetter regions following the conversion of grassland. There is evidence that greater SOC accumulation may have occurred in warmer regions following the establishment of SRC willow on arable land. This may indicate that where SOC losses occur these are accentuated in warmer and wetter regions where conditions favour microbial activity. Where SOC accumulation occurs the C inputs may have a greater effect on the SOC balance than decomposition, with larger inputs in warmer regions due to higher net primary production [36,57,58]. LUC This study utilised a large chronosequence dataset of 93 sites from across England and Wales to develop empirical models to assess the general trajectory of short term SOC stock changes following biomass crop establishment. Two model approaches were employed to assess the post-conversion trajectory of SOC stocks following biomass crop establishment and to put these changes in a context of typical pre-conversion SOC stocks. Estimates of SOC stock changes for each site were calculated by comparing against mean pre-conversion SOC stocks for major soil groups [43]. A paired sites approach can provide a more accurate baseline against which to measure changes in SOC stocks. However, it would also have compromised the number of sites that could be sampled since suitable reference sites may not have been available at all selected locations. Furthermore, while providing a potential baseline for change, it is rare that two fields will share the same site history before and since LUC, or the exact same soil properties. The CRF gen models represent the overall net effect of LUC on SOC, rather than an estimate of the likely incurred changes in SOC under any particular set of circumstances. This provides a useful indication of the general impact of the recent commercial deployment of biomass crops in Britain and the future short term net effect on SOC stocks if biomass crop planting were to continue on similar types of land. Since the resolution of agricultural land classification maps in Britain is not suitable for the assessment of single fields, we were unable to verify the quality of land for our study sites. However, research from another study using focus groups of farmers [59], as well as communication with the growers within this study, both suggest a tendency to select the least productive agricultural land for biomass crop establishment. Therefore, this study may better reflect the impact of targeting lower rather than higher grade agricultural land. Although a substantial amount of land has been identified in Britain as 'available' for biomass crop production, any future expansion is likely to be contingent upon increased social acceptance, economic feasibility and, for the production of biofuels, technological advancements. To address important sustainability criteria, it may be more favourable to target lower grade agricultural or unproductive land for biomass crop production to limit the impact on the food supply [7,12,13]. If the results presented here are indicative of such a planting strategy, the potential benefits of soil C sequestration on a commercial scale may have been over-emphasised. Although there does not appear to have been an overall net increase in SOC from the recent commercial planting of biomass crops in Britain, evidence suggests that increases are likely to have occurred under certain conditions. CRF spec models were developed to investigate the causes for the variability that has been observed on a landscape scale. Whilst in most cases the addition of explanatory variables improved the model fit, suggesting that SOC trajectory is sensitive to soil texture and climate, the low explanatory power of the models appears to provide limited justification for policy use in targeting future LUC for lignocellulosic biomass crop production to increase SOC stocks. While a more targeted LUC policy that incorporates the potential effects on SOC would be unlikely, an improved understanding of the short and longer term impact of LUC under different conditions is important nonetheless, even if this proves more useful for C accounting than for C abatement purposes. There are various options for future research efforts to further extend and test the outcomes of the current study. Our objective was to determine the general effect of LUC to biomass crops by sampling a large number of commercial plantations. The uncertainty in model estimates is high, particularly for Miscanthus plantations and former grassland sites for both Miscanthus and SRC willow. To reduce the uncertainty in these empirical models, the sensitivity of SOC trajectory to a range of factors has to be addressed. This could be achieved by targeted sampling of additional field sites and, as others have previously suggested, combining datasets from different studies to form an 'improved reporting scheme' [36]. In addition to reducing the uncertainty surrounding estimates derived from the CRF gen models, the CRF spec model fits could also be enhanced by further sampling and data collection. Additional data should include information on factors affecting SOC, in particular soil and climate. For example, in this study climate data was summarised by mean annual precipitation and temperature for the nearest Met Office weather station to each study site. It is possible that more site-specific climatological data, that includes additional variables such as slope aspect, at a higher spatial resolution could improve the model fits. However, there are obvious challenges facing further empirical data collection as additional biomass crops, particularly those established on grassland, may be limited in number and such studies are resource-intensive in terms of field and laboratory work. In these circumstances, finding synergies between statistical and process-based modelling is particularly important. Site-specific testing of simulations can be evaluated against the generality of a statistical model; statistical models can be explored for sensitivities apparent in process-based models. Process-based models can then be used to more confidently extrapolate beyond the time-frame of observational data, where for novel LUCs only relatively short term effects can be directly examined using the CRFs. In this instance, such models may be particularly useful for determining if any future increases in SOC stocks are likely to occur as any losses incurred by LUC are usually rapid and should have been captured by the present study [36]. Conclusions The results presented here indicate that commercial planting of SRC willow on arable land had no net effect on SOC stocks, while planting on grassland incurred a net loss of SOC. For Miscanthus, there was no demonstrable net effect on SOC stocks following commercial planting on arable or grassland. Further research would be required to reduce this uncertainty and determine the likely effects of LUC on the overall GHG mitigation potential of Miscanthus. The data presented here suggests that C sequestration benefits of lignocellulosic biomass crops may have been overemphasised and that crop performance in a commercial setting may not reflect that of experimental field trials. It is likely that increases in SOC can occur for both SRC willow and Miscanthus under certain conditions and the effects of environmental parameters on SOC trajectory require further investigation. Since SOC stock changes generally follow a 'slow in, fast out' trend, further increases may occur outside of the time-frame of this study. For more reliable longer term predictions, process-based models can be used in conjunction with the experimental data presented here.

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Cereal cystatins delay sprouting and nutrient loss in tubers of potato, Solanum tuberosum Background Recent studies have reported agronomically useful ectopic effects for recombinant protease inhibitors expressed in leaves of transgenic plants, including improved tolerance to abiotic stress conditions and partial resistance to necrotrophic pathogens. Here we assessed the effects of these proteins on the post-dormancy sprouting of storage organs, using as a model potato tubers expressing cysteine protease inhibitors of the cystatin protein superfamily. Results Sprout emergence and distribution, soluble proteins, starch and soluble sugars were monitored in tubers of cereal cystatin-expressing clones stored for several months at 4 °C. Cystatin expression had a strong repressing effect on sprout growth, associated with an apparent loss of apical dominance and an increased number of small buds at the skin surface. Soluble protein content remained high for up to 48 weeks in cystatin-expressing tubers compared to control (untransformed) tubers, likely explained by a significant stabilization of the major storage protein patatin, decreased hydrolysis of the endogenous protease inhibitor multicystatin and low cystatin-sensitive cysteine protease activity in tuber tissue. Starch content decreased after several months in cystatin-expressing tubers but remained higher than in control tubers, unlike sucrose showing a slower accumulation in the transgenics. Plantlet emergence, storage protein processing and height of growing plants showed similar time-course patterns for control and transgenic tubers, except for a systematic delay of 2 or 3 d in the latter group likely due to limited sprout size at sowing. Conclusions Our data point overall to the onset of metabolic interference effects for cereal cystatins in sprouting potato tubers. They suggest, in practice, the potential of endogenous cysteine proteases as relevant targets for the development of potato varieties with longer storage capabilities. Electronic supplementary material The online version of this article (doi:10.1186/s12870-015-0683-2) contains supplementary material, which is available to authorized users. Background Dozens of papers have discussed the potential of plant protease inhibitors as effective antidigestive compounds to engineer herbivore pest resistance in food and commodity crops [1,2]. Recent studies have also assessed their usefulness as ectopic modulators of endogenous proteases to introduce traits of agronomical value such as pathogen resistance or abiotic stress tolerance in leaf tissues [2], or to prevent unintended proteolysis of ectopically expressed biopharmaceuticals in plants used as vehicles for recombinant protein production [3]. Cysteine (Cys) protease inhibitors of the cystatin protein superfamily [4] were shown for instance to provide host plants with partial resistance to necrotrophic fungi [5] and broad tolerance to drought, chilling, oxidation or salt stress [6][7][8]. Recombinant cystatins were shown also to prevent degradation of recombinant proteins in leaf tissue when coexpressed as accessory proteins to inhibit endogenous proteolysis in the cytosol [9] or along the cell secretory pathway [10,11]. Little is known about the endogenous targets of cystatins in planta, but the ectopic effects reported for these proteins, the large numbers of Cys protease-encoding genes in plant genomes [10] and the well established roles of Cys proteases in such key physiological processes as programmed cell death, senescence, defense and storage protein mobilization [12] now make the regulation of these enzymes an interesting route for crop improvement. In this study we assessed the potential of recombinant cystatins to downregulate Cys protease activity and prevent protein loss in non-dormant storage organs, using cereal cystatin-expressing potato tubers as an example of economic value. Gene expression studies have established clear correlations between storage protein deposition or mobilization, cystatin content, and Cys protease activity in seeds or vegetative storage organs of different plants [4,12]. For instance, a positive link was established between deposition of the major storage protein patatin, high transcript numbers for the 88-kDa Cys protease inhibitor potato multicystatin (PMC) and low Cys protease activity in developing potato tubers [13]. Patatin mobilization was shown by contrast to correlate with low numbers of PMC transcripts, Cys protease upregulation and increased protease activity in sprouting tubers [14]. A simple working model was proposed to predict the fate of storage proteins in reproductive organs, involving the cystatin::Cys protease stoechiometric balance as a key determinant of the resulting output [4]. Inhibitory cystatins are actively synthesized in developing storage organs to eventually outnumber Cys proteases and promote storage protein deposition. An elevated, abscisic acid-dependent cystatin::Cys protease balance in dormant tissues allows for the pool of storage proteins to be maintained over dormancy and remain available to the growing plantlets upon germination or sprouting [13,[15][16][17]. A gibberellin-induced up-regulation of Cys protease genes concomitant with the repression of cystatin genes leads, finally, to a low cystatin::Cys protease balance favoring storage protein mobilization and plantlet growth [14,[17][18][19][20]. The physiological significance of a low cystatin::Cys protease balance upon germination was supported empirically with transgenic Arabidopsis lines overexpressing AtCYS6, a seed endogenous cystatin naturally responsive to gibberellins and abscisic acid [17]; or with Arabidopsis lines expressing BrCYS1, a heterologous cystatin from Chinese cabbage also responsive to these hormones [21]. In line with the assumed repressing effect of cystatins on germination, transgenic seeds constitutively expressing either cystatins exhibited low Cys protease activity and delayed germination compared to non-transgenic seeds [17,21], in sharp contrast with AtCYS6 knockout seeds showing higher Cys protease activity and early germination [17]. Here we assessed the impact of two ectopically expressed cereal seed cystatins, oryzacystatin I (OCI) [15] and corn cystatin II (CCII) [22], on the sprouting behaviour, protein catabolism and growing potential of potato tubers stored for several months at 4°C. OCI accumulates at low levels in potato tubers Two OCI-expressing potato lines, K52 and K53, were selected for the experiments from a collection of independent transformants established earlier in our laboratory [23]. OCI in these lines bears no signal peptide at the N terminus and accumulates in the cytosol under the control of the Cauliflower mosaic virus (CaMV) 35S constitutive promoter. Reverse transcriptase (RT) polymerase chain reaction (PCR) assays were performed to estimate levels of OCI-encoding transcripts in leaves and tubers. Comparable levels of OCI transcripts were found in the 5 th leaf of lines K52 and K53, compared to undetectable levels in the 5 th leaf of control line K used as parent for genetic transformation (Fig. 1a). Roughly similar transcript levels were found in cDNA samples prepared from 1-cm 3 tuber flesh pieces collected~5 cm down from the apical buds of tubers stored for 36 weeks at 4°C (Fig. 1a), indicating comparable ability of the viral promoter to drive OCI transgene expression in sprouting tubers and mature leaves. Despite similar transcript signals, recombinant OCI was detected at low relative levels of~0.08-0.15 % of total soluble proteins in tuber extracts of both lines K52 and K53, five to ten times less than levels measured in mature leaves [24] (Fig. 1b). One explanation for this could be a rapid turnover of the inhibitor in heterologous cellular environments harbouring distinct proteolytic machineries, as discussed earlier for a number of recombinant proteins reported to be unstable in plantbased expression systems [3]. A more likely explanation given the documented stability of plant cystatins in protease-rich heterologous environments such as plant leaf and microbial cells [10,11,25] would be a natural sink effect of highly abundant storage proteins on amino acid resources, inherently unfavorable to heterologous protein accumulation [26]. Studies have reported higher levels of recombinant protein in reproductive organs of storage protein-depleted mutants [27,28] or transformants [29][30][31][32], presumably associated with increased energy and/or amino acid resources in planta. Such observations, while strengthening the idea of a limited proteome plasticity in storage organs, could also explain the low levels of recombinant proteins such as OCI (this study) or tomato cathepsin D inhibitor in transgenic potato tubers [26] compared to steady-state levels in leaves of the same plants. OCI expression delays tuber sprouting at 4°C Distribution patterns of growing buds and sprouts were recorded on stored tubers of line K52, line K53 and control line K to look for eventual macroscopic effects of OCI on tuber sprouting despite a limited accumulation in tuber flesh tissue (Fig. 2). At least six~10 cm-long tubers harvested from different plants of each line were stored in the dark at 4°C for 48 weeks (ca. 11 months) prior to visual inspection. Following a resting period of several weeks after harvest, endodormancy is released in potato tubers and one, or a few, apical buds start growing and using storage nutrients [33]. Accordingly, three to five well developed,~10 mm-long sprouts were found at the apex of stored control tubers after 48 weeks, associated with visible signs of skin dehydration (Fig. 2). By comparison, about 12 small,~2.5 mm-long buds were counted on tubers of lines K52 and K53 (Fig. 2b, c), associated with a visually unaltered skin surface similar to that of freshly harvested tubers (see Fig. 2a for line K52). Similar observations were made with a number of additional lines expressing OCI at comparable or lower levels (Additional file 1), suggesting a sprout-repressing effect of this protein even at very low levels in tuber tissue. As expected given the establishment of apical dominance at early sprouting [34], most, if not all, growing sprouts on control line K tubers were found at-or around-the apex, on the upper (apical) half of the tuber (Fig. 2c). Buds on K52 and K53 tubers were also found mostly on the apical half, but a consistent number of two or three buds were also observed on the basal half (Fig. 2c). These observations pointed, overall, to an alteration of the apical dominance pattern in OCI-expressing tubers and to a significant retarding effect of recombinant OCI on sprout growth presumably associated with limited metabolic activity in the tubers and/or apical buds. These data suggest, in practice, the feasibility of preventing early tuber sprouting during long-term storage by the ectopic expression of recombinant cystatins at low levels, with a minimal demand in endogenous amino acid resources and a likely negligible effect on the tuber protein complement. Storage protein catabolism is delayed in OCI-expressing tubers Soluble proteins were assayed in tubers of lines K, K52 and K53 to assess the overall impact of OCI expression on storage protein deposition in developing tubers and protein catabolism during early sprouting, after storage for 36 or 48 weeks at 4°C (Fig. 3). One-cm 3 tuber tissue pieces were collected at 0 cm ('edge' tissue containing skin cells) or 4.5 cm ('flesh' tissue) down from the apex, beneath the emerging apical sprouts (Fig. 3a). Soluble protein content in flesh tissue samples of freshly harvested tubers (time 0) was estimated at~0.65 % fresh weight in both transgenic and control lines, suggesting a null impact of OCI on total storage protein deposition in growing tubers (ANOVA; P = 0.398, F = 1.077) (Fig. 3b). By contrast, protein content in flesh tissue of line K decreased to 0.55 % fresh weight after 36 weeks and then to 0.39 % after 48 weeks, while remaining significantly higher in flesh tissue of K52 and K53 tubers after both 36 (P = 0.014, F = 9.447) and 48 weeks (P = 0.010, F = 11.13), at~0.55-0.62 % fresh weight. Immunodetections were carried out to compare PMC levels in control and transgenic line protein samples. PMC accumulates in growing potato tubers along with the major storage protein patatin, and then disappears upon sprouting to release free endogenous Cys proteases and help provide free amino acids for plantlet growth [13,14]. Similar to total soluble proteins, PMC was found at comparable levels in OCI-expressing and control effect of OCI on tuber protein deposition. Extensive degradation of the endogenous cystatin was observed in line K after 48 weeks, down to a residual level of 40 % compared to time 0 in edge tissue, and to barely detectable levels in flesh tissue. By comparison, PMC content in edge tissue remained unchanged in both lines K52 and K53 after 48 weeks (ANOVA; P > 0.05), and was still detectable in flesh tissue despite significant degradation (Fig. 3c). Gelatin-polyacrylamide gel electrophoresis (PAGE) zymograms [35] were produced to visualize major protease (gelatinase) forms in stored tuber protein samples (Fig. 3d). In accordance with the anti-papain inhibitory activity of OCI [15] and the predominance of papainlike Cys proteases in sprouting tubers [36], protease activity after 48 weeks was less important in OCIcontaining protein samples than in line K samples. rOCI, a recombinant form of OCI produced in E. coli [36], was incubated with line K samples prior to gelatin-PAGE to confirm the occurrence of OCI-sensitive Cys protease(s) in sprouting tubers (Fig. 3d). As shown for the major gelatinase P1, rOCI had a sharp inhibitory effect on endogenous proteases causing an almost complete loss of gelatinase activity compared to the non-inhibited control. These findings, although not excluding alternative effects in vivo, support the hypothesis of a protease inhibition-mediated mechanism for the sprouting repression effect of OCI in potato tubers, in line with the protease regulatory role of this inhibitor in rice seeds [15] and as also suggested for the germinationretarding effect of recombinant cystatins in Arabidopsis seeds [17,21]. Starch processing is delayed in OCI-expressing tubers Sugar assays were conducted to assess the impact of OCI expression on starch processing and soluble sugar content in transgenic tubers (Table 1). Starch hydrolysis is known to occur in sprouting tubers, typically associated with an increase in soluble sugars [37]. Starch content in flesh samples of freshly harvested tubers was here estimated at~14.5 % of total fresh weight for the three tested lines (ANOVA; P = 0.594, F = 0.569), comparable to starch levels observed earlier in tubers of the same cultivar [26,38]. By contrast, starch content in line K decreased to 11.8 % after 36 weeks of storage, compared to a greater, almost unchanged mean value of 14 % in the OCI-expressing tubers (P = 0.002, F = 19.40). Starch content further decreased in control tubers after 36 weeks to reach 6.3 % of total fresh weight at 48 weeks, lower than the levels of about 8 % measured in OCI-expressing tubers (P = 0.012, F = 10.12). Similar to starch, comparable levels of sucrose (ANOVA; P = 0.969, F = 0.032) and glucose (P = 0.304, F = 1.463) were found in tubers of control and transgenic lines upon harvesting, estimated at~0.20 and~0.14 % of tuber fresh weight, respectively, similar to previously reported contents [26,38]. Glucose content gradually increased in tubers of all three lines, to reach about 1 % of tuber fresh weight at 48 weeks (P = 0.736, F = 0.323). Sucrose content also increased during storage but remained lower in the OCI tubers, about half the levels observed in control tubers after storage for 48 weeks (P = 0.002, F = 22.40). These observations suggest on the one hand a negligible effect of OCI expression on the deposition of starch and soluble sugars in growing tubers. They indicate on the other hand a significant interfering effect of the cystatin on sugar catabolism during long-term storage, concomitant with the abovedescribed repressing effects of this protein on sprouting and endogenous protease activity. Germination and plantlet growth are delayed, but not compromised, in OCI-expressing tubers A sowing assay was conducted with tubers of lines K and K52 stored for 48 weeks at 4°C to assess the ability of OCI-expressing tubers of producing viable plantlets despite limited bud outgrowth after long-term storage, and hence of being of eventual interest to produce seed tubers for vegetative propagation (Fig. 4). In brief, plantlets started to emerge from control tubers 5 d after sowing to reach an overall emergence rate of 80 % (i.e. 16 tubers emerged out of 20 sown) after 17 d, comparable to line K52 tubers exhibiting a 90 % emergence rate (18 tubers out of 20) recorded from 6 to 15 d post-sowing (Fig. 4a). Soluble protein content showed a gradual decrease after sowing in flesh tissue of both control and K52 tubers, down to a low, negligible residual level after 18 d (Fig. 4a). Overall plantlet growth -as estimated by mean plant height after emergence-showed a similar trend over time for the two lines, let apart a systematic delay of 2-3 d for the OCI-expressing tubers (Fig. 4b). Likewise, total cumulative numbers of leaves and stems on emerged plantlets showed similar incremental patterns for the two lines, except for a 2-3 d delay with the K52 line (data not shown). These observations suggest overall no clear effect of recombinant OCI on plantlet emergence and growth, with the notable exception of a short delay for the transgenic tubers likely explained by limited sprout size after storage. Corn cystatin II also delays storage protein breakdown in potato tubers Patatin, PMC and the serine (Ser) protease inhibitors Kunitz trypsin inhibitors and proteinase inhibitor II [26] were monitored in transgenic potato tubers engineered to express a maize functional homologue of OCI, corn cystatin CCII [22], to gain further confirmation for the retarding effect of ectopic cystatin expression on storage protein catabolism (Fig. 5). Two CCII-expressing potato lines, line 9.4 and line 10.4, were selected for the experiments among a collection of stable transformants derived from control parental line K [5]. [26] for MS/MS identification of the tuber test proteins following electrophoresis bears no N-terminal signal peptide and accumulates in the cytosol under the control of the CaMV 35S constitutive promoter. Ten cm-long tubers produced from greenhouse-acclimated plantlets were harvested for each line and stored at 4°C for 0 ('harvest' control) or 30 weeks (ca. 7 months) prior to storage protein analysis. Soluble proteins extracted from flesh tissue were resolved by sodium dodecyl sulfate (SDS)-PAGE and stained with Coomassie blue (Fig. 5a), and the relative amounts of patatin, PMC and Ser protease inhibitors estimated by densitometric analysis of the corresponding bands (Fig. 5b). Unlike the Ser protease inhibitors still found at their initial levels upon harvest, patatin and PMC underwent extensive degradation in tubers of control line K after 30 weeks, down to residual levels of~35 and~15 % the amounts found in freshly harvested tubers, respectively. By contrast, patatin and PMC showed respective residual levels of up to 60 % and about 100 % after the same period in CCII-expressing tubers, well above the corresponding levels in control tubers (ANOVA; P < 0.05). These observations indicate overall a significant delaying effect of CCII on storage protein catabolism in stored tubers, as observed above with OCI in lines K52 and K53. Here we document the retarding effects of these proteins on storage protein catabolism and bud outgrowth in a vegetative reproductive organ. Cystatin ectopic expression was shown previously to delay seed germination in Arabidopsis, presumably via an inhibition of endogenous Cys proteases involved in storage protein breakdown [17,21]. We report similar effects, and the practical potential of such effects, for cereal cystatins ectopically expressed in vegetative storage organs, using potato tubers as a model of economic value. Potato is an important staple crop worldwide and sustained efforts have been made over the years to improve its attributes as a food, using both classical breeding and biotechnological approaches [46]. The potato tuber is a rich source of valuable nutrients, such as starch and proteins [47], that need to be preserved between harvesting and eventual human consumption or use as 'seed' material for vegetative propagation [48]. Towards this goal, our data provide empirical evidence for the potential of recombinant cystatin expression as an 'in-built' strategy to control sprouting and nutrient depletion during storage, in complement to current approaches involving chemical sprouting inhibitors [49][50][51] or physical means such as postharvest irradiation or pressure treatments [52,53]. Work is underway to characterize interactions between endogenous Cys proteases and recombinant cystatins in cystatin-expressing tubers, taking into account the complex hormone-and sugar-mediated signaling networks shaping the dormancy and sprouting processes of potato tubers [54,55]. Work is also underway to compare the anti-sprouting effects of OCI and CCII with the eventual effects of improved cystatin functional variants developed earlier in our laboratory [56]. Plant lines Transgenic potato lines (Solanum tuberosum cv. Kennebec) expressing OCI or CCII were selected from in-house collections of independent transformants maintained in vitro at 20°C in 25 mm-wide x 15-cm long glass tubes, under a 16 h:8 h day/night photoperiod [23,57]. Gene constructs for transformation included either the OCI-encoding sequence (GenBank Accession No. J03469) or a CCII-encoding sequence (GenBank Accession No. D38130) without the native N-terminal peptide signals for cellular secretion. They also included an original CaMV 35S promoter (OCI lines) or a duplicated version of this promoter (CCII lines); a tobacco etch virus enhancer sequence (CCII lines); and a nopaline synthase (OCI lines) or a CaMV 35S (CCII lines) terminator sequence (see refs. [23] and [57] for details on gene constructs). Line K used as parental line for transformation was taken as a control for comparative purposes [57]. Tubers Tubers were produced from in vitro-grown plantlets acclimated in greenhouse for 2 weeks under a 16 h/8 h light-day photoperiod, and then let to grow for 20 weeks in 25-cm (10-inch) wide pots filled with vermiculite. Three to six tubers of similar size (ca. 10 cm-long) were harvested from different plants and used for cystatin expression monitoring, sprout inspection, sowing assays or compositional analyses after storage in the dark at 4°C for 0, 30, 36 and/or 48 weeks. Tuber samples for cystatin monitoring and compositional analyses were ground to a fine powder in liquid nitrogen, and the resulting powder lyophilized to dryness and stored at −80°C until use. OCI expression OCI transcripts were monitored by RT PCR using total RNA samples and the following oligonucleotide primers: 5'-GAACGACCTCCACCTCGTCGACCTC and 3'-GTACAAAGTGCCAGCGACAACTTGCT. Total RNA was extracted from tuber tissue powder (see above) or from the fifth leaf of~30 cm-tall plants (down from the apex [24]), using the Concert Plant RNA Reagent™ kit (Life Technologies, Burlington ON, Canada). After precipitation in isopropanol, total RNA was washed in 75 % (v/v) ethanol, dissolved in RNAse-free water, and assessed for quantity and quality by the monitoring of A 260 /A 280 and A 260 /A 230 absorbance ratios with a Nano-Drop™ 1000 spectrophotometer (Thermo Fisher Scientific, Mississauga ON, Canada) according to manufacturer's instructions. cDNA populations were synthesized using the Superscript kit for cDNA synthesis (Life Technologies) and used as templates for PCR. The PCR amplicons were resolved by 1 % (w/v) agarose gel electrophoresis and visualized by ethidium bromide staining. For quantitation, the gels were digitalized with an Amersham Image Scanner (GE Healthcare, Baie d'Urfé QC, Canada) prior to computer processing and image analysis using the Phoretix 2D Expression software, v. 2005 (NonLinear Dynamics, Durham NC, USA) [42]. At least three tuber or leaf replicates were used for each line to allow for statistical assessment of the data (ANOVA; α = 0.05). OCI content OCI in leaf and tuber extracts was titrated using the colorimetric protein substrate azocasein and E-64calibrated papain (EC 3.4.22.2) as a target protease [58]. Soluble proteins were extracted from tuber [or leaf] powder (see above) in 100 mM sodium phosphate, pH 6.5, containing 1 mM EDTA. Papain activity was monitored in 100 mM sodium phosphate buffer, pH 6.5, containing 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 5 mM L-cysteine and 0.1 % (v/v) Triton X-100 (Sigma-Aldrich, Mississauga ON, Canada). An arbitrary value of 100 % residual activity (0 % inhibition) was assigned to papain activities in leaf or tuber extracts of control line K. Soluble proteins and sugars Soluble proteins were assayed according to Bradford [59] with chicken egg white albumin as a standard, after extracting proteins from lyophilized tuber powder as described above. Soluble sugars in powder samples were extracted in 80 % (v/v) ethanol for 30 min at 80°C. After centrifugation for 5 min at 3000 g, the supernatant was recovered for glucose and sucrose enzymatic quantitation in the presence of ATP and NAD [60]. The pellet was resuspended in 0.2 M KOH for starch quantitation. PMC, storage proteins and endogenous proteases PMC, patatin, Kunitz inhibitors and potato proteinase II in tuber protein extracts were resolved by 10 % (w/v) SDS-PAGE, and their relative content determined by densitometry of Coomassie blue-stained gels as described above for the OCI amplicons. PMC was also quantified on nitrocellulose sheets following immunoblotting, after detection with polyclonal anti-PMC primary antibodies. Protease activities in sprouting tubers were visualized by mildly denaturing gelatin/SDS-PAGE [35], with or without prior incubation of the protein extracts with rOCI, a recombinant form of OCI expressed in E. coli using the glutathione S-transferase system [36]. Sowing bioassay A sowing ('germination') assay was conducted with tubers of line K52 and control line K to detect eventual macroscopic effects of OCI expression on growth and development of the emerging plantlets. Twenty~10 cmlong tubers stored at 4°C for 48 weeks (see Fig. 1a) were sown in vermiculite-containing, 25-cm (10-inch) wide pots, and the plantlets were left to grow for three weeks in greenhouse. Sprouting emergence rates and stem height of the growing plants were monitored daily over 20 days. About 30 additional tubers of each line were sown in parallel, and three of them collected daily, to determine total soluble protein content in flesh tissue over the same period. Tuber processing and protein determinations were performed as described above for freshly harvested and stored tubers. Competing interests The authors declare that they have no competing interests. Authors' contributions AM and M-AS contributed to the experimental design, performed the experiments and wrote a first draft of the manuscript. MK performed the sugar determinations and contributed to the analysis of the data. M-CG contributed to the experimental design and writing of the manuscript. DM conceived the study, contributed to the experimental design, coordinated the experiments, and prepared the last version of the manuscript. All authors read and approved the final manuscript.

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2017-04-19T01:37:17.842Z

2015-04-17T00:00:00.000Z

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QTL Mapping of Agronomic Waterlogging Tolerance Using Recombinant Inbred Lines Derived from Tropical Maize (Zea mays L) Germplasm Waterlogging is an important abiotic stress constraint that causes significant yield losses in maize grown throughout south and south-east Asia due to erratic rainfall patterns. The most economic option to offset the damage caused by waterlogging is to genetically incorporate tolerance in cultivars that are grown widely in the target agro-ecologies. We assessed the genetic variation in a population of recombinant inbred lines (RILs) derived from crossing a waterlogging tolerant line (CAWL-46-3-1) to an elite but sensitive line (CML311-2-1-3) and observed significant range of variation for grain yield (GY) under waterlogging stress along with a number of other secondary traits such as brace roots (BR), chlorophyll content (SPAD), % stem and root lodging (S&RL) among the RILs. Significant positive correlation of GY with BR and SPAD and negative correlation with S&RL indicated the potential use of these secondary traits in selection indices under waterlogged conditions. RILs were genotyped with 331 polymorphic single nucleotide polymorphism (SNP) markers using KASP (Kompetitive Allele Specific PCR) Platform. QTL mapping revealed five QTL on chromosomes 1, 3, 5, 7 and 10, which together explained approximately 30% of phenotypic variance for GY based on evaluation of RIL families under waterlogged conditions, with effects ranging from 520 to 640 kg/ha for individual genomic regions. 13 QTL were identified for various secondary traits associated with waterlogging tolerance, each individually explaining from 3 to 14% of phenotypic variance. Of the 22 candidate genes with known functional domains identified within the physical intervals delimited by the flanking markers of the QTL influencing GY and other secondary traits, six have previously been demonstrated to be associated with anaerobic responses in either maize or other model species. A pair of flanking SNP markers has been identified for each of the QTL and high throughput marker assays were developed to facilitate rapid introgression of waterlogging tolerance in tropical maize breeding programs. Introduction Rainfed maize crop grown during the summer-rainy season, occupying over 80% of the total maize area in the Asian tropics, occasionally face extreme weather conditions that limit crop establishment and yield potential. Moisture availability is seldom optimal for rainfed maize in this region. Inadequate distribution, especially in higher-rainfall areas of Eastern India and many parts of Southeast Asia, causes transient/intermittent water-logging, which is one of the most important constraints for maize production in Asian tropics. Over 18% of the total maize production area in South and Southeast Asia are affected by temporary floods and waterlogging, causing annual production losses of 25-30% [1]. The increasing demand for maize in Asian region is rapidly transforming cropping systems in certain parts of Asia from rice monoculture to more profitable rice-maize systems [2], which currently occupies approximately 3.5M ha area in Asia [3]. However, maize production in this emerging rice-maize cropping sequence often faces the problem of early stage excessive soil moisture, as soils of paddy fields are frequently saturated due to late rains. Being a non-wetland crop species, maize is highly susceptible to anaerobic soil conditions during germination and early growth stages [4,5]. However, the extent of damage due to waterlogging stress varies significantly with developmental stage of the crop, and past studies have shown that the maize crop is comparatively more susceptible at germination and early seedling to tasseling stage [6]. Seeds with carbohydrate reserves, including maize seeds, are generally more tolerant of hypoxia (limited oxygen) or even anoxia (no oxygen) than seeds with fatty acid reserves [7,8]. Therefore, maize seed can germinate under wet soil conditions in the presence of nominal amounts of oxygen [9], but further growth is crippled, if subjected to excess soil moisture stress. At later growth stages some genotypes, with an inbuilt ability to produce adventitious roots at above-ground nodes and to form aerenchyma in the cortical region of adventitious roots have been shown to tolerate excessive moisture to certain extent [10,11]. Considerable genetic variability has been observed in maize for tolerance to excessive moisture stress [4,10,12,13,14], which could be favorably exploited in developing maize varieties that can tolerate such hypoxia/anoxia conditions. As described by Mano and Omori [15], three primary physiological mechanisms conditioning water logging tolerance are 1) the ability to grow adventitious/brace roots at the soil surface during flooding conditions; 2) the capacity to form root aerenchyma and (3) tolerance to toxins (e.g., Fe 2+ , H 2 S) under reduced soil conditions. Understanding the genetics of such physiological traits will certainly aid in more precise manipulation of waterlogging tolerance in the maize breeding programs. Waterlogging tolerance is predominantly a polygenic trait in many crop species including maize. Both additive and non-additive effects are important in the inheritance of flooding tolerance in maize and transgressive segregation has been observed for grain yield in F 2 population involving waterlogging tolerant and sensitive genotypes, thereby indicating the possibility of favorable alleles coming from both the parental lines [16]. Several subspecies (ssp.) of teosinte germplasm have been used as source of flooding tolerance in maize [16] and a number of genetic studies identified genomic regions for several rootrelated traits under flooding and drained conditions. Mano et al. [17] mapped QTL for adventitious root formation under waterlogged conditions on chromosome 4 and 8, using a maize by teosinte (Zea mays ssp. huehuetenangensis) cross, in which the favorable alleles were contributed by teosinte. Exploring the capacity to form aerenchyma in the cortex of adventitious roots, Mano et al. [18] identified three QTL on chromosomes 1, 5 and 8 that together explained close to 45% of phenotypic variance for aerenchyma under non-flooding, drained water conditions in a population derived from B64 x teosinte (Zea mays ssp. nicaraguensis). A similar study involving another ssp. of teosinte (Zea mays luxurians), idenitified different set of QTL for constitutive aerenchyma formation thereby indicating the possibility of pyramiding multiple genomic regions from the different ssp. of teosinte into cultivated maize [19]. Recently, introgression lines tolerant to flooded conditions were developed by transferring genomic regions from Z. mays ssp nicaraguensis into maize inbred line (Mi29), which demonstrated the effect of a specific chromosomal segment on the long arm of chromosome 4 donated by teosinte [20]. Construction of high density linkage map in maize by teosinte (Z. mays ssp nicaraguensis) aided in identification of another QTL on chromosome 1 (Qaer1.06) that was found to regulate constitutive aerenchyma formation [21,22]. Besides teosinte germplasm, flooding tolerance has also been frequently observed and subsequently mapped on several instances in cultivated maize (Zea mays ssp mays). In a study involving a maize F 2 population, Mano et al. [23] detected QTL for the ability to form adventitious roots on soil surface on chromosomes 3, 7 and 8, in which the QTL on chromosome 8 was common with that of teosinte germplasm. A QTL of moderate effect size was reported by Mano et al. [24] on bin 3 and 4 of chromosome 1 (1.03/1.04), that explained around 14 and 10% of phenotypic variance for leaf injury and dry weight production respectively under flooded conditions. Flooding tolerance has also been a major focus in many Brazilian maize breeding programs and Anjos e Silva et al. [25] identified three loci corresponding to glutamine synthetase (on chromosome 5), zein (on chromosome 4) and triosephosphate isomerase (on chromosome 3) which together explained up to 30% of phenotypic variance for shoot and root dry matter under flooded conditions. A recent genome-wide association study in Chinese germplasm identified four interesting signals on chromosome 5, 6 and 9, each explaining around 15% of phenotypic variance for root/shoot length and fresh weight under waterlogged conditions [26]. From the above-mentioned genetic studies, it is apparent that waterlogging tolerance does exist in elite maize germplasm and the inheritance is likely governed by several QTL. As the heritability of GY and other associated traits under flooded conditions generally tend to be low, the evaluation of tolerance can be easily influenced by environmental conditions, hence the use of marker-assisted selection (MAS) would likely be very effective strategy in these breeding programs [27]. However, the effectiveness of MAS relies on the precise localization of the QTL using the representative breeding germplasm and identification of tightly linked, easy-to use molecular markers. Objectives of the present investigation were to 1) obtain heritability estimates for GY and other secondary traits associated with waterlogging tolerance; 2) determine the extent of association between GY and secondary traits under waterlogged conditions; 3) identify genomic regions influencing waterlogging stress related traits and estimate their effect sizes; 4) determine the degree of overlap between QTL identified from per se and TC (test cross) evaluations for waterlogging stress related traits and 5) propose a set of SNP markers and easy to use genotyping assays that physically delimit the identified QTL to enable integrating marker assisted selection for waterlogging tolerance in tropical maize improvement programs. Germplasm The two parental lines used in the study were contrasting for their responses to waterlogging stress. CML311-2-1-3 is an elite but highly sensitive line, whereas CAWL-46-3-1 was tolerant for waterlogging stress. These two parental lines were selected on the basis of their consistent responses in line evaluation trials conducted during 1998-2003 during rainy season at Directorate of Maize Research (DMR), New Delhi, India (28.48N, 77.18E, 228.2 masl). CML-311-2-1-3 was derived from selected segregants in the CIMMYT inbred line CML-311, which was derived from a synthetic population, S89500. CAWL-46-3-1 was derived from a local population (Jaunpur Local) tolerant to vegetative stage waterlogging stress. The segregating population involving these two parental lines was developed during the dry season of 2005/2006 at DMR Maize Research Station in Hyderabad, India. The population was advanced to F 3 generation, and thereafter a single-seed descent approach was followed for developing the RILs. A total of 211 lines were developed. In the dry season of 2009/2010, the RILs (S6 stage of inbreeding) were test-crossed with CML451, and the F 1 seeds (RILs-TC) were harvested for further evaluations. CML451 is a late maturing yellow line, which is used as tester in the tropical maize breeding program of CIMMYT. The RILs and test crosses (TCs) were evaluated under managed waterlogging stress conditions during the rainy season of 2009 at experimental farm of International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, India (17°53' N latitude,78°27' E longitude and 545 masl). Managed stress phenotyping Soil of the experiment field was vertisol with a pH of 7.5. Planting was done in dry soil condition on prepared ridges in field using an alpha lattice design [28] with two replications. All the entries were over sown and thinned to one plant per hill at the V2 growth stage to give a population density of 66,000 plants per hectare. Each entry was planted in one row, 3.0 m long, with 0.20 m spacing within and 0.75 m between rows. Before planting, 60 kg nitrogen (N) per hectare in the form of urea, 60 kg phosphorous per hectare as single super phosphate, 40 kg potassium per hectare as muriate of potash and 10 kg zinc as zinc sulfate were applied as a basal dressing. Second and third doses of N (each 30 kg N per hectare) were side-dressed at kneehigh and tasseling stages. Weeds were controlled with pre-emergence application of pendimethalin and atrazine (both at 0.75 kg per hectare) Experiments were kept free from insect, weeds and diseases using recommended post-emergence chemical measures, and managed under optimal agronomic practices. Water logging treatment was applied through flooding at the knee-high stage (V7-8 growth stage) continuously for seven days. At the beginning of the treatment, plots were completely filled with water, and maintained thereafter for 7 days at a depth of 10±0.5cm by supplying water at a rate that exceeded infiltration and evaporation. After completion of the stress treatment the field was completely drained-out. The TC progenies were screened for water-logging tolerance in a waterlogging-pit facility. The size of pits for this study is 1096cm x 396cm x 43cm that can accommodate 300 pots of the size 30 x 30 cm and volume 14930 cm 3 . Each pot was filled with a mixture of sieved vertisol, alfisol type of soils and farm-yard manure (FYM), in the ratio of 2:2:1. Each entry was planted with two seeds in each pot with five replications using completely randomized design (CRD) following recommended crop management practices. At V2 growth stage, thinning was done and one plant per pot was maintained for further study. At V7-8 stage, the pits were filled with water in a way that water rise above the pot surface at least by 5.0 cm. Water level lost by evaporation was replenished by filling the pits every day to maintain the same water level for 7 days. After 7 days, water level in the pits was reduced to 3-4 cm depth in order to maintain high moisture condition for another 48 hours, following which pits were completely drained-out and plants were kept for recovery and data recording. Data observations Plant height was measured at male flowering, as the average distance from the base to the node bearing the flag leaf in the plot. Leaf senescence was scored at one week after release of the stress, using a 1-10 scale (1 = 10% and 10 = 100% dead leaf area). The in vivo chlorophyll concentration of the uppermost fully expanded leaf was determined on all plants per plot/pot at the same time as the senescence score, using a Minolta SPAD-502 chlorophyll meter. Results were averaged to give single plot values. Plant lodging was measured a week after imposing the stress, and measured as percentage of the total number of lodged plants to the total number of plants. Similarly, plant mortality (%) was calculated by counting the number of dead plants per entry, one week after completion of water-logging stress and dividing by the total number of plants. Data on brace root was recorded at 50% male flowering by counting the number of above-ground nodes bearing brace roots. Days from planting to anthesis and silking, indicated by when 50% of plants had one extruded anther or produced one visible silk, were recorded by daily visual observations during the flowering period. Anthesis-silking interval (ASI) was calculated as the difference between number of days to 50% silking and 50% anthesis. Under stress, some of the highly susceptible lines failed to reach 50% silking, resulting in barren plants. In such cases, the maximum days to 50% silking of the trial was considered as days to 50% silking of those genotypes for calculation of ASI. At maturity, ears were harvested and dried to a constant moisture level and grain yield was recorded on a shelled grain basis and adjusted to 15% grain moisture. Analysis of phenotypic data The data from each trait observed were analyzed with Proc Mixed in SAS (9.2) [29]. Blocks were treated as random and entry as fixed effect to obtain the best linear unbiased estimates of the mean from the RIL data set. The mean of each RIL test cross and the best linear unbiased estimates from RILs were then used for QTL analysis. Linkage and QTL mapping Genomic DNA was extracted from young leaves collected in a bulk of 10 plants per RIL entry and according to CIMMYT's laboratory protocols [30]. The parental lines were genotyped with a set of 1250 SNP markers, for which KASP assays [31] were designed at LGC genomics (formerly Kbiosciences) facility in London, UK. These 1250 SNPs were a subset of 1536 SNPs from Yan et al. [32]. Genotyping of the RILs were carried out with 331 polymorphic SNP markers using the KASP platform. Linkage map was constructed using QTL IciMapping ver3.2 software (http://www.isbreeding.net) using the twin criterion of more than 3.0 LOD and a maximum distance of 37.2 cM between two loci [33]. Recombination frequencies between linked loci were transformed into cM distances using Kosambi's mapping function [34]. The QTL were identified for the adjusted means for each trait using Inclusive Composite Interval Mapping (ICIM) [33] as implemented in QTL IciMapping v.3.2. The walking step in QTL scanning was 1 cM and a likelihood odds (LOD) threshold of 2.5 based on 1000 permutations was chosen for declaring potentially significant QTL for secondary traits associated with drought tolerance [35,36]. The sign of the additive effects of each QTL was used to identify the origin of the favorable alleles in accordance with Lubberstedt et al. [37]. Alleles coming from CML311-2-1-3 (waterlogging sensitive) were coded as "2" and from CAWL-46-3-1 (waterlogging tolerant) as "0". Additive effect was calculated by deducting the phenotypic average of the individuals carrying "0" allele from that of individuals carrying "2" allele. Hence, negative sign of the additive effect of QTL for Grain Yield, Brace Root and Chlorophyll and positive sign for root lodging (%), stem lodging (%), plant mortality (%) and ASI indicates that the favorable allele for the respective traits originated from the waterlogging tolerant line and vice-versa. The proportion of observed phenotypic variation explained (PVE) due to a particular QTL was estimated by the coefficient of determination (R2) from the corresponding linear model (single marker) analysis, and using the maximum likelihood estimates [38]. For each marker that was closest to a QTL, the RILs were grouped into two classes belonging to the two parental alleles of this marker locus. The trait means of two allele classes were compared using a t-test both for significance and for identification of the parent to which the allele having a positive effect on the target trait belonged. A pair of flanking markers that fall in the confidence interval for each of the QTL detected was provided to facilitate marker-assisted selection of the target QTL. Confidence interval was empirically determined by identifying positions on the right and left side of each detected QTL that decay by one LOD unit from that of the QTL and used to identify overlap intervals. Mean performance Analysis of variance (ANOVA) revealed that mean squares and variance components for genotypes were significant at P < = 0.01 (F-test) for all the observed traits evaluated under waterlogging stress in both per se (Table 1) as well as TC experiments. Except ASI, most of the traits exhibited moderate to high heritability estimates indicating the repeatable nature of the traits under waterlogged conditions. Among the various pairs of correlations generated, significant positive associations between brace roots and chlorophyll content with GY and significant negative associations between root and stem lodging with GY were most notable, suggesting their importance in indirect selection for water logging tolerance (S1 Table). Linkage mapping All the ten maize chromosomes were represented in the linkage map constructed with ten linkage groups (Corresponding to ten chromosomes), spanning a total length of 2,008.2 cM at an average marker interval of 12.4 cM. Almost all bin locations in the maize genome were represented in this linkage map except for some such as 1.06, 2.04, 7.02 and 8.04 due to lack of polymorphic loci obtained from these regions. Wherever discrepancies were observed with respect to order and position of markers between linkage and physical distances, forced reordering of markers were carried out according to the physical locations based on AGP v2 of maize reference sequence [39,40]. Missing data in the genotyping of mapping population across 331 SNP loci were below 5%. Chi-square tests showed that 68 markers deviated from the expected ratio of 1:1 and were subsequently removed for the QTL analyses. Allele frequencies of CML311 were slightly higher than CAWL-46-3-1. The contribution of alleles from the susceptible parent (CML311) in the QTL for waterlogging tolerance Grain yield (GY). The RIL evaluations revealed five QTL for GY under waterlogging stress on chromosomes 1, 3, 5, 7 and 10 and one QTL on chromosome 5 using RIL TC dataset (Tables 2 and 3, Fig 1). The position of the QTL identified in the RIL-TC experiment on chromosome 5 was close to that of GY in RIL and accounted for 3.4 and 8% of the phenotypic variance respectively. The trait enhancing alleles in both the cases were contributed by the water logging tolerant parent line. The five QTL detected in RIL experiment together explained close to 30% of the phenotypic variance. The favorable alleles at QTL on chromosomes 1, 3 and 5 were contributed by the waterlogging tolerant parent, while the susceptible parent donated the favorable alleles at the rest of the two loci. The additive effects of the QTL from water logging tolerant parental line were significant and substantial which ranged from 520 to 640 Kg/ha). Interestingly, the favorable allele at QTL on chromosome 7 was contributed by the waterlogging susceptible parent (CML311), which had an additive effect of around 500 Kg/ha. Secondary traits associated with waterlogging tolerance. A total of 13 QTL were detected across all the chromosomes for the 10 waterlogging component traits using RIL and three traits using TC dataset (Tables 2 and 3, Fig 1). The detected QTL individually accounted for 3-13.6% of the phenotypic variance. Two QTL were detected on chromosomes 7 and 8 for brace root trait, favorable alleles for which were contributed by the tolerant parent. A QTL on chromosome 7 was detected for brace root in RIL-TC experiment, which however differed considerably in terms of physical location from that of RIL evaluations. It was interesting to note that the physical location of brace root QTL identified on chromosome 7 in TC experiment overlapped with the GY QTL detected based on RIL evaluations. A relatively large effect QTL was identified on chromosome 2 for chlorophyll content, which explained close to 14% of phenotypic variance and the favorable allele was contributed by the waterlogging susceptible but elite parent (CML311). Seven QTL with moderate additive effects were detected for root and stem lodging percentage under waterlogged conditions especially in per se evaluations for which the favorable alleles were entirely contributed by the waterlogging tolerant parent ( Table 2). Another relatively large effect QTL was notable on chromosome 5 for plant mortality percentage in TC evaluations, which is an indication of early stage seedling tolerance to waterlogged conditions in contrast to stem and root lodging percent that correspond to later stages of adult plant tolerance. For ASI, a QTL on chromosome 3 accounted for around 7% of phenotypic variance in TC evaluations. As expected, at both these loci for plant mortality percentage and ASI, the favorable alleles ('trait-reducing') were contributed by the waterlogging tolerant parent. Candidate genes underlying QTL intervals. Based on the 'Named Genes' annotation track (http://www.plantgdb.org/ZmGDB), 22 candidate genes with known functions were identified within the physical intervals delimited by the confidence interval of the QTL influencing GY and other secondary traits associated with waterlogging tolerance, of which six of them have previously been demonstrated to be associated with anaerobic responses in either maize or other model species like Arabidopsis and Tobacco ( Table 4). The range of putative functions varied from conditioning metabolic responses such as biosynthesis of lipophilic compounds to early development of brace roots under flooded conditions. The delimited interval on chromosome 3 for ASI harbored a MADS domain transcription factor (zmm16), which has been implicated in reproductive organ development. Discussion Agronomically, waterlogging tolerance is the maintenance of relatively high grain yields under waterlogged as compared to non-waterlogged conditions. Physiological mechanisms controlling such tolerance vary from whole plant regulation to intracellular signaling and programmed cell death. Identification of key donor lines conferring consistent waterlogging tolerance and unravelling genetic factors that influence vital physiological regulators under anaerobic/hypoxic conditions will enable not only gaining better understanding of critical mechanisms but also efficient incorporation of waterlogging tolerance in the maize breeding pipeline. The previous genetic investigations on waterlogging tolerance revealed that the trait is likely polygenic in maize and affected by several mechanisms and complicated by confounding factors such as temperature, plant development stage, nutrient availability, soil type and sub-soil topography [27]. Direct selection on GY under waterlogged conditions is the most favored strategy by the breeders, which however suffers from low heritability estimates for the reasons stated above. Secondary traits such as anthesis-silking Interval (ASI), number of nodes with brace roots (BR), root and shoot biomass, chlorophyll content, plant and ear height, etc. have the potential to be used as indirect selection indices along with grain yield for waterlogging tolerance in maize. Here, we have presented optimized trait measurement approaches especially for such secondary traits that yielded moderate to high heritability (h 2 ) estimates. Some of the traits like BR and stem and root lodging percent not only recorded high h 2 estimates but also robust favorable correlations with GY, thereby warranting their inclusion in the selection strategy for enhanced waterlogging tolerance. Similar strategies of using adventitious root and aerenchyma formation, leaf injury, dry matter production as indicators of waterlogging tolerance have been reported in maize [17,23,24,41]. In the current study, we have identified additive QTL with relatively moderate effects for grain yield under waterlogged conditions, for which favorable alleles were contributed by the water logging tolerant parent on chromosomes 1, 3 and 5. It is interesting to note that QTL for root lodging and ASI were mapped on the overlapping intervals with that of GY QTL on chromosomes 1 and 3. Investigating the ability to form root aerenchyma formation in a cross involving Zea nicaraguensis, Mano et al. [18] identified two QTL in the overlapping physical interval of GY QTL identified in the current study on chromosome 1 and 5. Similarly, a mapping study on the ability to form adventitious roots identified a QTL on chromosome 3, which co-located with the GY QTL in the present study. Contrary to the above-mentioned three QTL regions, favorable alleles at the QTL on chromosome 7 and 10 were contributed by the elite but waterlogging sensitive parental line. In the absence of optimal performance data, it will however be difficult to point out whether the identified QTL in these locations actually correspond to waterlogging tolerance or general GY performance. Nevertheless, identification of such genomic regions along with pairs of flanking markers will facilitate marker-aided selection of superior transgressive segregants in the early filial generations of breeding programs targeted towards developing improved maize germplasm that perform equally well under optimal and waterlogged conditions. Recently, Zhang et al. [26] carried out a genome-wide scan of seedling waterlogging tolerance in a diverse collection of Chinese maize germplasm which revealed an interesting region on chromosome 5.04 (~159-165 Mb as indicated by PZE-105105668) that explained 15 to 20% of phenotypic variance for root/shoot fresh & dry weights under waterlogged conditions and overlapped with the QTL identified for GY in the current study. Zhang et al. [26] further validated this region on 5.04 in a population that was backcross-derived from a cross involving waterlogging tolerant (HZ32) and sensitive (K12) lines, thereby indicating the potential of this region to be further explored in marker assisted introgressions. Analysis of candidate genes in the delimited interval of 112-160 Mb on chromosome 5 revealed a cysteine protease gene (ccp1), which has been previously demonstrated to be associated with anoxia-induced root tip death in maize [42]. Two QTL regions were identified on chromosomes 7 and 8 for brace root development which had significant positive association with GY under waterlogged conditions (Tables 2 and 3). The favorable alleles at both the QTL were contributed by waterlogging tolerant parent and together explained close to 10% of phenotypic variance. Independently, for root lodging, three QTL were detected on chromosomes 1, 3 and 10 at which the favorable allele were contributed by waterlogging tolerant parent (Table 2). Based on the evaluation of immortalized F 2 populations derived from recombinant inbred lines of CML288 (tropical) and Huanzao4 (temperate Chinese), Ku et al. [43] reported QTL for total and effective brace root tier number at the same physical interval (~8-15 Mb) on chromosome 8, where QTL for brace root were identified in the current study. Root and stem lodging in maize are considered critical attributes for maintaining crop productivity especially under waterlogged conditions. A total of 7 QTL were identified in the present study for root and shoot lodging and the favorable alleles were solely contributed by waterlogging tolerant parent at all the 7 loci. The QTL identified on chromosome 7 for brace root overlapped with that of genomic region responsible for stem lodging (Fig 1). Reference to functional annotation of the delimited genomic interval on chromosome 7 (137-155 Mb) identified a glutathione S-transferase16 (gst16) gene, which is predicted to play a key role in the metabolic processes leading to early stage brace root development [44]. ASI was found to be more relevant in the hybrid (Test cross) trial and a strong QTL on chromosome 3 was identified (Table 3), wherein the favorable allele coming from the waterlogging parent decreased the interval between male and female flowering by~2 days. Recently, Almeida et al. [45,46] performed metaQTL analyses across three tropical populations for GY, ASI and other secondary traits, which identified a hotspot QTL region on chromosome 3 (~170-214 Mb) that contained an important candidate gene, zmm16 (MADS box domain transcription factor), which is associated with reproductive organ development in maize [47] Waterlogging tolerance especially at the early stages of plant development such as seedling growth phase can provide substantial advantage for subsequent survival under flooded conditions during the later stages of growth. The QTL identified on chromosome 5 (~7-23Mb) for plant mortality % based on TC experiment explained more than 10% of phenotypic variance and merits attention for marker-assisted introgressions. This physical interval harbored two important genes related to waterlogging tolerance 1) a cytochrome b6 gene (GRMZM2G463640), which is oxygen-dependent and is known to change sub-unit structure and holoenzyme levels, especially during low oxygen conditions [48]. The Arabidopsis genome contains as many as 286 different cytochrome P450 genes, some of which play crucial roles in the biosynthesis of a variety of endogenous lipophilic compounds and cross talk in the responses to abiotic and biotic stresses [49,50]; 2) single myb histone 6 gene (GRMZM2G095239), whose related family member (myb2) has been demonstrated to play a strong regulatory role in the induction of alcohol dehydrogenase (adh1) during low oxygen conditions [51]. The partial mismatch between QTL identified based on per se and TC phenotypes is likely because of one or combination of below mentioned factors-strong tester effects, which may mask the parental allele performance [52,53], smaller population size [54,55], field versus pit phenotyping of per se and TC individuals, high QTL x Environment interaction [56] and genetic architecture of the trait (such as epistatic interactions as reported by [52,57,58]). Reliable screening methods, consistent sources of tolerance and breeding critical information such as number and effect sizes of various genomic regions influencing tolerance traits will help in enhancing the success of breeding programs. The screening methods reported and set of QTL identified in the current study for GY and various other secondary traits will be helpful in designing marker-aided pyramiding or introgressions in the tropical maize breeding program aimed at efficient incorporation of waterlogging tolerance.

v3-fos

2016-05-17T20:52:01.748Z

2015-11-09T00:00:00.000Z

16182387

s2

Farmers knowledge and perception on maize stem borers and their indigenous control methods in south western region of Cameroon Background Agriculture is a major contributor to the Gross Domestic Product (GDP) of Cameroon, The South West region of Cameroon is known for its potential in the production of major agricultural commodities, but farmers’ yields from various speculations are low, dwindling over time due to some major constraints. Maize production is hampered by adverse socio-economic factors, several pests and diseases as well as high rainfall with low solar radiation. Lepidopterous maize stem borers are a major threat to increase maize production. Therefore we hypothesized that the farmers of the South West region: (1) also perceived stem borers as an important pest of maize; (2) they have their own indigenous methods of control; (3) they use chemical pesticides because they have no alternative, but would prefer plant materials if these were standardized. Methods A semi-structured questionnaire survey was administered in four villages: Maumu, Lower Bokova, Ekona and Bonduma. A total of 151 (male and female) farmers were randomly interviewed to document farmers’ perception on stem borers, and their use of indigenous knowledge to manage key pests of maize. Results Stem borers were present throughout the maize growing areas in the Fako division and ranked as one of the most important pests of the crop. Most farmers (82.1 %) perceived that stem borers caused significant damage on maize and were responsible for yield reductions in the crop. The increased impact of these pests was due to improper/untimely use of expensive conventional insecticides given the lack of a cheaper alternative method of control. About 50 % of respondent admitted not having any indigenous knowledge of stem borer control, while only 20 % had tried plant products. The most relevant indigenous stem borer control was the use of wood ash. Most (90 %) of the respondent would prefer plant-based insecticides in future because they are safer, cheaper and readily available. Conclusions Farmers’ knowledge would contribute in understanding the activities of stem borers and use of plant insecticides. Research is therefore needed to standardize the methods of using plant-based products and also identify the active ingredients of these plants to ensure their effectiveness against maize stem borers and other pests. Background Agriculture is the primary production sector that contributes to the Gross Domestic Product (GDP) of Cameroon [1]. It is also the main source of employment in the country. Maize production is the main source of income for more than three million small-scale farmers in Cameroon. Though the government subsidizes maize production, this support falls far short of farmers' needs due to the myriads of production constraints including persistent pest problems such as stem borers. Lepidopterous stem borers seriously limit potentially attainable maize yields by infesting the crop throughout its growth stages from seedling to maturity [2]. In Cameroon, maize is grown across all agro-ecological zones, from sea level to the highlands at 2000 m a.s.l., and the stem borers are present in all these zones. However, the pest densities and plant damage vary greatly between fields [3]. The most important species that reduce maize yield in West and Central Africa are the pink stalk borer, Sesamia calamistis Hampson (Lepidoptera: Noctuidae), the African sugarcane stalk borer, Eldana saccharina Walker (Lepidoptera: Pyralidae) and the maize stalk borer, Busseola fusca Fuller (Lepidoptera: Noctuidae) [4][5][6]. Yield losses of food grain in areas with severe borer problems vary between 10-70 % [7][8][9][10]. In most of Cameroon, these stem borers are often controlled using conventional insecticides. However, these Synthetic insecticides are unacceptable since they may lead to problems of toxic residues, health and environmental hazards [9,11] when inappropriately used. As stem borers burrow into the stem, they are often protected from contact insecticides [10]. Various methods of cultural control of stem borers in Africa have been reviewed [12][13][14][15], these are most relevant and economical to African resource-poor farmers. Though many of these methods are labor intensive, they have little adverse environmental effects and are readily applicable without extra investment in expensive equipment In Tanzania farmers have greatly relied on indigenous knowledge and/or plant pesticides to meet their daily needs [16]. This knowledge is most relevant to the rural poor and marginalized population. Several indigenous plant based pest management options used for the control of field and storage insect pests have been identified by research in parts of Tanazania [17]. The studies indicated that botanical formulations reduced stem-borer load by more than 55 % and increased maize yield by more than 60 % compared to the control. Over several decades indigenous knowledge practices have been incorporated into scientific knowledge for development and conservation of natural resources [18]. Hegazy et al. [19] and Mugisha-Kamatenesi et al. [20], found that if thoroughly investigated, the current knowledge gained from indigenous plant species may provide more goods and services for local use. In many parts of Africa, derivatives of indigenous plants like Piper guineense and Tephrosia vogelii gained attention because of their insect pest control potential [21,22]. More so, Jatropha curcas (native to the American tropics, but widely distributed from Senegal to Cameroon) is also widely used for pest control. Entire or powdered fruits of Piper spp. have insecticidal and/or repulsive effects against many pests [23][24][25]. Despite the enormous potential that has existed for generations, plant based indigenous pest control practices have remained largely unexploited due to limited research intervention and resources committed. However, current interest in reducing environmental contamination and global warming are serving as added impetus for the re-evaluation and intensification of environmentally friendly and cost-effective pest management technologies such as the use of traditional botanical pest control agents [26]. Many studies carried out in parts of Africa found that plant derived ash including those of wood and cocoa pod increased P, K, Ca, Mg status of soil and pH and yield of vegetables, rice, millet and maize [27][28][29]. Farmers' knowledge of various constraints varies qualitatively/quantitatively depending on their interest in the subject, the environment, and its relevance to their lives. Use of indigenous and plant-based insecticides has been greatly neglected in Fako division and this may partly explain why farmers rely solely on synthetic pesticides. In order to improve food security and alleviate poverty in this region, indigenous pest control measures need to be documented and scientifically validated. Their methods of use also need to be standardized in order to popularize these age-old practices. Therefore, these studies were conducted to investigate farmers' knowledge and perception on maize stem borers and their indigenous control methods in South Western region of Cameroon. We hypothesized that the farmers: (1) perceived stem borers as important pest of maize; (2) they have their own indigenous methods of control and only (3) use chemical pesticides because they have no alternative, but would prefer plant material if these are standardized since they are safer and cheaper. We therefore sought to know whether or not farmers in the South West Region of Cameroon know about maize stem borers, if they rate them as important pests of maize and how they combat this problem. Farmers were also asked whether or not they used any indigenous methods and/or plant-based products against stem borers, if they could recognize the plants used and how effective these methods were vis-à-vis chemical/synthetic products as well as whether they will prefer to use indigenous or conventional methods to mitigate their maize pest problems in future? We also sought to know if there were differences in knowledge and practices in controlling maize pest problems between men and women as well as among the different villages studied. Study site The survey was conducted in the rainforest agroecological zone of Cameroon. A total of 151 farmers from four villages (Maumu and Ekona for Muyuka subdivision and Lower Bokova, and Bonduma for Buea sub division) in the Fako division of the South West Region were interviewed. Farmers were selected on the bases that each has been involved in maize cultivation for at least one year and were willing to participate in the study. The villages used in the study are in Buea (4°08' 036"N, 9°25' 826"E; 573 m asl) with rich volcanic rocky soils and temperature ranges of 20-25°C and Muyuka (4°150' 45"N, 9°28' 431"E; 599 m asl) with sandy soil and high temperatures ranges from 20-28.1°C, an altitude of 378 m sub-divisions. The location of these villages in a predominantly agrarian area and gender heterogeneity of the participants was a strong driving force on the farmers' perceptions. Buea is more cosmopolitan, with mountainous rich volcanic soils and favorable climatic conditions for maize cultivation. Muyuka, being warmer, favors the rapid buildup of pest populations. The farming community of the Maumu village in Muyuka is inaccessible due to poor road infrastructures and heavy rains, particularly during the rainy season (June to September). The poor roads prevent proper functioning of markets and lack of agricultural inputs. Most of the farmers being females have no direct contact with extension workers. The main food crops in the region are maize Zea mays, cassava Manihot spp, cocoyam Colocasia esculentum, groundnuts Arachis hypogaea, beans Phaseolus vulgaris, banana/plantains Musa spp, with vegetables and few spices as secondary crops while oil palm Elaeis guineensis, cocoa Theobroma cacao and coffee are the main cash crops. General characteristics of respondents Generally, small-scale (subsistence) farming (86.09 %) was the primary economic activity of most of the respondents while the remaining, (9.93 %) practiced farming as a part time job. Within the different villages, 100 % of respondents in Maumu; 40.63 % in Bonduma; 97.73 % in Ekona and 97.50 % in Lower Bokova respectively practiced farming as their main occupation. Maize farming experience ranged from 1-50 years. All of the respondent (100 %) grow food crops, 32.45 % of these also grow cash crops, while 13.33 % kept animals in addition. In all, 76.82 % of the respondents grow their maize in mixed cropping system, 17.22 % in mono cropping while 5.96 % practiced both. Besides the growing of food crops, most of the farmers in Muyuka subdivision also grew cash crops, especially coffee. Most (94.04 %) of the farmers plant maize twice a year, while 2.65 % and 3.31 % respectively grow maize once and three times a year. Most (92.05 %) of the harvested maize is used for household feeding; while 7.95 % is for sale or animal feed. The majority of the respondents (58.94 %) had completed primary education, 16.56 % had no formal education while 24.51 % had secondary and pre-university education. Within the four villages, 28.13 % in Bonduma, 61.36 % in Ekona, 62.50 % in Lower Bokova and 80.00 % in Maumu of the respondents had completed primary education. The study revealed that more farmers in Buea were educated, and involved in business than those in Muyuka. In Muyuka most of the farmers had no formal education; therefore farming was their main source of living (Table 1). Survey A semi-structured questionnaire was used in the survey. A total of 151 farmers (72.85 % females and 27.15 % males) 35 in Maumu, 40 in Lower Bokova, 44 in Ekona and 32 in Bonduma, were interviewed separately within their farming areas or around their residence. Farmers were selected on the bases that each has been involved in maize cultivation for at least one year and were willing to participate in the study. Interviews were done in English or local language (pidgin) with the assistance of local agricultural extension workers. The questionnaire sought to know: (a) the kind of indigenous methods and plant products used by farmers for maize stem borer control and their main constraints, as well as their knowledge on stem borer problems (b) if they use chemical/synthetic products to control pests, their names, the frequency of use and the constraints linked to their use; (c) whether they had contacts with agricultural extension workers, and their future preference between indigenous and conventional methods in dealing with pests and disease problems in maize fields. Data were also collected on the socio-economic characteristics of respondents. Statistical analysis For each variable, statistical comparison between the two sexes, the two locations and four villages were done based on the procedure of the software SAS ('Statistical Analysis Systems' version 9.1). The frequencies of respondent were also analysed with Chi-square test using PROC PREQ while the Kruskal-Wallis and Wilcoxon two sample tests using the 'Nonparametric One Way' ('NPAR1WAY WILCOXON') procedure were used in the absence of normality to compare the means of quantitative variables. All probabilities were appreciated at the 5 % confidence level. Main constraints and major pest problems in maize production Participants revealed that the major constraints to increased maize production were: land, labour, finance as well as pest and diseases (Table 2). Pests and diseases were the major constraints (85.43 %), followed by finances (55.63 %), land (37.09 %) and labour (28.48 %). There were significant differences between males and females in each village with regard to pests and disease problems. In Maumu, 100 % of the males and 48.15 % of females considered pest and disease as a problem. The same trend was observed in Ekona (100 % and 93.75 %) and Lower Bokova (88.10 % and 70.83 %), where males and females respectively considered pest and diseases as their major constraint. On the contrary, in Bonduma more females (94.12 %) than males (53.33 %) considered pests and diseases as main constraints. Stem borers (82.12 %) ranked the highest among the major field pest and disease problems of maize in the study areas. Male and female perceptions were significantly different in all four villages (Table 3). In Maumu, 100 % of female farmers considered stem borers as their major field pest, as against 75 % males. In Bonduma and Ekona, the trend was the same, while in Lower Bokova more males (100 %) than females (95.83 %) perceived stem borer as a major threat to maize production. Some of the farmers described that the larvae of the pest bore into the stems and cobs, causing wilting of the plants. Cobs with borer tunnel holes when taken to the market sold at cheaper prices, due to their lower aesthetic values. Apart from the stem borers, some farmers in Muyuka also considered snails (6.62 %) as a very important pest, especially during the heavy rains (Table 1). Other pest problems included weevils (16.56 %), white grubs (11.92 %), birds (8.61 %) as well as theft (1.99 %). Due to their meager earnings, most farmers also complained of high prices of pesticides as one of their major constraint. Zones not sharing a common letter in a row are significantly different at P = 0.05. 'p-value' is the significance level of Kruskal-Wallis (NA no significance, P >0.05) Indigenous knowledge and efficacy of plant-based control The survey showed some knowledge on use of indigenous methods for maize pests and diseases control ( Some respondent used wood ash as an indigenous control method (Ekona), and rated it to be very effective. The wood ash was sometimes mixed with Mocap (ethopropos), which is a synthetic product. The only problem was that most of them could not recognize the particular plant or the parts of the plant being used or even the proportion and formulations. Indigenous methods of stem borer control in the Region Various indigenous control methods were enumerated by the farmers. The main ingredient in most of them was ash collected from household kitchens or burnt wood (Fig. 1). Some used only the dry wood ash while others mixed fine dry soil with wood ash. Some others mixed their ash with conventional insecticides such as Mocap (ethopropos), Sevin, Gamalin or Kerosene. Some mixed the ash with water or Kerosene and used as sprays. In all of these methods, treatment was applied within the leaf whorl of the plant without any personal Zones not sharing a common letter in a row are significantly different at P = 0.05. 'p-value' is the significance level of Kruskal-Wallis (NA no significance, P >0.05) protective equipment (PPE). This raises safety concern for persons doing the applications. To keep away other larger insects and birds, farmers hang torn plastic bags, old plastic buckets in and around their fields especially just after planting (Fig. 2). Other farmers just simply let go and face the consequences of their maize being destroyed by these pests thus reducing average yields. Sometimes the birds fed on the cobs when the maize was matured. To remedy such situations, some farmers tied the leaves closest to the cobs around the cob to prevent the birds from feeding on the grains (Fig. 3). Use of pesticides In all four villages surveyed, 70.20 % of respondents used pesticides to treat their maize farms and 29.80 % did not (Table 5). More farmers in Buea used conventional pesticides, especially Cypercal 12 than those in Muyuka; most of their treatments were applied in the month of September More farmers in Ekona (79.55 %) and Lower Bokova (95 %) depended on pesticide compared to Bonduma (40.63 %) and Maumu (57.14 %). In Maumu, more males (87.50 %) than females (48.15 %) used pesticides while in Ekona and Lower Bokova, all males (100 %) used pesticides as against 91.67 % and 78.57 % of females respectively (Table 5). Cypercal 12 was the main pesticide used in Lower Bokova (52.50 %), while Mocap (Ethopropos) was widely used by all farmers, especially in Ekona (43.18 %) (Fig. 4). Zones not sharing a common letter in a row are significantly different at P = 0.05. 'p-value' is the significance level of Kruskal-Wallis (NA no significance, P >0.05) Fig. 1 Typical local kitchen with three-stone fire place where wood ash is collected widely used because it was cheaper and easy to get, and is also a contact/repellent product. About 53.13 % of farmers in Bonduma did not use any of the pesticides, though sometimes they got advice from friends on the type of pesticide to use. Some farmers admitted not using any pesticides against stem borers, but sometimes used weed killers, since maize does not tolerate high weed population. Some farmers mixed Mocap (Ethopropos) and wood ash, where the wood ash serves as a carrier substance for the insecticide. The main constraints reported by the farmers regarding the use of pesticides were high prices (72.19 %), while 27.15 % were indifferent (Fig. 5). In Lower Bokova, 82.50 % of farmers complained of high prices, since most of them depended on pesticides. Some of their proposed solutions to these constraints were; need for government subsidies, prices should be moderated in the market and the need for alternative methods of stem borer control (Fig. 6). In Ekona, 61.36 % of farmers believed government subsidies would help resolve the problem of high prices. Others believed pesticides were easier and quicker to administer on farm. More than 50 % of respondents in the four villages proposed government subsidies and price reduction as solutions to the pesticide constraints. Farmers' future preference Regarding farmers' future preferences, more of them in Muyuka would prefer the continued use of conventional methods of stem borer control than in Buea. Due to the high level of education and the cosmopolitan nature of Buea, most of the farmers had lost their indigenous knowledge than in Muyuka. They had no basic knowledge on indigenous methods of plants used for pest control. Most farmers did not have contacts with agricultural extension workers because the few extension workers could hardly visit all the farmers in their areas. This was a major constraint to the farmers since they were not well informed about insecticides and common indigenous methods of pest control. Most of the farmers (88.74 %) expressed interest in the use of plant-based products (Fig. 7). More farmers in Lower Bokova (97.50 %) were willing to try plant-based insecticides than in Bonduma (84.38 %), Ekona (88.64 %) and Maumu (82.86 %). Some of the reasons mentioned were that the plants were safer than synthetic insecticides and also readily available. Others sought to know whether such plants could have long-term effects and were not time-consuming. The remaining 11.26 % did not show any interest in the use of plant products. When questioned on their future preferences, 60.26 % declared that they would prefer plant products in place of synthetic insecticides, 8.61 % preferred both synthetic and indigenous, 26.49 % still preferred conventional insecticides while 4.64 % were undecided (Fig. 8). Lower Bokova again showed that more farmers (80 %) would prefer plant-based insecticides as compared to Ekona (47.73 %), Maumu (60 %) and Bonduma (53.13 %). Those who preferred synthetic insecticides believed that if the government could subsidize or reduce the cost of these products in the market, they will be able to afford them. The role of the Ministry of Agriculture is still preponderant as it grants the import authorization and approves the distribution and sales of the agricultural inputs. Some of the study villages are far from the sales point. Consequently, transportation costs rendered costs of inputs out of the reach of many farmers. Discussion Apart from food crops, farmers in Fako division of South West Region of Cameroon also grow cash crops and also rare animals for home consumption. Most of the crops are attacked by field pests during the rainy season when crops are in the vegetative and reproductive stages; this greatly reduces the farmers' harvests. Most of the pests problems encountered are on cereals, particularly maize. Crop products are stored as food reserves in these areas, while some of the produce are used for income generation. Considering that the maize yields are always low, partly due to pests/diseases and other allied constraints, it has serious implications on their food security needs. Land, labour, finance, pests and diseases were the most important constraints to maize production in the Fako division of the South West region of Cameroon. As is the case in most parts of Africa [30], most of the smallholders' farmers in Cameroon are women with twothirds of their farms being below 2 hectares. Many practice low-resource agriculture based primarily on the use of local resources with modest external inputs. Increased urbanization causes a shift of farmland into urban areas, thus reducing available space for farming [31]. The Zones not sharing a common letter in a row are significantly different at P = 0.05. 'p-value' is the significance level of Kruskal-Wallis (NA no significance, P >0.05) Fig. 4 Percentage (±i) respondent relative to main pesticides used in Fako division of the South Western Cameroon quantity of available land and farm inputs determine maize output. Similar results were obtained by [32] in the West region of Cameroon, stating that, maize yields were low due to reduced farm sizes, low quality of maize seeds planted, inadequate labour, fertilizer and agrochemicals inputs. Most of the food production burden falls on women and children because of rural urban migration and reduction in active work force wreaked by various diseases and ill-health [33]. Children are sometimes denied the chance to go to school to assist in weeding because of labour scarcity, resulting in low educational performance [34]. Pest and diseases were the major constraints limiting attainable Similar results were reported by [32] in the forest and humid forest of Cameroon, which corroborates with the findings of [35]. Increase in pest and disease may partly be as a result of increased use of pesticides by larger corporations which makes the pest become resistant and later shift into the untreated fields. The study showed that the farmers regarded stem borers as important pests of maize in Fako division of the South West Cameroon. Stem borers interfere with the movement of water and metabolites through the plant's vascular system, which stunts its growth and development. Attacks during the first eight weeks after sowing result in "dead heart" and late damage (beyond eight weeks after sowing) leads to stem lodging. Both types of damage to the crop cause drastic loss in maize yield [36]. The farmers reported an increased damage during the dry season when pest populations are higher. Farmers' perceived that, the insect pests had economic implications, given that the insects caused significant damage that warranted the implementation of control measures. These perceptions contribute to the understanding of various aspects of the bio-ecology of insect pests [36,37]. Similar results were also obtained in the humid forest and Western highlands of Cameroon [38][39][40]. The results indicated that only 45.70 % of the farmers used one indigenous method or another, while 54.30 % depended solely on conventional control methods, which are expensive. Some reported that indigenous methods were time-consuming and they were not sure of the results. Those who used indigenous methods believed they were cheaper and they faced no problems with their use. Cultural/indigenous practices are not expensive for the farmers and do not necessitate in general, supplementary material investments to control insect pests [41]. A large proportion of the farmers believed indigenous control methods are not effective similar to the findings of [41], which stated that the development of traditional/indigenous control methods is very limited. The results showed that very few farmers were using plants as insect pest control methods in their fields. Farmers perceived plant derivatives could not give the desired results achieved when conventional methods are used. Possibly integrating the use of resistant plants with plant derivatives could be a better option for replacing synthetic chemicals, given that they are simple, economical and important strategies in insect pest control. They are also not dangerous to the environment and are generally compatible with other pest control methods [11,41]. Farmers' knowledge and perception of their use can accelerate and facilitate their adoption in the local communities. More farmers depended on pesticides than on botanical control, although not adequately informed about their proper use similar to studies carried out by the Ministry of Agriculture which showed that more than 42 % of farmers use pesticides [42]. Increased use of pesticides is due to the proliferation and accessibility of unlicensed dealers' shops that are only out to make money but care less about the consequences of pesticides. Farmers relied mostly on estimations for the amount and concentration required for a given botanical pesticide formulation because most of them are illiterate. Therefore, there might be risk of overdose, since most of them do not have frequent contacts with extension workers. The most mentioned pesticides used by farmers are Mocap (Ethopropos) and Cypercal. The former, being the cheapest and readily available while the latter, classified as "1b" by the World Health Organization (WHO) and qualified as highly dangerous [42] is no longer recommended [43]. Mocap (Ethopropos) is an organophosphate (classified as IA), extremely hazardous to eyes, and the body when inhaled especially as farmers donot use personal protective equipments (PPE) during pesticide applications. Most of these insecticides are also highly dangerous to the environment and pollute water bodies. Substances classified in these category by WHO should not be applied by untrained or inadequately protected people [44][45][46]. Indigenous control methods were important because most of the farming in these areas are subsistence. Despite the fact that most of the farmers acknowledge the effectiveness of plant-based insecticides, most of them would still prefer conventional products in future if they are affordable. This may be because those who use the indigenous controls and botanicals do not know the right formulations and amounts to apply, as well as the time of application. The main component of this indigenous control was ash from burnt wood collected from local kitchens. Owolabi et al. [29] studied the effect of liming materials such as plant derived ash on maize yield, and found that it increased soil pH and maize yield. The findings showed that there was heterogeneity in knowledge between the two locations as well as in gender respectively. In most of the responses, the women seemed to perceive the pest incidences with equal importance. Most of the males interviewed were from Lower Bokova (17), followed by Bonduma (14) which are villages in Buea with a higher level of education. While most of the females were found in Ekona (42) and Maumu (27) and farming was their main occupation without any formal education. This confirms why their perceptions were different, as more women than men reported increased incidence of stem borers in their fields. Perception differences in gender have also been observed in Nepal, where men generally used more vague attributes like harmful or harmless, while women were more specific, regarding the depredatory insects [37]. Gender differences in perception may also be due to division of labour, as most of the women in this region spend equal time in fields as well as household tasks while the men are involved in farming and community works. In absence of a standardized protocol on preparation and application, the indigenous plant-based formulations will have varied efficacies at different times even with the same farmer. Without the standardization of the specific amount of a product used, exposure time and way of preparation and no proper application rate and method, efficacy rating of any pesticide will be compromised. There is therefore need to increase productivity through the development of alternative low-cost plantderived technologies for fighting pests and diseases in crop fields. Studies in China showed that some of these plants, such as the leaves and twigs of Tephrosia vogelii do possess strong antifeedant stomach poison and growth inhibiting effects against many insect pests, including the stem borers [47]. Therefore, if the use of ash and other plant derivatives are exploited further and their qualities improved and quantified these will be of great use to the resource-poor farming communities in this region. Most especially if the particular plant is known for its insect control potentials. Conclusions Findings from the survey reveal that farmers in Fako division practiced subsistence agriculture with maize as one of the major crops. The maize is always grown in a mixed cropping system and stem borers are the major field pests limiting attainable yield. The pest burden is greatest during the dry season. Besides maize and other food crops, some farmers also grow cash crops and rare animals for home consumption. Most farmers in the study villages depend on the use of synthetic pesticides to control stem borers. Farmers have limited knowledge of indigenous methods of stem borer control over synthetic, but would prefer it in the future if well informed. This is because, the indigenous and/or plant based pest control methods would be cheaper and readily available as well as being safer if accompanied by standardized methods as well bio-safety and environmental guidelines for efficacy. Farmers would prefer an integrated approach to pest control since it contributes all possible strategies to reduce the pest burden. There was a wide perceptional difference in gender and location similar to most studies. This may differ from scientific studies, but having significant implications for development [32]. To ensure quality and safety, biosafety and quality studies are required for quality assessment of resulting product for human health.

v3-fos

2019-03-15T13:07:11.952Z

2015-10-12T00:00:00.000Z

196454877

s2

Effect of Season on the Some Blood Hormones and Enzymes of Iraqi Bull Buffalo The present study was conducted to investigate some aspects of the reproductive system in Iraqi bull buffaloes and the effects of seasonal changes on physiological parameters. 96 blood samples and male reproductive system of buffalo’s bull were collected from the slaughter house during August 2013 to August 2014. Blood samples were analyzed to study the effect of season on the blood hormones and enzymes during the different seasons of the year, the results of the hormonal assays showed that the highest level of the Follicular Stimulation Hormone (FSH) and Lutenizing Hormone (LH) record in autumn 0.66 ml U/ML, 0.91, mu/ ml, respectively, while it was 0.46 µ/ml, 0.23 ml U/ML in winter and 0.40 ml U/ ML, 0.18, mu/ml in spring, while the lowest level showed in summer 0.32 ml U/ ML, 0.16, mu/ml, respectively. The results of the of the hormones testosterone and estradiol-17β showed that in autumn were 2.732 m.moL, 108.77 p.mol/L, respectively, in winter were 2.339 m.moL, 70.69 p.mol/L and in spring were 1.675m.mol, 40,80 p.mol, while the lowest rate were in summer 1.516 m.mol, 23.37p.mol/L. Study results showed that the levels of enzymes to the privation of significant difference between the different seasons in the enzyme lactic Dehydrogenase and has the highest rate recorded in autumn 1552.85 IU/L and 1510.33 IU/L in winter and then spring and summer 1471.66 IU/L, 1379.36 IU/L. While there was a significant difference p (<0.05) during the seasons (autumn, Spring, Summer) for the enzyme acid phosphatase and the highest level was in autumn 2.732 U/L, while the lowest level was in summer 1.496 U/L. The difference between the seasons of the year in the enzyme alkaline phosphatase, the highest rate has been recorded in autumn, 224 U/L and then winter, spring and summer, respectively (211.66, 210.75, 198.66 U/L). The results of the study also showed that there is no significant differences between the seasons of the year in the amylase enzyme and the highest rate were showed in autumn and winter 11.62, 13.5 U/L while the lowest rate in spring and summer 11.4 U/L, 8.6 U/L respectively. gonadotropin hormones, enzymes in male Introduction The water buffalo has been associated with people since prehistoric times. It is one of the oldest species of domesticated livestock and continues to be used as a source of milk and meat, and as a draft animal. Water buffaloes have been classified into the river and swamp types [1]. The river type is larger, used for milk, wallows in fresh-water and originates, the swamp type is smaller, used for draft and meat, wallows in muddy water and is indigenous to most Asian countries [1]. The Iraqi Buffalo contribute in supplying the local market with high nutritional value and to fill part of the shortfall in the case of dairy products in scarce during some months of the year, noting that most of the dairy products in the local markets is of buffalo milk during a period of drought, with cows male as well as the superiority of some local animals in the amount of milk production and content of fat [2]. The environmental factors associated with heat stress which affects the physiological systems governing thermal regulation and the maintenance of positive heat loss, high ambient temperature is the major constraint on animal productivity [3]. Therefore, care must be taken because it represents animal the Twenty-first century for being resistant to diseases and producer of milk and has the ability fattening outweigh all other farm animals. For these reasons and the fact that male buffalo being responsible for raise reproductive efficiency by selecting the right season for the breeding to reduce the economic losses, especially since the female buffalo characterized silent estrus, weak estrus signs, short period of ovulation [4], and the problem of reduced libido in male during the non-breeding season. So through the study, we will determine the appropriate season for pollination by studying the level of hormone and differences between the Research Article gonadotropin hormones, enzymes in mature male buffalo, and by this study we can raise the reproductive efficiency of female buffaloes and thus raise production and increased income for owner and increase development and economic activity of the country and prevent import from abroad. Materials and Methods Ninety six blood samples of healthy bulls buffalo, aged (3)(4)(5) years, were obtained from the slaughterhouse from August 2013 to August 2014, 3visits/week before slaughter immediately, (8 samples) for each month from the (Jugular vein) then empties in test tube (Gel tube) size (8 ml) contains Gelatin substance which help to isolate the serum from the blood, then transported to the lab, the samples have been centrifuged at 3000/rpm for 10 minutes for determination the levels of hormones (estrogen, testosterone, follicle stimulating hormone, luteinizing hormone) and enzymes (lactic dehydrogenase, acid phosphates, alkaline phosphatase, and amylase)with specific kits and according to the instructions of the company. Statistical analyses Data were analyzed statistically by SPSS program, version 17 software 2010. Testing method used include one way ANOVA for comparisons among season followed by least significant differences (LSD) test for comparison between two groups. P valve of p< 0.05 were considered to record statistical significances. Testosterone hormone The results showed that the mean of the level of the hormone testosterone in blood serum in autumn (2.732±0.323) m.mol/L, winter (2.339±0.199) m.mol/L, sprig (1.675±0.249) m.mol/L and summer (1.516±0.122 ) m.mol/L The statistical analysis showed significant variation (p<0.05) between the level of the hormone through the different seasons of the year (autumn, summer) and that the highest level of the testosterone in the autumn, no significant variation between (winter, spring ) Estradiol hormone The mean level of estradiol hormone in blood serum in autumn (108.77±17.23) p.mol/L, winter (70.69±7.21) p.mol/L, sprig (40.8±8.37) p.mol/L and summer (23.37±3.27) p.mol/L The statistical analysis showed significant variation (p<0.05) between the level of the hormone through the different seasons of the year (autumn, winter, summer).that the highest level of the estradiol in the autumn, there are no signification variation between (winter, spring). Follicular stimulation hormone The results showed that the level of hormone FSH in blood serum in autumn was (0.66± 0.08) m.l U/ML, winter (0.46±0.07) m.l U/ML, Sprig (0.40±0.04) m.l U/ML and summer (0.32±0.03) m.l U/ML. The statistical analysis showed significant variation (p<0.05) between the level of the hormone in autumn and the other of season (winter, spring, summer). And that the highest level of the FSH in the autumn, there are no significant variation between winter, spring and summer. Luteinizing hormone The mean level of LH hormone in blood serum in autumn was (0.91±0.26) m.u/ml, winter (0.23±0.06) m.u/ml, sprig (0.18±0.02) m.u/ml and summer (0.16±0.02) m.u/ml. The statistical analysis showed signification variation (p<0.05) between the level of the hormone in autumn and the other of season (winter, spring, summer).that the highest level of the LH in the autumn, there are no significant variation between winter, spring and summer. Lactic dehydrogenase In Figure 5 showed the level of Lactic dehydrogenase in blood serum in autumn was (1552.85±82.55) U/L, winter (1510.33±140.39) U/L, sprig (1471.66±146) U/L and summer (1379.36±57.9) U/L. The statistical analysis showed no significant variation between the level of the lactic dehydrogenase through the different seasons of the year and that the highest level of the lactic dehydrogenase was in autumn, summer and followed by spring and the lowest level was in winter. Acid phosphatase In Figure 6 showed the level of Acid Phosphatase in blood serum in autumn was (2.732±0.323) U/L, winter (2.337±0.195) U/L, sprig (1.533±0.185) U/L and summer (1.496±0.132) U/L. The statistical analysis showed significant variation (p<0.05) between the level of the Acid Phosphatase through different seasons of the year (autumn, spring, summer) and that the highest level of the Acid Phosphatase was in spring, autumn and followed by winter and the lowest level was in the summer. Alkaline phosphatase In Table 2 Amylase In Table 2 & Figure 8 showed the level of Amylase in blood serum in autumn was (13.5±1.95) U/L, winter (11.62±1.66) U/L, sprig (11.4±1.42) U/L and summer (8.6±3.2) U/L. The statistical analysis showed no significant variation between the level of the amylase through the different seasons of the year and that the highest level of the amylase was in autumn and the lowest in summer. 5/6 Copyright: *The similar letters refers to the non-significant differences among months while different letters refers to the significant differences at (p<0.05). Discussion There were no available references of the hormonal and enzymes levels in the blood bull buffalo during different seasons except for the seminal fluid. The Hormonal study The present study showed the mean of hormone (Testosterone, Estradiol, Follicular stimulation hormone, Luteinizing hormone) was Table 1. This results agreement with [5] which indicated that an initial rise in Follicle Stimulating Hormone (FSH) results in a proliferation of Sertoli cells, a lengthening of the seminiferous tubules and an increase in tubule diameter. At the same time, there is a rise in Luteinizing Hormone (LH) secretion resulting in increased testosterone production by the Leydig cells and agree with as show by Simanainen et al. [6] which indicate that the weight of the testis was one of the markers of a possible alteration in androgen status; A decrease in testicular weight is most likely due to a decrease in the level of serum testosterone. The results of present study agree with Tekepetery et al. [7] which shown that the Increase in the activity of the testes and epididymis in moderate and cold season and these activities are regulated by the increases testosterone hormone levels in these seasons, that increased in mating seasons and decreased in nonmating seasons as shown by Arrighi et al. [8], and in bull [9]. while Wu et al. [10] Al-Sahaf et al. [11] refer that the testes are affected by the increase of temperature lead to decreasing the number of the receptors of Luteinizing Hormone (LH) that presents on Leydig cells and then decreasing in testosterone hormone and activity of the testes in hot season, Al-Sahaf et al. [11] indicated to an increase in testosterone, FSH and LH hormones caused an increase in diameter of testicular. This results of present study similar with [12] which shown that the level of hormone testosterone and LH-receptors were higher in the breeding season (autumn, winter) compared to those in the low breeding season (spring, summer), and agree with Hochereau-de-Rievers et al. [13]. The increase in the measures of epididymis in moderate and cold months as compared with hot months is a result of an increase in length and diameter of the seminiferous tubules. This result supports with Gundogun et al. [14] refer to presence of the enzyme activities follows reproductive seasonality, Abdel-Samee et al. [15] indicated that the liver function may be partially affected by heat stress that not agreement with present study. Kataria et al. [16] found that ALP and ACP were significantly higher during extremely hot (May -June) than in extreme cold (December-January) conditions, in India not supported present study, and also not agree with Litwack [17]. The increase in ALP activity in summer and winter may be due to increase secretion adrenocorticotrophic hormone (ACTH) due to environmental stress. perhaps to different the breed of buffalo in Indian or by the environment condition in this region, The possible source of these enzymes is thought to be the testes or epididymitis because they show a positive correlation with sperm concentration and a negative correlation with semen volume [18]. Table 2 & Figure 8 showed that the means level of amylase in different season of the year's were13.5±1.95 U/L, winter11.62±1.66 U/L, sprig 11.4±1.42 U/L and summer 8.6±3.2 U/L respectively, this study supports by Hafs HD et al. [19] have reported separate trials, both of which showed that crude preparations of α-amylase (50-100 units/mg), when added to extenders for bull semen at 10 μg/ml significantly increased conception rates. The former trial also showed that the addition of β-amylase increased of conception rates, α-amylase, and enzymes involved in the degradation glycogen, sperm capacitation [20]. Increase amylase leads to increase enrage which effected on reproduction and growth of animals. Amylase: In Lactic Dehydrogenase: In present study showed Increase in LDH in autumn was 1552.85±82.55 IU/L, winter 1510.33±140.39 IU/L, sprig 1471.66±146 IU/L and summer 1379.36±57.9 IU/L respectively. The present study agree with Duan et al. [21] which shows that the Lactic dehydrogenase plays an important metabolic role in sperm capacitation and fertilization, the results of present study supported by Kareskoski et al. [18] which shows that the source of enzyme Lactic dehydrogenase is thought to be the testes or epididymides because they show a positive correlation with sperm concentration and a negative correlation with semen volume, and the present study agree with Gundogun et al. [14] found presence of enzyme activities follows reproductive seasonality, Jones et al. [22] indicate reduced Lactic dehydrogenase activity in seminal plasma might indicate disturbed spermatozoal function and metabolism, may be duo to hot conditions changes in the biological functions that include depression in feed intake, efficiency and utilization, disturbances in metabolism of water, energy and enzymatic reactions. Such changes result in impairment of reproduction and production performances.

v3-fos

2018-04-03T01:40:17.715Z

2015-02-18T00:00:00.000Z

17632087

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Isolation, Characterization, and RP-HPLC Estimation of P-Coumaric Acid from Methanolic Extract of Durva Grass (Cynodon dactylon Linn.) (Pers.). P-coumaric acid is a nonflavonoid phenolic acid and is a major constituent of the species Cynodon dactylon Linn. (Pers.). In this study isolation of P-coumaric acid was achieved by preparative TLC and the compound thus isolated was characterised by UV, mass, and H(1) NMR spectral analysis. An isocratic RP-HPLC method was developed for the estimation of P-coumaric acid from methanolic extracts of durva grass. The chromatographic separations were achieved by RP-C18 column (250 mm × 4.6 mm, 5 μ), Shimadzu LC-20AT Prominence liquid chromatograph, and a mobile phase composed of water : methanol : glacial acetic acid (65 : 34 : 1 v/v). The flow rate was 1.0 mL/min and the analyses of column effluents were performed using UV-visible detector at 310 nm. Retention time of P-coumaric acid was found to be 6.617 min. This method has obeyed linearity over the concentration range of 2-10 μg/mL and the regression coefficient obtained from linearity plot for P-coumaric acid was found to be 0.999. RP-HPLC method was validated in pursuance of ICH guidelines. Introduction It has been estimated that there are approximately 8,000 naturally occurring phenolic compounds. Phenolic antioxidants are believed to provide a protective effect against oxidative damage diseases such as cancer, coronary heart disease, and stroke [1][2][3][4] and it has antidiabetic action [5,6]. P-coumaric acid is a hydroxy cinnamic acid, an organic compound that is a hydroxy derivative of cinnamic acid [7,8]. P-coumaric acid can be found in Gnetum cleistostachyum [9]. In food Pcoumaric acid can be found in a wide variety of edible plants such as peanuts, navy beans, tomatoes, carrots, and garlic. It is found in wine and vinegar [10]. It is also found in barley grain [11]. P-coumaric acid has antioxidant properties and is believed to reduce the risk of stomach cancer [12], by reducing the formation of carcinogenic nitrosamines [13]. They have excellent antioxidant activities, which are higher than those of vitamins C and E against reactive oxygen species [14]. They have a wide range of biological activities, such as protection against coronary heart diseases, antiinflammatory, antitumour, antimutagenic, and antimicrobial activities [15]. A thorough literature survey was done to study analytical methods developed so far; it reveals that there were few HPTLC densitometry methods for the determination of Pcoumaric acid with other phenolic acids in flowers and roots of the plant sources [16][17][18], few HPLC-UV detection methods using different mobile phase combinations for the pharmacokinetic study of P-coumaric acid in mouse after oral administration and determination of P-coumaric acid along with other phenolic acids present in various herbs [19][20][21][22][23][24]. There were few miscellaneous methods reported for determination of phenolic acid content [25][26][27]. Though require tedious extraction and sample preparation for analysis in biological fluids and other sample matrices. This emolliates the author to develop an accurate and specific reversed phase high performance liquid chromatography method for determination of P-coumaric acid content in methanolic extract of durva grass with good linearity, and less solvent consumption resulted from the less run time set in the method. This method has been validated according to ICH guidelines [28]. This study was aimed at development of suitable extraction and isolation procedure which aids in getting the purified material and in developing an isocratic RP-HPLC method for estimation of P-coumaric acid in methanolic extract of durva grass. Further the study also deals with characterisation of P-coumaric acid in the extract by mass and H 1 NMR spectral analysis for structure identification. Preparation of Standard Solutions. A standard stock solution was prepared by dissolving 100 mg of P-coumaric acid in 100 mL volumetric flask containing 60 mL mobile phase and then sonicated for about 10 minutes and made up to 100 mL with mobile phase to get the primary standard stock solution containing 1000 g/mL of P-coumaric acid. Working standard solutions were prepared by further dilution with mobile phase. Preparation of Plant Extract. The whole plant Cynodon dactylon was used in this study. The material is cleaned and set free from moulds, insects, animal faecal matter and other contaminations such as earth, stones, and extraneous materials. The specimen was shade-dried and protected from sun light for several days not less than one month. It was ground to a fine powder using mortar and pestle without any loss of powdered drug. Then it was passed through a sieve of 40 meshes and the material passed by the sieve was collected and stored in a well tight amber coloured container and it was used for further study. Coarsely powdered aerial parts of the plant (about 1 Kg) are successively extracted with continuous Soxhlet apparatus with methanol for 48 hours. The homogenate was filtered using Whatman's filter paper and the volume of the filtrate was recorded. The filtrate was centrifuged at 100 ×g for 15 min under cooling (4-6 ∘ C) conditions. The clear supernatant was taken on rotary evaporator and the extracts were concentrated, dried, and stored in vacuum desiccators. Further the extracts were used for different studies. Preliminary Phytochemical Screening. The main objective of the preliminary phytochemical screening is to investigate the plant extract in terms of its active constituents. It involves the partial isolation of active constituents and identifies them International Journal of Analytical Chemistry 3 qualitatively. In this screening various types of identification tests for a variety of chemical classes have been performed according to CCRAS guidelines [29]. Isolation and Purification of Active Compounds Analytical TLC was carried out on preparative TLC plates (5 × 5 cm with 0.2 mm thickness, silica gel GF 254 , Merck, Darmstadt, Germany) cut from the commercially available sheets. An aliquot of standard solution of P-coumaric acid and a sample solution of crude extract are spotted onto the silica gel plate and allowed to dry for a few minutes. Afterwards, the chromatoplate was developed with chloroform : methanol : formic acid (85 : 10 : 5 v/v) as mobile phase in a previously saturated glass chamber with eluting solvents for some time at room temperature. The developed plate was dried under normal air and the spots were visualized by spraying with a solution of 0.5% w/v ferric chloride and dried under oven. The Rf (retention factor) values of isolated compounds and standard were calculated and compared. Preparative TLC for Purification. A streak of crude extract was applied manually on a preparative TLC glass plate (20 cm × 20 cm; 1500 m thickness) with inorganic fluorescent indicator binder (Analtech, Sigma-Aldrich, Steinheim, Germany). After air drying, the plate was developed, using the same mobile phase as used in the analytical TLC, in a presaturated glass chamber. In each experiment, two plates were used in parallel. One of the plates from each set of experiment was sprayed as described above, and the bands were scraped off carefully from the plate. The scratched sample was dissolved in HPLC grade methanol and centrifuged at 12000 rpm for 15 min in order to remove silica. The supernatant was collected, filtered from 0.22 m filter, and dried under reduced pressure. Further, all the dried samples were passed under nitrogen gas for 5 min and then dissolved in methanol for further characterization and quantitative HPLC analysis. The entire purification process was carried out under dark or dim light conditions. Characterization of Purified Compound. The UV spectrum of the purified compound was recorded from 190 to 600 nm on an ELICO double beam UV-visible spectrophotometer. ESI mass spectra were acquired from isolated compound and characterised. Proton nuclear magnetic resonance spectra were acquired using NMR spectrometer (400 MH Z ) employing TMS as an internal standard and deuterated methanol was used as solvent. Determination of P-Coumaric Acid in Methanolic Extract of Cynodon dactylon by RP-HPLC. The chromatograph was stabilised for about 45 minutes with mobile phase consisting of water : methanol : glacial acetic acid (65 : 34 : 1 v/v). The flow rate was 1.0 mL/min phase at the required flow rate to get a steady base line. Aliquots of standard solution containing P-coumaric acid (0.2-1.0 mL, 100 g/mL) were transferred to a series of 10 mL capacity volumetric flasks to get the concentrations ranging 2-10 g/mL. Accurately about 20 L of each calibration standard was injected into the chromatogram. Peak areas of each solution were recorded. A calibration curve was plotted between concentration and peak area response. 20 L of sample solution prepared from methanolic extract was injected and the area of peak was recorded duly maintaining the ambient experimental conditions as followed by the standard drug solutions. The amount of P-coumaric acid present in the sample of extract was computed from its calibration graph. Validation of the Developed Method System Suitability. The chromatograph was stabilised for about 45 minutes with mobile phase at the required flow rate to get a steady base line. System suitability was ascertained by six replicate analyses of the drugs at concentrations of 10 g/mL of P-coumaric acid. The percentage of RSD of the three injections of the same quantity of standard drug solutions of P-coumaric acid in terms of their peak areas, retention time, efficiency, or number of theoretical plates and asymmetry factor ascertains its system suitability for compatibility of analysis and for obtaining reproducible value. The effect of wide range of other constituents and other additives usually present in the extract was investigated to know the specificity of the method. It shows no interference from other compounds. For linearity, aliquots of primary working standard solutions containing P-coumaric acid were diluted in a way such that the final concentrations of Pcoumaric acid are in the range of 2-10 g/mL. A calibration curve was plotted between concentration and peak area response and statistical analysis of the calibration curve was performed. Method of least square analysis was carried out for getting the slope, intercept and correlation coefficient, and regression data values. Precision was determined by intraday and interday study. Precision of the method was evaluated by carrying out the assay and analyzing corresponding responses 6 times on the same day and on different days for the sample solution. Accuracy studies were performed for P-coumaric acid at three different levels (25%, 50%, and 100%) and the mixtures were analyzed in triplicate by the proposed method. A known amount of standard P-coumaric acid at 25%, 50%, and 100% of sample (which was previously analysed) was added and it was reanalysed by the proposed method. And the percentage recovery was evaluated. The robustness of the developed method was evaluated by small deliberate changes in flow rate (±0.1 mL/min), detection wavelength (±5 nm), and mobile phase composition (±2%). The effect of these variables on the developed method was determined. Limit of detection and limit of quantification were calculated using the following formula LOD = 3.3 ( )/ and LOQ = 10 ( )/ , where ( ) = standard deviation of response (peak area) and = slope of the calibration curve. Results and Discussion The extraction procedure was optimized regarding extraction solvent and recovery. Methanol was used for extraction of the P-coumaric acid from Cynodon dactylon L. Complete extraction of P-coumaric acid was achieved by successive solvent extraction with methanol. Methanol has a protective role. It can prevent phenolic compounds from being oxidized by enzymes, such as phenoloxidases [30,31]. Preliminary phytochemical study reveals that the extract may contain phenolic compounds which may be non-flavonoid in nature. Several mobile phase combinations were tried and chloroform : methanol : formic acid (85 : 10 : 5 v/v) was found optimum for separation of P-coumaric acid from methanolic extract of durva grass. The RF values of standard and sample compound match each other and the RF value was found as 0.52. This compound is structurally related to the investigated compounds and behaves similarly on the column as analyte. TLC profile of compound was represented in Figure 1. Isolation of P-coumaric acid from the extract was achieved by preparative thin layer chromatography using the same chromatographic conditions followed by identification of active constituent. Characterisation of isolated compound was done by studying ultraviolet, mass, and H 1 NMR spectra. P-coumaric acid shows UV absorption at about 345 nm in methanol indicates the presence of conjugation and hydroxyl auxochrome which shifts the absorption RF maximum towards visible side of the spectrum and it was represented in Figure 2. Mass spectral data shows molecular ion peak / = 164.2 which has a moderate abundance (Table 1). Spectrum obtained from the H 1 NMR shows different kinds of protons and its assignment corresponds to type of hydrogen in the complete structure of P-coumaric acid and it was reported in Table 2. An accurate isocratic RP-HPLC method was developed and validated by optimised chromatographic conditions. The conditions and system suitability were presented in Table 3. Chromatograms showed a peak of P-coumaric acid at retention time of 6.617 min. The regression coefficient obtained from linearity plot for P-coumaric acid was found as 0.999, which indicates this method had good linearity and the linearity data was given in Table 4. The representative chromatograms of this method were given in Figures 3 and 4, for calibration standard and sample of methanolic extract, respectively. The calibration plot for P-coumaric acid was shown in Figure 5. The amount of P-coumaric acid was found as 0.48 mg/100 mL extract. The method validation parameters were established in this work, LOD and LOQ of the Pcoumaric acid were found as 0.302 g/mL and 0.99 g/mL, and the proposed method was found to be precise for the determination. The percentage of RSD for the proposed method was found to be less than 2.0 which indicate the method's precision. Results of the precision study are shown in Table 5. Recovery studies of the method were found to be good and percentage of recovery was represented in Table 3. Robustness was done by small changes in the chromatographic conditions like mobile phase flow rate, max , and mobile phase composition. The proposed method was found to be robust as there were no marked changes in the performance characteristics of the method. Conclusion Isolation, identification, and characterisation of P-coumaric acid was achieved successfully which will be helpful for the standardization of herbal formulations containing this

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Transcriptome sequencing reveals the roles of transcription factors in modulating genotype by nitrogen interaction in maize Key message Global transcriptome analysis in maize revealed differential nitrogen response between genotypes and implicate a crucial role of transcription factors in driving genotype by nitrogen interactions at gene expression level. Abstract Developing nitrogen-efficient cultivars are essential for sustainable and productive agriculture. Nitrogen use efficiency of plants is highly dependent on the interaction of environmental and genetic variation and results in adaptive phenotypes. This study used transcriptome sequencing to perform a comprehensive genotype by nitrogen (G × N) interaction analysis for two elite Chinese maize inbreds grown at normal and low nitrogen levels in field conditions. We demonstrated that the two maize inbreds showed contrasting agronomic and transcriptomic responses to changes in nitrogen availability. A total of 96 genes with a significant G × N interaction were detected. After characterizing the expression patterns of G × N interaction genes, we found that the G × N interaction genes tended to show condition-specific differential expression. The functional annotations of G × N interaction genes revealed that many different kinds of genes were involved in G × N interactions, but a significant enrichment for transcription factors was detected, particularly the AP2/EREBP and WRKY family, suggesting that transcription factors might play important roles in driving G × N interaction at gene expression level for nitrogen response in maize. Taken together, these results not only provide novel insights into the mechanism of nitrogen response in maize and set important basis for further characterization but also have important implications for other genotype by stress interaction. Electronic supplementary material The online version of this article (doi:10.1007/s00299-015-1822-9) contains supplementary material, which is available to authorized users. Introduction Nitrogen (N) is a major nutritional factor limiting plant growth. Over the past few decades, heavy use of nitrogen fertilizers have played a key role in increasing crop yields, however, only 30-40 % of the applied N was actually utilized by crops (Kant et al. 2011;Xu et al. 2012). More than 60 % of the soil N is lost through surface runoff, denitrification, volatilization and microbial consumption (Kant et al. 2011;Xu et al. 2012). This loss is costly and detrimental to the environment (Kant et al. 2011;Xu et al. 2012). Thus, improving the nitrogen use efficiency (NUE) of crops is of key importance for sustainable and productive agriculture. NUE, defined as the total biomass or grain yield produced per unit of applied fertilizer N, is a complex quantitative trait that depends on a number of internal and external factors, including soil nitrogen availability, nitrogen uptake, assimilation, transportation and remobilization (Kant et al. 2011;Masclaux-Daubresse et al. 2010 (Kant et al. 2011;Simons et al. 2014;Stitt et al. 2002;Xu et al. 2012). A number of biosynthetic enzymes, transcription factors and kinases have been found to be involved in nitrogen uptake, assimilation and remobilization (Kant et al. 2011;Masclaux-Daubresse et al. 2010). The nitrate transporters NRT1.1, NRT1.2, NRT2.1, and NRT2.2 are responsible for nitrate uptake from the environment (Ho et al. 2009;Miller et al. 2007). Glutamine synthetase (GS)/glutamate synthase (GOGAT) cycle is predominantly responsible for assimilating ammonium into amino acids (Lam et al. 1996;Xu et al. 2012). Notably, overexpression of GS1-3 in maize can lead to an increase of 30 % in kernel number (Martin et al. 2006). A large number of quantitative trait loci (QTLs) for physiological and agronomic traits have been identified in maize using quantitative genetic approaches to associate metabolic functions and agronomic traits to DNA markers (Agrama et al. 1999;Hirel et al. 2007;Kant et al. 2011). Previous studies have found QTL for grain yield and yield components overlapping the location of genes for N metabolism (Gallais and Hirel 2004;Hirel et al. 2001). Next generation sequencing technology provides an unprecedented opportunity to characterize transcriptomewide responses to environmental changes. An increasing number of transcriptome sequencing studies on maize development under different N conditions have been performed to identify N-responsive genes and regulatory control of the expression patterns (Amiour et al. 2012;Humbert et al. 2013;Simons et al. 2014). Results from these studies have shown that the transcriptional response to nitrogen availability is highly complex, contingent on a variety of developmental, metabolic, and regulatory factors (Amiour et al. 2012;Humbert et al. 2013;Simons et al. 2014). The recent transcriptome-wide studies further showed that different maize genotypes responded differently to nitrogen availability (Bi et al. 2014;Zamboni et al. 2014). These results suggested that there is wide variation of genotype by nitrogen (G 9 N) interaction at gene expression level. However, a further understanding of how maize genotypes interact with different N levels at transcriptional level is lacking. Studies that are specifically designed to identify genes with significant G 9 N interaction and characterize their regulatory features are needed in maize. Dissecting genotype by environment interactions at the transcriptional level has started to become an important approach for dissecting complex traits and understanding traits evolution (Cubillos et al. 2014;Degenkolbe et al. 2009;Des Marais et al. 2012Geng et al. 2013;Grishkevich and Yanai 2013;Idaghdour and Awadalla 2012;Lasky et al. 2014;Laudencia-Chingcuanco et al. 2011;Lowry et al. 2013;Richards et al. 2012;Snoek et al. 2013). In this study, using transcriptome sequencing, we performed a comprehensive genotype by nitrogen (G 9 N) analysis for two maize inbreds Zheng58 and Chang7-2, the parents of Zhengdan958, a maize hybrid with the largest planting area in China. The previous investigation of nitrogen use efficiency for 27 representative Chinese inbreds has shown that both Zheng58 and Chang7-2 are nitrogen-efficient inbreds at both normal and low nitrogen levels compared to other inbreds (Cui et al. 2013). However, in the response sensitivity, Chang7-2 showed a relatively greater differential response between nitrogen conditions than Zheng58 (Cui et al. 2013). The objectives of this study were to examine the transcriptomic responses to nitrogen changes in Zheng58 and Chang7-2, and further identify genes with significant G 9 N effects and characterize their expression patterns and functional features. We showed that Zheng58 and Chang7-2 showed a contrasting agronomic and transcriptomic responses to the nitrogen treatments. Transcription factors were significantly enriched among genes with significant G 9 N interactions, which implicates that transcription factors might play a crucial role in modulating the G 9 N interactions at transcriptional level. Plant materials Zheng58 and Chang7-2 were grown in 2011 at the Shangzhuang experimental station of China Agricultural University in Beijing under normal nitrogen (NN) and low nitrogen (LN) conditions. The NN treatment indicates the application of the general agronomic fertility treatment (450 kg/ha urea). While for the LN treatment, no nitrogen fertilizer was applied. The LN experiments were conducted in locations where nitrogen fertilizer was not applied during the preceding 2 years. A total of four genotype-condition combinations, namely NN_Zheng58, NN_Chang7-2, LN_Zheng58 and LN_Chang7-2, were tested. In NN and LN field, Zheng58 and Chang7-2 were planted in seven replications. In each replication, Zheng58 and Chang7-2 were adjacently planted in single-row plot, with 10 plants per row, 25 cm between plants within each row and 50 cm between rows. Previous studies have shown that flowering time is a critical period bridging N uptake and N assimilation during vegetative growth to post-flowering N absorption and remobilization (Hirel et al. 2007). Leaves above the primary ear act as one of the main N source for grain-filling (Crawford et al. 1982). For each genotype-treatment combination, when 80 % of plants in the plot flowered, the leaf above the primary ear was sampled for RNA sequencing. Of the seven field plot replications, for each genotype-nitrogen combination, two plot replications were randomly selected and sampled to make biological replications for RNA sequencing. The leaves above the primary ear from four randomly selected plants in the same plot replications were pooled together to make a biological replication. Samples were collected and stored at -80°C in preparation for RNA extraction. At the same time, a total of 15 agronomic traits were measured for each genotype-treatment combination, including plant height, ear height, leaf length, leaf width, tassel length, tassel branch number, days to anthesis, days to silking, cob length, ear diameter, kernel row number, cob diameter, cob weight, total kernel weight and hundred kernel weight. For each genotype-nitrogen combination, all seven filed plot replicates were measured for each trait, with each biological replicate having five randomly selected plants scored. The phenotypic mean of the five plants was used as the phenotype of each replication for the phenotypic difference comparison. T test was performed to test the significance of phenotypic difference. RNA sequencing and data analysis Total RNA was isolated and purified using RNAprep pure Plant Kits (TIANGEN BIOTECH). Approximately 15 lg of total RNA was used for library construction following a standard procedure. Libraries were sequenced with a read length of 100 bp (paired-end) and an insertion size of 300 bp on an Illumina HiSeq 2000 at Berry Genomics, Beijing. Read quality was evaluated using FastQC software (Andrews 2010). 3 0 reads with quality less than 20 were first trimmed by NGS QC Toolkit (v2.3) (Patel and Jain 2012). Only reads with a read length greater than 50 bp were kept for downstream analysis. The high-quality reads were then aligned to the B73 reference sequence (AGPv2) (Schnable et al. 2009) using Tophat2/Bowtie1 (Kim et al. 2013). Five mismatches, a minimum intron size of 5 bp and a maximum intron size of 60,000 bp were used for alignment. For each sample, the number of reads covering the gene model (filtered gene set 5b) was calculated using htseq-count with the intersection-strict option (Anders et al. 2014). Identifying genes with significant G 3 N interactions To identify genes with a differential nitrogen response between genotypes (namely G 9 N interaction) in expression level, the R-bioconductor package ''edgeR'' (v3.4.0) (Robinson et al. 2010) was used to conduct the differential expression analysis. Compared to other differential expression analysis software packages, edgeR employs a robust negative binominal distribution to account for biological variation and dispersion from all genes (Rapaport et al. 2013). Only genes with at least one read count in each sample were kept for further analysis. edgeR first calculates scaling factors for the library sizes that enter into the statistical model for normalizations computed by calcNormFactors function. Then, edgeR uses the model.matrix function to construct the design matrix and estimate the BCVs and dispersions of the negative binomial model by estimateGLMCommonDisp and esti-mateGLMTagwiseDisp function. At last, edgeR uses glmFit function to fit the model and uses glmLRT to test the significance of differential expression for different contrasts. The G 9 N interaction contrast that can be simply expressed as ''(LN_Zheng58-NN_Zheng58)-(LN_Chang7-2-NN_Chang7-2)'' was tested for each expressed gene using edgeR. The contrasts for the expression difference between genotypes under each treatment (NN_Zheng58-NN_Chang7-2; LN_Zheng58-LN_Chang7-2) and the expression difference between treatments for each genotype (LN_Zheng58-NN_Zheng58; LN_Chang7-2-NN_Chang7-2) were also conducted for characterizing the expression patterns of G 9 N interaction genes. The P value of differential expression was converted to false discovery rate (FDR) using Benjamini and Hochberg's algorithm (Benjamini and Hochberg 1995). Expression was considered to be significantly different at a threshold of FDR \0.1. Principle component analysis (PCA) was performed by prcomp and plotted by plot3d function in R. Functional annotation of G 3 N interaction genes G 9 N interaction genes were evaluated for common functions using GO term enrichment test in AgriGO (Du et al. 2010) (http://bioinfo.cau.edu.cn/agriGO/). GO categories were considered significantly enriched with a FDR \0.05 and at least five genes in the category. The potential functions of the identified G 9 N interaction genes were first analyzed using the annotation information from maizeGDB database (Lawrence et al. 2004) (http://www.maizegdb.org/) and then using the TAIR database (Swarbreck et al. 2008) (http://arabidopsis. org/) by protein BLAST. MapMan (Thimm et al. 2004) was also used to examine metabolic pathways and other biological processes. Maize transcription factors were downloaded from the transcription factor database GrassTFDB (Yilmaz et al. 2009) (http://www.grassius.org/grasstfdb.html). Fisher's exact test was used to test if G 9 N interaction genes showed significant overrepresentation of transcription factors compared to the global expressed gene sets. Quantitative real-time PCR analysis First-strand cDNA synthesis was synthesized using TransScript Ò One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGene Biotech) and then stored at -20°C for subsequent analysis. qRT-PCR was performed with the Toolkit for SYBR Ò Green I with ROX Reference Dye II (Takara Biotechnology). Each PCR reaction contained 10 ll mixture, consisting of 1 ll cDNA, 5 ll of SYBR Ò Green Premix Ex Taq II, 0.2 ll of ROX Reference Dye II, and 1 ll of the forward and reverse primers. All qRT-PCRs were performed in three technical replicates in 7500 Real-Time PCR System and performed in two steps: pre-denaturation for 30 s at 95°C and 40 cycles of denaturation for 15 s at 95°C, and annealing/extension for 34 s at 60°C. After the PCR, a melting curve was generated by gradually increasing the temperature to 95°C to test the amplification specificity. Outliers were manually discarded and the housekeeping gene Actin was used as internal standard to calculate the relative expression level for all target genes using comparative C T 2 ÀDDC T À Á method (Schmittgen and Livak 2008). Results and discussion Transcriptional and phenotypic response for nitrogen changes About 10.3 million clean paired-end reads were generated for each of the eight RNA-seq samples and aligned to B73 reference genome (AGPv2) (Schnable et al. 2009). On average, 81.4 % of reads were mapped to the reference genome and 82.4 % of them could be uniquely mapped (Table 1). Only uniquely mapped reads were used in subsequent analyses. For each gene model, read counts were calculated using htseq-count (Anders et al. 2014). A total of 20,685 genes with at least one read for each sample were retained for downstream analyses. Comparisons of biological replicates showed that their expression values across all expressed genes were highly correlated (average R 2 = 0.97). A multidimensional scaling (MDS) analysis was conducted using expression levels normalized by edgeR to evaluate the repeatability of biological replicates (Robinson et al. 2010). As shown in Fig. 1, biological replicates for the same genotype-treatment combination clustered together, indicating that the transcriptional variation between replicates was low relative to the variation due to genotype and treatment. The first MDS dimension separated the samples by genotype (Zheng58 and Chang7-2) and then by the nitrogen condition (NN and LN) in the second dimension. The principle component analysis (PCA) further showed that the experiment was well controlled (Fig. S1). Interestingly, the MDS analysis suggested that Zheng58 and Chang7-2 showed different sensitivities to the nitrogen treatments. The distance between Chang7-2 samples at NN and LN was much larger than that of Zheng58, suggesting Chang7-2 exhibited a greater transcriptional response to differences in nitrogen availability. This is consistent with the organismic-level phenotypic response of the inbreds. The plots of Zheng58 and Chang7-2 from which the RNAseq samples were collected were evaluated for 15 agronomic traits (Fig. 2). Five traits showed significant treatment effects for Chang7-2, while only one trait differed for Zheng58. Gene expression is an important molecular phenotype that links genetic variant and organismic phenotype. The consistent environmental response pattern between gene expression level and organismic-level agronomic traits suggest that the transcriptional level response might play key roles in determining the organismic-level phenotypic response in response to environmental changes. Therefore, the global transcriptional response of genotypes can be used as a robust predicator of their phenotypic changes in response to environmental cues. Despite the consistent global environmental response pattern between gene expression level and organismic-level agronomic traits, it is hard to construct specific links between G 9 N genes and the associated agronomic traits with current limited information. Further genetic dissection in segregating population is needed to establish the causal link. It is worth noting that our study is with limitation because only one tissue and one developmental stage from a single field season were examined. The further investigations across multiple tissues and developmental stages from multiple field seasons will provide a full picture of how transcriptional variation interacts with environment to produce organismic-level nitrogen response. Contrasting phenotypic differences in response to nitrogen conditions between Zheng58 and Chang7-2. The phenotypic values represent mean ± SD (n = 7). NN and LN are indicated by light grey and dark grey, respectively. Red asterisks indicate a significant phenotypic difference between NN and LN (Student's t test; *P \ 0.05). Z58: Zheng58; C7-2: Chang7-2. PH plant height, EH ear height, LL leaf length, LW leaf width, TL tassel length, TBN, tassel branch number, DTA days to anthesis, DTS days to silking, CL cob length, ED ear diameter, KRN, kernel row number, CD cob diameter, CW cob weight, KW total kernel weight, HKW hundred kernel weight Genes with significant G 3 N interactions and their expression patterns A total of 96 genes were identified with a significant G 9 N interaction at FDR \0.1 (Table S1) using edgeR (Robinson et al. 2010) and their overall expression patterns are shown in Fig. 3. G 9 N interactions can be attributable to changes in magnitude or direction of effect. The expression profiles of the 96 G 9 N interaction genes across genotypes and treatments could be classified into three main patterns (Fig. 4). In pattern 1, which includes 60 genes (62.5 %), the expression difference between genotypes was only observed under either the NN or the LN treatment (Fig. 4a). For pattern 2, the expression difference between genotypes is in the same direction at both N levels (Fig. 4b), whereas for pattern 3, the expression difference between genotypes is opposite in the two conditions (Fig. 4c). A total of 13 (13.5 %) and 23 (24.0 %) genes fall into pattern 2 and 3, respectively. Of the 96 G 9 N interaction genes, 80 (83.3 %) genes exhibited a greater expression difference between N levels in Chang7-2 compared to Zheng58. This observation is consistent with the above transcriptome-wide analysis that showed a greater sensitivity of the Chang7-2 transcriptome to nitrogen availability. It is worth noting that, compared to 20,685 investigated genes, the number of genes showing G 9 N interaction is small. This is mainly because (1) both Zheng58 and Chang7-2 are nitrogen-efficient inbreds (Cui et al. 2013). Therefore, the genetic difference in nitrogen response between Zheng58 and Chang7-2 might not be substantial, and (2) only two biological replicates were included for each genotype-condition combination. This limitation might significantly affect the statistical power to identify more G 9 N genes. Further investigations on inbreds with substantial nitrogen response difference and including sufficient biological replications will help identify more G 9 N interaction genes. To validate the RNA-seq results, a total of six genes were selected for qRT-PCR analysis using the same samples as RNA-seq. The primer sequences used in qRT-PCR were listed in Table S2. The comparative CT method relies upon the assumption that the efficiency of the PCR is close to 1, and the target gene and internal control gene have similar PCR efficiencies (Schmittgen and Livak 2008). The very similar shapes of PCR amplification plots (Fig. S2) Fig. 3 The expression heat map of 96 G 9 N interaction genes with dendrogram added. Rows and columns correspond to log 2 (expression) of genes and samples, respectively. Red and blue indicate lower and higher expression levels, respectively suggested that the investigated genes and the internal control gene Actin have similar PCR efficiency. As shown in Figure S3, G 9 N interactions detected by RNA-seq demonstrated correspondence with results obtained by qRT-PCR (R 2 = 0.6, P = 0.067). Functional features of G 3 N interaction genes The functional features of 96 G 9 N interaction genes were annotated based on the annotation information from maizeGDB, MapMan and AgriGO (Table S3-S5). The analysis showed that the 96 G 9 N interaction genes belong to a wide range of functional categories, including biosynthetic enzymes, transcription factors, genes involved in hormone metabolism and stress-responsive genes, which is consistent with the diverse functions previously found to underlie genotype by environment interaction (Des Marais et al. 2013). Of the 96 G 9 N genes, 24 genes encode biosynthetic enzymes that are involved in a number of primary and secondary metabolic processes, such as amino acid, lipid, photosynthesis, hormone metabolism and protein degradation. Similarly, Bi et al. (2014) also detected numerous genes involved in various metabolic pathways that contribute to the differential nitrogen response among three genotypes. Hormone genes that are involved in the metabolism of abscisic acid, auxin, and cytokinins have been frequently identified as important N-responsive genes in previous studies (Kiba et al. 2011). These results suggested that the changes in nitrogen limitation have triggered complex transcriptional response at diverse biological processes. Despite the wide range of functional classes of G 9 N genes, of 96 G 9 N interaction genes, 21 genes are transcription factors, which is a significant enrichment compared to the background gene set (P = 3.20e-05; Table 2). These transcription factors belong to 15 different types of transcription factor families. Of them, five genes belong to AP2/EREBP family and three genes belong to WRKY family and these two families showed significant enrichments ( Table 2). The Gene Ontology analysis has been widely used as an important approach to characterize the biological process, cellular component and molecular function of differentially expressed genes. The 96 G 9 N genes were found to be involved in 42, 15 and 11 GO terms in biological process, cellular component and molecular function, respectively. These wide GO categories of G 9 N genes are consistent Fig. 4 Expression patterns of G 9 N interaction genes. Two genotypes are indicated by black and red lines. a The expression difference between the genotypes is condition-specific, namely significant expression difference between the genotypes is only detected in one condition. b The expression differences between the genotypes are detected in both conditions and the effect direction is in the same direction in the two conditions. c The expression differences between the genotypes are detected in both conditions but the effect direction is in the opposite direction in the two conditions with the above annotation of G 9 N genes. The enrichment analyses of GO terms revealed some common functional features shared by the G 9 N interaction genes. A total of 22 GO terms were found to be significantly enriched for the 96 G 9 N interaction genes at FDR \0.05, such as ''regulation of metabolic process'', ''regulation of nitrogen compound metabolic process'', ''regulation of transcription'', ''transcription factor activity'' and ''transcription regulator activity''. Of these significant GO terms, the most significant term is ''transcription factor activity'' (P = 6.50e-05, Fig. 5; Table S4). Taken together, these results suggest that a number of genes with different functions have been involved in modulating the G 9 N interaction at transcriptional level, indicating the complexity of the molecular mechanism of G 9 N interaction. However, the overrepresented transcription factors in G 9 N genes suggested that transcription factors might play an important role in mediating the transcriptional changes in response to changes in nitrogen availability. The important roles of transcription factors in regulating plant responses to various stresses have been well demonstrated in numerous studies (Chen and Zhu 2004). Transcription factors generally contain multiple cis-regulatory elements as well as multiple DNA-binding domains. This feature provides sufficient flexibility for environmental context-dependent regulations, thus enabling transcription factors to be more easily disposed to form G 9 E interactions. The most significantly enriched GO terms for 96 G 9 N interaction genes. Boxes in the graph represent GO terms labeled by their GO ID, term definition and statistical information different biotic and abiotic stresses (Eulgem et al. 2000;Kizis et al. 2001). GRMZM2G177110 is a homolog of the LBD (LATERAL ORGAN BOUNDARIES DOMAIN) transcription factor LBD37 in Arabidopsis. LBD37 and other two close homologs, LBD38 and LBD39, have been shown to be negative regulators of N availability signals, as well as of anthocyanin biosynthesis in Arabidopsis (Rubin et al. 2009). The LBD genes also repress many other known N-responsive genes, including key genes required for NO 3 uptake and assimilation (Rubin et al. 2009). Further characterizing GRMZM2G177110 will provide cues for understanding the roles of the LBD gene family in maize nitrogen response. Four G 9 N interaction genes encode ubiquitin E3 ligases. The ubiquitin-26S proteasome pathway has been shown to play an important role in N remobilization during leaf senescence for grain-filling (Liu et al. 2008). E3 ligases 'ubiquitinate' target genes and thus determine substrate specificity (Zhang and Xie 2007). Nitrogen limitation adaptation (NLA), a RING-type ubiquitin E3 ligase, is a well characterized gene which has been shown to control the adaptability of Arabidopsis to nitrogen limitation (Peng et al. 2007). GRMZM2G078472 encodes an asparagine synthetase (AsnS). Asparagine has been shown to play a central role in nitrogen transport and storage in plants due to its high nitrogen/carbon ratio and stability ). Three G 9 N interaction genes encode ABA-responsive protein. GRMZM2G471304 encodes an auxin responsive protein. GRMZM2G392101 is a cytokinin response regulator. These hormone genes are important for many plant growth, and developmental processes and response to environmental factors. It has also been shown that, amongst phytohormones, abscisic acid, auxin, and cytokinins have been closely linked to nitrogen signaling (Kiba et al. 2011). Four genes, including GRMZM2G429955, GRMZM2G155216, GRMZM2G1 34130 and GRMZM2G046092, are involved in photosynthesis. The alterations in the expression of genes encoding proteins involved in photosynthesis have been shown as an important differential response when N is limiting (Amiour et al. 2012). These genes are promising candidates for further investigations of the molecular basis of nitrogen response. Identification of the specific mutations that drive G 9 N interactions will provide a deeper understanding of basis of phenotypic changes for nitrogen response in maize. Conclusions We performed differential expression analysis of two elite maize inbreds under two field nitrogen conditions via transcriptome sequencing and identified a set of 96 genes that showed a significant genotype by nitrogen treatment interaction. Analysis of the expression patterns of these genes indicated that genes with G 9 N interactions were more likely to show condition-specific differential expression. Transcription factors, particularly the AP2/EREBP family and WRKY family, showed significant enrichments in G 9 N interaction genes, suggesting the importance of these transcription factor families in the differential nitrogen response between genotypes. Taken together, these results provide novel insights into the mechanism of nitrogen response in maize and provide a set of nitrogen responsive genes for further characterization. Availability of supporting data The data set supporting the results of this article is available in the Sequence Read Archive (http://www.ncbi.nlm. nih.gov/sra/) with the accession number 'SRP052559'. All data sets supporting the results of this article are included within the article. Author contribution statement QC carried out data analysis. ZL and BW carried out the field planting, management and phenotyping. ZL, QC and XW performed tissue sampling and RNA preparation. JL provided the materials. FT and JL conceived of and supervised the study. QC wrote the manuscript draft, FT edited and revised the manuscript. All authors read and approved the final manuscript.

v3-fos

2016-05-04T20:20:58.661Z

2015-05-19T00:00:00.000Z

12397727

s2

Nutritional Properties and Antinutritional Factors of Corn Paste (Kutukutu) Fermented by Different Strains of Lactic Acid Bacteria The aim of this study is to reduce antinutritional factors and to improve the nutritional properties of Kutukutu during fermentation with Lactic Acid Bacteria (LAB). For that, Kutukutu (700 g) was prepared in the laboratory and inoculated with pure cultures of LAB (109 CFU/mL). Then, preparation was incubated for 120 h. Every 24 h, Kutukutu were collected, dried at 45°C for 24 h, and analyzed. The results showed that Lactobacillus brevis G25 increased reducing sugars content to 80.7% in Kutukutu after 96 h of fermentation. Lactobacillus fermentum N33 reduced the starch content to 73.2%, while Lactobacillus brevis G11, L. brevis G25, and Lactobacillus cellobiosus M41 rather increased the protein content to 18.9%. The bioavailability of Mg and Fe increased, respectively, to 50.5% and 70.6% in the Kutukutu fermented with L. brevis G25. L. plantarum A6 reduced the tannin content to 98.8% and L. buchneri M11 reduced the phytate content to 95.5%. The principal component analysis (PCA) shows that, for a best reduction of antinutrients factors and improvement of protein content and minerals, Kutukutu must be fermented by L. brevis G25 and L. fermentum N33, respectively. These starter cultures could be used to ameliorate nutritional proprieties of Kutukutu during the fermentation. Introduction The production of fermented corn paste by natural fermentation of grains soaked in water and ground is an artisanal transformation process of maize commonly used in Africa [1]. Such fermented corn paste can take many denominations in different countries. In Nigeria, for example, the fermented paste is called "Ogi" while in South Africa the term commonly used is "Mawe" [2]. In Cameroon, particularly in North Region, they call it "Kutukutu" [3]. This Kutukutu has an important place in the sociocultural and nutritional plan. In the sociocultural plan, Kutukutu is taken regularly during fast periods and is frequently used as complementary foods for infants [4]. In Cameroon, 70% of mothers give porridge prepared with Kutukutu to infants during the weaning period [5]. Moreover, it is a major source of proteins, carbohydrates, and calories in the diets of large number of population [6]. However Kutukutu contains many antinutritional factors such as phytic acid, polyphenols, and tannins which reduce bioavailability and digestibility of proteins and carbohydrates through formation of complex with minerals and inhibition of enzymes [7]. The technological processes such as mechanical, thermal, chemical, and biological processes are used to reduce antinutritional factors content and to improve the bioavailability of nutriments. Unlike thermal, chemical, and mechanical processes which can deteriorate quality of food, fermentation is one of the processes that decreases the level of antinutrients in food grains and increases the starch digestibility, protein digestibility, and nutritive value [4]. Among the microorganisms used in food fermentation, the LAB represents the principal group found on various substrates [8]. LAB are a large group of closely related bacteria that have similar properties such as lactic acid production, which is an end product of the fermentation. This LAB group includes Lactobacillus, Lactococcus, Streptococcus, and Leuconostoc species. Lactic fermentation is a common way 2 International Journal of Food Science of preparing traditional fermented food in Africa like maize porridge, alcoholic beverages, and dairy products. Several studies reported that LAB improve the nutritional quality of foods during fermentation by increasing the protein content, reducing sugar content, reducing the antinutritional factors (phytates, tannins, and polyphenols), improving the bioavailability of minerals [9], and increasing the energy density by hydrolyzing starch into simpler compounds such as glucose and fructose [10]. Although natural fermentation improves nutritional value and organoleptic qualities of foods [9], it has a major problem of fluctuation in the quality of different foods obtained [11]. Indeed, the spontaneous fermentation process that is carried out by the development of epiphytic microflora can lead to undesirable products on the organoleptic, microbiological, or toxicological quality [11]. That is why the natural fermentation is often the main cause of diarrhea and malnutrition in children [12]. To solve this problem, there is a crucial need to isolate and identify LAB with specific physiological and metabolic properties, which can be used as starters in view to improve general food quality and nutritional value as suggested by few authors [13][14][15][16][17]. The aim of this study is to reduce antinutritional factors and to improve the nutritional properties of Kutukutu during fermentation with L. brevis G11, L. brevis G25, L. buchneri M11, L. cellobiosus M41, L. fermentum N33, Lactobacillus fermentum N25, and L. plantarum A6. Starters. The Kutukutu was obtained after individual fermentation with seven LAB under laboratory conditions. The strains like L. brevis G11, L. brevis G25, L. buchneri M11, L. cellobiosus M41, L. fermentum N33, and L. fermentum N25 were isolated from fermented corn and Kutukutu sampled in Northern Cameroon (Maroua, Garoua, and Ngaoundere). L. plantarum A6 was kindly provided by the Microbiology Laboratory of CIRAD Montpelier, France. These lactic starters stored at 4 ∘ C on agar slants were cultured by streaks on MRS agar and incubated anaerobically at 30 ∘ C for 72 h. The perfectly insulated colonies were inoculated in test tubes containing 10 mL of MRS broth and incubated at 30 ∘ C for 16 h. The resulting preparation was centrifuged at 3000 rpm for 10 min and the resulting pellet was washed in 10 mL of physiological peptone water (peptone 1 g in saline solution (0.85% NaCl), pH 7.2) and centrifuged again. The pellet obtained was suspended in 10 mL saline water. The concentration of viable cells was adjusted at 10 9 CFU/mL using McFarland Standard tube number 4. Production of Kutukutu. In order to evaluate the influence of LAB on the nutritional properties of the Kutukutu during fermentation with starters, the Kutukutu was produced under laboratory conditions following the traditional process with some modifications. Dry corn purchased from a local market in Ngaoundere (Adamaoua, Cameroon) was decontaminated in sterile distilled water containing benzoic acid 6% (w/v) (E210) for 24 h at room temperature. Then sterile corn was soaked in sterile distilled water for 48 h at room temperature. Grinding was proceeded after the determination of the water content (39.6%) using a metallic grinding mill. The paste obtained was mixed (1/3 w/v) with sterile distilled water and sieved through a sieve of mesh 200 m. After decantation for 24 h at room temperature, the paste was collected (water content 73%) in a sterile container and kept for inoculation and fermentation. Fermentation of Kutukutu. Flasks containing 700 g of previously described paste were inoculated separately with 1 mL containing 10 9 CFU of L. brevis G11, L. brevis G25, L. buchneri M11, L. cellobiosus M41, L. fermentum N11, L. fermentum N25, and L. plantarum A6. These flasks were covered and kept at 25 ∘ C for 120 h. The preparations were then homogenized on daily basis to enhance the distribution of bacteria in the medium. Aliquots were collected every 24 h, dried at 45 ∘ C for 24 h, and analyzed. The control sample was the same paste without LAB. Diagram of inoculation of Kutukutu with LAB in laboratory is reported in Figure 1. Changes of Physicochemical Parameters in Kutukutu. To assess the physicochemical parameters, the pH was measured according to the method described by Afoakwa et al. [18]. The lactic acid content was determined by titration according to Obadina et al. [19] and was expressed in grams of lactic acid per 100 g of sample. Reducing sugar was determined by the method described by Fischer and Stein [20] and the optical densities were read at 540 nm. The standard curve was drawn using a prepared aqueous solution of maltose. The starch was determined by Jarvis and Walker method [21]. The optical densities were read at 580 nm. Standard curve was obtained using an aqueous solution of starch. The total nitrogen content (N × 6.25) was determined after digestion of the samples according to the Kjeldahl method described by AFNOR [22] and the coloration was determined by the method of Devani et al. [23]. Standard curve was obtained using a solution of ammonium sulfate. The phytates content was determined by the colorimetric method described by Vaintraub and Lapteva [26], modified by Gao et al. [27], and the optical densities were read at 500 nm using a spectrophotometer. Standard curve was obtained using a solution of phytic acid. The total polyphenols content and tannins were determined by the method of Marigo [28]. The optical densities were read at 725 nm. The formula below was used to determine the tannin content: Statistical Analysis. The results were analyzed using Statgraphics 5.0 (1998) software for the analysis of variance (ANOVA), calculation of averages, and standard deviations. Differences between means were tested using the Duncan Multiple Range Test. Sigma plot 11.0 software was used to draw the curves. Changes in pH. Generally, the pH of Kutukutu fermented with the different LAB trains decreased with time compared to the control ( Figure 2). However, Kutukutu fermented with L. brevis G25 had the lowest pH (2.7) after 120 h. The decrease of pH is due to hydrolysis of carbohydrates during the fermentation which was followed by the production of organic acids [11]. Studies made by Ali and Mustafa [29] showed a similar reduction of pH from 4.3 to 3.4 in the sorghum dough fermented with the lactobacilli strains (L. fermentum, L brevis, and Lactobacillus amylovorus) after 6 h at 37 ∘ C. Changes in Lactic Acid. Contrarily to pH, acidity of Kutukutu increased significantly with time ( < 0.05) compared to the control (Figure 3). It was noted that L. brevis G25 had the highest acidity range (from 0.3 to 1.2%) during fermentation of Kutukutu. The increase of the acidity reflects the metabolism of sugars by LAB during fermentation [30]. From the organoleptic point of view, the acidity of Kutukutu makes it more appetizing for anorexic children and may also reduce bacterial contamination [31,32]. This result is in agreement with the study of Wedad et al. [33] who showed increase in acidity of sorghum cultivar "Mugud" and cultivar "Karamaka" from 0.36 to 1.6% and from 0.36 to 1.8%, respectively, after 16 h of spontaneous fermentation at 28 ∘ C. The work of Hounhouigan et al. [34] also showed similar increase in acidity (88%) of corn flour after 72 h of fermentation. Reducing Sugar. The quantity of reducing sugars increased from 0 to 48 h of fermentation and then decreased after 48 h (Table 1). An increase of 130% in reducing sugars (from 168.2 to 387.6 mg/100 g DM) of Kutukutu fermented with L. buchneri M11 after 48 h was observed. Contrarily to other LAB species, the reducing sugars were produced by L. brevis G25 over a long period (96 h). According to Osman [35], the increase of sugars during fermentation could be explained by the hydrolysis of starch due to amylases produced by the LAB. Osman [35] showed an increase of glucose in millet flour from 6.8 to 11.35 g/100 g after 20 h of fermentation at 30 ∘ C. Osman also portrayed an increase in fructose ranging from 1.17 to 1.20 g/100 g after 20 h of fermentation at 30 ∘ C. Reducing sugars can These results corroborate with those of Osman [35] who showed reduction of glucose and fructose from 11.35 to 7.3 g/100 g and 1.2 to 0.6/100 g, respectively, for fermented millet flour between 20 and 24 h. Starch. The majority of starchy compounds in the Kutukutu decreased significantly ( < 0.05) during fermentation as compared to the control (Figure 4). After 120 h of fermentation, we observed reduction of starch ranging from 1213.9 to 325.1 mg/100 g DM (73.2%) in the Kutukutu fermented with L. fermentum N33. The hydrolysis of starch by the LAB during fermentation reduces swelling of the starch granules and viscosity of the flours during the preparation of porridge [37]. The decrease of starch content in Kutukutu during fermentation could be due to the hydrolysis of starch due to amylases produced by the LAB into simple sugars [36]. Agati et al. [38] showed that LAB isolated from fermented maize could have a strong amylolytic activity. Hama et al. [39] showed a decrease of starch from 65.6 to 23.6 g/100 g (64.0%) after 72 h of spontaneous fermentation of Dégué. Crude Proteins Content. A slight increase of the crude proteins content was observed during the fermentation of Kutukutu with all selected strains excepted for L. fermentum N33 ( Figure 5). After 120 h of fermentation, crude proteins content in Kutukutu fermented with L. brevis G11, L. brevis G25, and L. cellobiosus M41 increased from 5.8 to 6.9 g/100 g DM (18.9%) for each one. However, L. fermentum N33 has a different behavior from the other bacteria. Initially, an increase in proteins content ranging from 5.8 to 6.3 g/100 g DM (8.6%) was observed after 48 h of fermentation, followed by a drop from 6.3 to 5.0 g/100 g DM (20%) after 120 h of fermentation. The increase of crude proteins content could be attributed to the use of carbohydrates by LAB [35]. These results are in agreement with those of Awade et al. [40], who showed an increase in crude proteins content by 14.63% after 14 h of fermentation of corn flour. However the decrease in proteins content observed in L. fermentum N33 fermented Kutukutu may be explained by the fact that the LAB used these proteins for their metabolic activities during fermentation [41]. Osman [35] observed a similar reduction of protein content by 4.5% after 20 h of fermentation of millet flour at 30 ∘ C. Minerals Availability. During the fermentation of Kutukutu, a significant increase ( < 0.05) in minerals was observed (Table 2), but minerals content was different between all the tested bacteria strains. The highest content of Mg, Fe, and Na was registered in Kutukutu fermented with L. brevis G25 varying between 25.9 and 39 mg/100 g DM (50.5%), 9.2 and 15.7 mg/100 g DM (70.6%), and 1.2 and 1.3 mg/100 g DM (8.3%), respectively. There was also an increase in the K and P from 82.6 to 118.8 mg/100 g DM (43.8%) and from 95.1 to 138.1 mg/100 g DM (45.2%), respectively, in Kutukutu fermented with L. brevis G11. L. fermentum N33 and L. brevis G25 increased the Zn content in Kutukutu with values ranging from 1.1 to 1.3 mg/100 g DM (18.2%). L. brevis G25, L. brevis G11, and L. buchneri increased the Cu content in Kutukutu from 0.1 to 0.2 mg/100 g DM (100%), while only L. brevis G25 and L. brevis G11 increased the Mn content in Kutukutu from 0.2 to 0.4 mg/100 g DM (100%). The increment in minerals could be explained by the reduction of antinutritional substances such as phytates and phenolic compounds which form complexes with minerals [10,42]. Eltayeb et al. [43] observed an increase of Fe and Zn from 5.8 to 5.9 mg/100 g and from 2.9 to 3 mg/100 g, respectively, in fermented millet flour of "Garira" variety after 24 h of spontaneous fermentation at 37 ∘ C. They also noticed an increase in P, Zn, and Fe content from 183.4 to 205.3 mg/100 g, 2.9 to 3.1 mg/100 g, and 6.5 to 10.2 mg/100 g, respectively, in the fermented millet flour variety "Gadarif'' after 12 h of fermentation at 37 ∘ C [43]. Figure 6. After 120 h of fermentation, the total polyphenols content was reduced from 425.8 to 66.3 mg/100g DM (84.5%) and from 425.8 to 86.8 mg/100g DM in the Kutukutu fermented with L. fermentum N33 and L. plantarum A6, respectively. The reduction in polyphenols content during fermentation could be attributed to the production of polyphenol oxidases by LAB [40]. Many studies on the improvement of nutritional quality of fermented grains such as millet showed a significant reduction of the levels of polyphenols [44,45]. Adam et al. [46] observed a reduction in polyphenols content ranging from 120.4 to 111.08 mg/100 g and from 125.1 to 107.2 mg/100 g, respectively, in millet cultivar "Ugandi" and "Dembi yellow'' after 14 h of fermentation at 37 ∘ C. Tannins. Tannins content was reduced significantly ( < 0.05) during the fermentation of Kutukutu compared to the control (Figure 7). L. plantarum A6 and L. fermentum N33 reduced the tannins content in Kutukutu from 215.1 to 2.5 mg/100 g DM (98.8%) and 215.1 to 4.6 mg/100 g DM (97.9%), respectively, after 120 h of fermentation. Indeed, some LAB such as L. plantarum, L. pentosus, and L. paraplantarum are able to degrade tannins through their acylhydrolase tannin activity [47]. This ability is often associated with the vegetable products and confers an ecological advantage to the LAB [47]. Antony and Chandra [48] showed 52% reduction of tannins in millet flour during fermentation. In the same way Onyango et al. [49] reported a significant ( < 0.05) reduction of tannins content after 8 days of fermentation of red sorghum flour, white sorghum and millet at 25 ∘ C. International Journal of Food Science 7 The values followed by the same letter on the same column are not significantly different ( > 0.05). 3.9. Phytates. The entire selected LAB reduced the phytates content after 120 h of fermentation ( Figure 8). Phytates content in Kutukutu fermented with L. buchneri M11 was reduced from 278.7 to 12.4 mg/100g DM (95.5%). This observed reduction of phytates can be due to phytases and phosphatases produced by LAB which hydrolyze phytates to inositol and orthophosphates [50]. Studies made by Ejigui et al. [50] also illustrated a reduction in phytates levels ranging from 9.87 to 3.8 mg/100 g in corn flour after 96 h of fermentation at 30 ∘ C. Similarly, Onyango et al. [49] reported a significant ( < 0.05) reduction of phytates in red sorghum flour, white sorghum, and millet after 8 days of fermentation at room temperature. Cui et al. [51] presented a decrease of phytates to 24.3% in 4 corn cultivars during the fermentation. Principal Component Analysis (PCA) The variables used to evaluate the improvement of the nutritional quality of Kutukutu with LAB were attached to a principal component analysis ( Figure 9). That helped to visualize correlations and to select among the 07 bacteria studied, ones who give the best results. These variables are organized in two principal components which express 79.4% of total variability. The axis F1 explains 47.29% of information and the second axis F2 explains 32.13% of information. The analysis of the correlations between the different variables and the principal axis shows that the variables such as minerals (Mg (0.95), Mn (0.89), iron (0.63), and Cu (0.82)), proteins (0.73), lactic acid (0.85), and pH (−0.94) contribute significantly to the formation of the F1 axis, while variables such as polyphenols (0.95), phytates (−0.91), starch (0.80), and some minerals like Zn (−0.81) contribute mainly to the formation of the F2 axis. The supplementary variables (reducing sugars (−0.60)) that are classified on the axis F1 also show a significant contribution on this axis. When LAB are also represented in the axis system F1 × F2 (Figure 10), axis F1 corresponds to the variables induced by L. brevis G25, L. fermentum N25, while the F2 axis variables are induced by L. plantarum A6, L. buchneri M11, L. fermentum N33, and L. cellobiosus M41. However, L. brevis G25 (55.3%) and L. fermentum N33 G25 (33%) are those LAB that contribute most to the formation of this axis system (F1 × F2). This arrangement of variables and observations on F1 and F2 axis shows that L. fermentum N33 helps to reduce antinutrients factors such as phytates and polyphenols, while L. brevis G25 contributes to increased bioavailability of minerals (Mg, Mn, Cu, and Fe), lactic acid, and protein contents. Conclusions The fermentation of the Kutukutu by selected LAB induced many changes in nutritional properties as well as antinutritional factors. L. brevis G25 increased (80.7%) reducing sugars content and increased the proteins content to 18.9%. It also increases availability of Mg and Fe, respectively, to 50.5% and 70.6%. L. plantarum A6 reduced the tannins content to 98.8% in Kutukutu and L. buchneri M11 reduced the phytates content (95.5%) in the Kutukutu, while, for a best reduction of phytates and polyphenols, Kutukutu must be fermented by L. brevis G25. To improve protein content and minerals (Mg, Mn, Cu, and Fe), Kutukutu must be fermented by L. fermentum N33. Both of these bacteria can be used for improving the nutritional quality of Kutukutu during fermentation.

v3-fos

2019-04-25T13:11:41.778Z

2015-02-01T00:00:00.000Z

131180417

s2

Eco-Friendly Weed Control Options for Sustainable Agriculture Background: Weeds are unwanted plants playing a very important role in different eco-systems and many of them cause enormous direct and indirect losses. The losses include interference with cultivation of crops, loss of biodiversity, loss of potentially productive lands, loss of grazing areas and livestock production, erosion following fires in heavily invaded areas, choking of navigational and irrigation canals and reduction of available water in water bodies. Weed management takes away nearly one third of total cost of production of field crops. In India, the manual method of weed control is quite popular and effective. Of late, labour has become non-availability and costly, due to intensification, diversification of agriculture and urbanization. The usage of herbicides in India and elsewhere in the world is increasing due to possible benefits to farmers and continuous use of the same group of herbicides over a period of time on a same piece of land leads to ecological imbalance in terms of weed shift and environmental pollution. The complexity of these situations has resulted in a need to develop a wholistic sustainable eco-friendly weed management programme throughout the farming period. Objectives: This study reviews the different approaches used in sustainable weed control options. Conclusion: Sustainable farming has the ability to save the natural resources for the future and develop the farm in the little expense, a transition to sustainable weed control is required for environmental, social and economic reasons and sustainable weed management is socially acceptable, environmentally benign and cost-effective. INTRODUCTION Weeds are considered to be a potential pest causing more than 45% loss in yields of field crops, when compared to 25% due to diseases, 20% due to insects, 15% due to storage and miscellaneous pests and 6% due to rodents. Weed management takes away nearly one third of total cost of production of field crops. In India, the manual method of weed control is quite popular and effective. Of late, labour has become non-availability and costly, due to intensification, diversification of agriculture and urbanization. The usage of herbicides in India and elsewhere in the world is increasing due to possible benefits to farmers. At the same time, the continuous use of the same group of herbicides over a period of time on a same piece of land leads to ecological imbalance in terms of weed shift, herbicide resistance in weeds and environmental pollutions. Treatments of herbicides for controlling aquatic weeds in a pond also reduce dissolved oxygen and pH and increase biological oxygen demand 1 . Herbicide application may also kill species of bacteria, fungi and protozoa that combat disease causing microorganisms, thereby upsetting the balance of pathogens and beneficial organisms and allowing the opportunist, disease causing organisms to become a problem 2 . The complexity of these situations has resulted in a need to develop a wholistic sustainable eco-friendly weed management programme throughout the farming period. Sustainable development is the management and conservation of the natural resource base and the orientation of technological and institutional change in such a manner as to ensure the attainment and continued satisfaction of human needs for present and future generations. Such sustainable development conserves land, water, plant and animal genetic resources, is environmentally non-degrading, technically appropriate, economically viable and socially acceptable 3 . With respect to the environment, society and economics, sustainable agriculture would, (1) Not harm the environment from pollution, (2) Not be reliant on non-renewable inputs or degrade renewable ones, (3) Nourish people with non-toxic, healthy food and other useful feed stocks and (4) Provide a fair, steady, return on effective investment in labor and capital. Sustainable weed management is the use of weed control methods that are socially acceptable, environmentally benign and cost-effective. An attempt has been made to review the different approaches used in sustainable weed control options, in this study. OBJECTIVES OF SUSTAINABLE WEED MANAGEMENT There are several basic objectives of the sustainable weed management. The main objectives are: C To make best use of the resources available for weed control C To develop cultivation methods that manage weeds and improve soil quality and to determine the impact of weed management systems C To minimize use of non-renewable resources like herbicides and to use of renewable energy and recycled mineral resources C To protect the health and safety of farm workers and animals, local communities and society from the application of chemicals C To protect and enhance the environment and natural resources C To protect the economic viability of farming operations C To provide sufficient financial reward to the farmer to enable continued production and contribute to the well-being of the community C To produce sufficient high-quality and safe food C To build on available weed control technology, knowledge and skills in ways that suit local conditions and capacity APPROACHES INVOLVED IN SUSTAINABLE WEED MANAGEMENT There are three different approaches involved in sustainable weed control management. The different approaches are reviewed in the context of the cultural, mechanical and biological methods, respectively. Cultural approaches Proper crop stand: Crop population, spatial arrangement, right method and time of sowing, adequate seed rate and the choice of cultivar (variety) are essential to limit the weed growth. Any crop variety that is able to quickly shade the soil between the rows and is able to grow more rapidly than the weeds will have an advantage in weed management. Studies have shown that narrow row widths and a higher seeding density will reduce the biomass of later-emerging weeds by reducing the amount of light available for weeds located below the crop canopy. Similarly, fast growing cultivars can have a competitive edge over the weeds. Planting pattern is a cost effective technique that modifies the crop canopy structure and micro-climate enhances crop competitiveness in weed suppression, improves the resource use efficiency and maximizes crop productivity 4 . It was reported that combination of early sowing (October 25) with quicker growing wheat var (PB 154, 343, 542) significantly smothered Phalaris minor 5 . Rice variety PR 108 exhibited greater smothering effect on weeds but PR 118 obtained maximum grain yield as compared to PR 108, 114, 116 grown under puddled conditions. The index of competition was lower in the cultivars Avarodhi and Pant G114 as compared to the cultivar Radhney in Chick pea 6 . Closer spacing, early planting and increasing the fertilizer rates are observed to increase crop yields and reduce weed populations in barley and wheat under small farming systems of semi-arid regions 7 . The plant population and dry matter production of weed Tagetes sp. were significantly lower in the narrow spacing than wider spacing and control 8 . The plant population of 50 plants mG 2 was found to be significantly superior to 33 and 25 plants mG 2 as it recorded significantly less weed dry matter and highest grain yield compared to other plant population levels 9 . Planting pattern with closer spacing of 60×20 cm with 83,333 plants haG 1 proved to be very effective in suppressing weeds, by recording the least density of grasses, sedges and broad leaved weeds in sweet corn 10 . Green manure in situ: A practice of ploughing or turning into the soil undecomposed green manure crops in the same field where the crop is grown. Green manure crops are commonly associated with organic agriculture and are considered essential for annual cropping systems that wish to be sustainable. Traditionally, the practice of green manuring can be traced back to the fallow cycle of crop rotation, which was used to allow soils to recover. Green manures usually perform multiple functions that include soil improvement and soil protection. In addition to soil improvement, green manuring is also used for weed suppression in cropping systems. Raising green manure Sesbania aculeata in the preceding off-season and ploughing in situ before puddling reduced the weed counts and increased the weed control index in the succeeding rice crops due to smothering effect of green manure on the emergence and growth of weeds 11 . Sowing of green manure seeds in between rice row, serves as a green manure and checks weed growth 12 . The weed control efficiency was higher when maize was raised with green manure (cowpea) as intercropping 13 . In rice-wheat cropping systems, inclusion of Sesbania in summer resulted in least grasses and sedges in the succeeding crops 14 . Intercropping: Growing of two or more generally dissimilar crops simultaneously on the same piece of land, in distinct row arrangement is known as intercropping. Intercropping and cover cropping are practices that increase diversity in the cropping system and enhance the utilization of resources such as light, heat and water. These practices can also help to suppress weeds and increase the likelihood of being able to reduce herbicide use in the cropping system. Alternatively, in organic or other systems where herbicides are not used, intercropping and cover cropping can reduce the yield loss potential and provide stability in the system. Research and experience from around the world have shown that intercropping and cover cropping systems tend to suppress weeds better than sole cropping systems 15 . Maize+Cowpea intercropping system recorded the highest weed control efficiency of 90.6% at 60 days after sowing. It was followed by maize+blackgram intercropping system 16 . The highest weed control efficiency, test weight and grain yield were found intercropping of blackgram with maize followed by manual weeding 17 . The grain yield, productivity ratio index, production efficiency and weed control efficiency were highest under maize+blackgram (2:1) for maize; however weed smothering efficiency of maize was highest under maize+blackgram (1:1) 18 . Dual cropping of Sesbania aculeata with drum seeded rice reduced total weed density and weed biomass as compared to other method of seeding 19 . Crop rotation: Crop rotation is an important component of integrated weed management. The choice and sequencing of crops affect long term weed population dynamics and consequently weed management. Crop rotation is a planned sequence of crops growing in the same field year after year. Rotating crops adds diversity to the cropping system, increasing the sustainability of the system. Crop rotation provides the foundation for long-term weed management. Planting a wide variety of crops with varied characteristics reduces the likelihood that specific weed species will become adapted to the system and become problematic. The success of rotation systems for weed suppression appears to be based on the use of crop sequences that employ varying patterns of resource competition, allelopathic interference, soil disturbance and mechanical damage to provide an unstable and frequently inhospitable environment that prevents the proliferation of a particular weed species 15 . Crop rotation can also slow the development of herbicide resistant weeds 20 . The crops like sorghum, maize, barley, rye, sweet clover, sunflower, rape seed, soybean, alfalfa, cowpeas and hemp has smothering effect on various weed species through crop interference. Soybean and sunflower planted without tillage into desiccated rye mulch give over 90% reduction in the biomass of Chenopodium album, Amaranthus retroflexus and Ambrosia artemisiifolia compared to tillage and no rye. Mungbean-mustard cropping sequence resulted in high return and benefit-cost ratio than fallow mustard, by recoding least weed counts and weed biomass 21 . Organic manures: A byproduct of the processing of plant and animal matter that has sufficient nutrient capacity to have value as fertilizer. Pressmud is one of the byproducts of the sugar industry. Pressmud is obtained in sugar factories to a tune of 2% of the weight of sugarcane crushed. Pressmud contains sizable quantity of macro and micro nutrients, besides 20-25% of organic carbon. In addition to the manurial value of pressmud, it destroys the weed seeds and seedlings due to reduced soil pH and allelochemicals produced from the native microbes of pressmud. Significant weed control and increased the yields of rice were reported from pressmud 10 t haG 1 applied alone and the same was reported to synergistically interact with herbicide 22 . Application of pressmud at higher dose of 20 t haG 1 performed superior by suppressing weed growth and favourably influencing growth and yield characters of rice 23 . Pressmud incorporation at 10 t haG 1 before puddling and azolla inoculation at 1 t haG 1 on 7 days after transplanting contributed lesser weed counts and highest weed control index in succeeding rice crops due to the destruction of weed seeds and seedling 11 . Application cane pressmud and neem cake reduced the weed seed bank of Cyperus rotundus, Echinochloa colonum and Trianthema portulacastrum in maize, due to reduced pH and phytonicidal properties of organic manures 24,25 . Mechanical approaches Off-season ploughing: Ploughing operations carried out in the off-season with the help of tractors or bullock drawn implements known as off-season ploughing, before the crops are sown or transplanted. Off-season ploughing was very effective in reducing the weed population in succeeding rice crop as tubers and weed seeds are exposed to scorching sun and a highly unfavourable environment, with eventual destruction of their perennation 26 . Summer ploughing increased the total buried weed seed population by 3-4 times compared to no-ploughing 27 . Off-season ploughing twice at 45 days interval was found to be superior in reducing the population of weeds; Cyperus rotundus, C. difformis, Sphenoclea zeylanica and Fimbristylis littoralis and highest weed control index in succeeding rice crops. Mechanical destruction of existing weed vegetation in the summer and exposure of reserves of weed seeds or propagules and subsequent scorching contributed for superior performance of summer ploughing in controlling weeds during succeeding crop seasons 11 . Soil solarization: Soil solarization is a method of hydrothermal disinfection accomplished by covering moist soil with transparent polyethylene (TPE) film during the hot summer months. Solarization during the hot summer months can increase soil temperature to levels that kill many disease-causing organisms (pathogens), nematodes and weed seed and seedlings. It leaves no toxic residues and can be easily used on a small or large scale. Soil solarization also improves soil structure and increases the availability of nitrogen and other essential plant nutrients. The basic phenomenon helping weed control upon soil solarization is build up of lethally high temperatures in top soil where most of the dormant and viable weed seeds are present. The possible mechanisms of weed control by soil solarization are breaking dormancy of weed seeds and solar scorching of emerged weeds and direct killing of weed seeds by heat. Soil solarization increases soil temperatures by 8-12°C over the corresponding non-mulched soil 28 . Rhizomes of perennial weeds may be controlled by solarization, if they are not deeply buried. Solarization for two successive years was most effective in suppressing the perennial weeds. Soil solarization with the use of 0.05 mm transparent polyethylene sheets for 40 days was effective in controlling weeds than the use of 0.1 mm thickness polyethylene sheet and the lesser duration of soil solarization. Soil solarization with 0.05 mm thickness for 40 days recorded significantly higher pod yield of ground nut and least weed seed reserves in the top 5 cm soil 29 . Stale seed bed: It is the technique in which the weed seeds are allowed to germinate by rain or wetting and killing them (at 1-2 flushes of the weeds) before sowing seeds of main crops. At this stage a shallow tillage or a non-residual herbicide like paraquat may be used to destroy the dense flush of young weed seedlings. This may be followed immediately by sowing a desired crop. The main objective with this technique is that most of the weeds that have the potential to germinate, because of their placement in the upper 1" to 2" of the soil, will usually do so within two weeks after the soil is prepared. Adequate soil moisture and temperature (at least 50°F at a depth of 2") must be present. The technique can be utilized in early spring, when the weather is still too cold for proper seed germination. Several passes are made with a rototiller or plow and then weed seeds are allowed to germinate as weather permits. By tilling, the farmer increases the chance of weed seed germination by the same method as one would for favorable vegetable/crops. The fine soil allows weed seed to grow rapidly by allowing the seed to open and the roots to spread easier than in compacted soil. Deep tilling will also bring dormant seed to the surface for germination. Some species of plant are known for seeds that can lay deeply buried in the soil for years before favorable conditions allow germination. Spike tooth harrow is a very useful implement for destroying the emerging weeds during the preparation of stale-beds. Soybean sowing, using stale seedbed techniques, by killing the first or second flush of weeds resulted in higher soybean yield 30 . Adopting stale seedbed techniques either for 7 or 14 days (by keeping field drained and destruction of weeds by letting in water on 14th day) significantly reduced the population of grassy and broad leaved weeds and improved grain and straw yield of wet seeded rice compared to normal seed bed 31 . Use of weeders: Now a days, use of mechanical weeders in agricultural operations is increasing because of non-availability of labours for weeding. The cost of the weeding operations is also reduced by using the machineries for weeding. The machineries like mini-weeders, power tillers, mini-tractor drawn rotavator are used for weeding in wider spaced crops like sugarcane, cotton and orchards. Since the wider spacing of 5-6 feet is practiced Sustainable Sugarcane Initiatives (SSI), mini-tractor drawn rotavator can be used for effective controlling all types of weeds in sugarcane. Cono weeder is used for controlling the wet land weeds and getting more yields in the System of Rice Intensification (SRI). The mini weeder and power tillers are used for controlling different types of weeds in cotton crop. Moreover, different types of weeding implements are available for weeding operations in various field and horticultural crops. Small farm implements and machine i.e., power tiller, marker and cono weeder played very imperative role in controlling weeds, enhancement of productivity and reduction in drudgery in SRI 32 . The cono weeder incorporation of dhaincha and azolla resulted in higher weed control during early stages of rice crop. Mulching: Mulches are coverings placed on the surface of the soil. Mulching smothers the weeds by excluding light and providing a physical barrier to impede their emergence. Any material such as straw, plant residues, leaves, loose soil or plastic film can be used as a mulching material. Such materials as straw, bark and composted material can provide effective weed control. Producing the material on the farm is recommended since the cost of purchased mulches can be prohibitive, depending on the amount needed to suppress weed emergence. An effective but labour-intensive system uses newspaper and straw. Two layers of newspaper are placed on the ground, followed by a layer of hay. It is important to make sure the hay does not contain any weeds seeds. Organic mulches have the advantage of being biodegradable. Cut rye grass mulch spread between planted rows of tomatoes and peppers was more economic than cultivation. Materials such as black polyethylene have been used for weed control in a range of crops in organic production systems. Plastic mulches have been developed that filter out photosynthetically active radiation but let through infrared light to warm the soil. These infrared transmitting mulches have been shown to be effective at controlling weeds. The new approach of using rice straw for controlling weeds in different crops indicated that rice straw can be used for mulch, which benefits in preventing weed growth as well as supplies organic matter for N-fixation by heterotrophic N-fixing microorganism 33 . News papers and black polythene are recommended for the environmental friendly and sustainable control of weeds and realizing good yields of edible pea 34 . Surface application of rice residues at 6 and 7 t haG 1 significantly reduced population, dry matter production and leaf area index of Phalaris minor as compared to straw removal and incorporation treatments, in wheat 35,36 . Biological approaches Allelopathic plants: The concept of allelopathy is receiving increased attention in the search for weed control strategies. Allelopathy is any direct or indirect effect by one plant, including micro-organisms, on another through production of chemical compounds that escapes into the environment to influence the growth and development of neighboring plants 37 . Plant releases chemicals that show allelopathic potentiality are called allelochemicals or allochemicals 38 . It covers a wide range of chemicals used by plants or organisms. Generally different plant organ such as plant tissues, including leaves, flowers, fruits, stems, roots , rhizomes, seeds and pollen are the main sources of allelochemicals of donor plants are in stressed or competing with neighboring plants, that released through crop-environmental ecological process 39 . Allelochemicals or natural compounds have more benefits over synthetic compounds as they have novel structure and short half-life, therefore considered safe of environmental toxic 40 . Therefore, allelopathy mechanism can be applicable as a component of sustainable weed management. There are many plant species have allelopathic potential to control the aquatic weeds effectively. Rice cultivar ADT 36 was moderately allelopathic and reduced the weed biomass by 33.4 and 32.0% in laboratory bioassay and micro pond, respectively. Allelopathic cultivars of rice can control both monocot and dicot weeds under field conditions with some selectivity observed amongst such weeds, suggesting that certain compounds with selective action might be implicated in rice allelopathy 41,42 . Weed population was lower at all doses of rice straw incorporated and it can also be utilized for producing new group of natural herbicides 43 . Dry leaf powder and flower powder of Parthenium hysterophorus at 0.5% (w/v) kills water hyacinth within one month 44 . An Indian medicinal herb Coleus amboinicus/aromaticus shows remarkable allelopathic inhibition of water hyacinth. The aquatic weed of Eichhornia crassipes can be effectively controlled by the integrated approach of releasing the insect agents Neochetina spp., with an adequate inoculation loads of 2 insects plantG 1 followed by the spraying of aqueous leaf powder extract of C. amboinicus/aromaticus at 25% concentration, 10 days later on the weed canopy 45 . A number of crop plants with allelopathic potential can be used as cover, smother and green manure crops for managing weeds by making desired manipulations in the cultural practices and cropping patterns. These can be suitably rotated or intercropped with main crops to manage the target weeds selectively. Sunflower was reported to inhibit the growth of weeds Sinapis arvensis and Setaria viridis in terms of root and shoot length and seedling dry weight 46 . The list of allelopathic crops and weeds to interfere with different weeds are given in Table 1 and 2. uptake of nutrients by their interactions in the rhizosphere when applied through seed or soil. They accelerate certain microbial processes in the soil which augment the extent of availability of nutrients in a form easily assimilated by plants. Azolla is a free-floating water fern that floats in water and fixes atmospheric nitrogen in association with nitrogen fixing blue green alga Anabaena azollae. Azolla fronds consist of sporophyte with a floating rhizome and small overlapping bi-lobed leaves and roots. Dual culturing of azolla in rice fields had the added benefit of suppressing weed growth besides fixing atmospheric nitrogen. Since it formed a mat over the surface, it reduced the entry of sunlight and aeration into soil thereby suppressing weed growth. The addition of azolla in rice fields suppressed the weeds of Eichinochloa crusgalli and Cyperus difformis and the degree of suppression increased with increase in per cent of azolla cover and water depth 47 . Application of pressmud at 10 t haG 1 +azolla at 1 t haG 1 recorded the least weed count and highest weed control index in rice crop, as the thallus growth formed a thick mat on the surface of water, curtailing the interception of light by weed seeds and seedlings 11 . Insect bio-control agents: Bio-control of weeds is the deliberate use of natural enemies to reduce the densities of the weeds economically or aesthetically tolerable limits. Insects are important in biological control because of their, (1) Great variety and numbers, (2) High degree of host specialization, (3) Intimate adaption to their host plants, (4) Availability of a range of natural enemies suited to particular ecological situations and (5) Ease with which they can be handled. There are two kinds of biological control: Classical and inundative. In classical biological, once the agents are well established there is no need to make further releases as they persist forever. But, in inundative biological control large quantities of agents are released to control the target weeds. Biological agents are increasingly being seen as a feasible solution to the problem. The research effort in the use of fish to control excessive aquatic weed growth in irrigation canal has steadily gained ground in recent years 48 . The list of weed species controlled by insect agents is given in Table 3. Bio-herbicides: Weeds can be controlled by pathogens like fungi, bacteria, viruses and virus like agents. Among the classes of plant pathogens, fungi have been used to a larger extent than bacteria and virus or nematode pathogens. A bio-herbicide is a preparation of living inoculums of plant pathogens formulated and applied in a manner analogous to that of an herbicide in an effort to control or suppress the growth of weed species. The development of a bio-herbicide involves three major phases, (1) Discovery, (2) Development and (3) Deployment 49 . The discovery phase involves the collection of diseased plant material, isolation of the causal organism, demonstration of Koch's postulates, identification of the pathogens, culture of the pathogens on artificial media and maintenance of the pathogen culture in short-term and long-term storage. The development phase involves the determination of optimum conditions for spore production, determination of optimum conditions for infection and disease development, determination of host range, elucidation of mechanism of action of the pathogen and/or toxin and quantification of the efficacy of the bio-herbicide as control option. The final phase, deployment, often involves close collaboration between researchers, farmers and the industrial sector for the production, possible commercialization and use of bio-herbicides, formulation, fermentation, regulating aspects, marketing and implementation are essential aspects of this phase. Herbicide-resistant weed biotypes will eventually develop after repeated applications of the same herbicides in a given field. For example, glyphosate resistant Lolium rigitum developed after repeated use of glyphosate in an orchard to control grass weeds 50 as herbicide resistant becomes more problematic with many common weeds, strategies using bio-herbicides will become more important in maintaining adequate weed control in conventional systems. The potential for successful use of bio-herbicides in managing herbicides-resistant biotypes was demonstrated where growth of an imazaquin-resistant common cockleber biotype originating soybean field was suppressed with the mycoherbicides, Alternaria helianthi 51 . The fungus Colletotrichum gleosporioides attack cuscutta 52 and has been used to control cuscuta selectively in soybean 53 . Fusarium oxysporum was found to be the best resulting in killing of inoculated water hyacinth in about 15 days 54 . The list different bio-herbicides available for controlling weeds are given in the Table 4. Herbicide resistant crops: Herbicide resistance is the inherited ability of the plant to survive and reproduce following exposure to a dose of herbicide that would normally be lethal to the wild type. In a plant, resistance may occur naturally due to selection or it may be induced through such techniques as genetic engineering. The adoption of Genetically ModiWed (GM) crops has increased dramatically during the last 10 years and currently over 52 million hectares of GM crops are planted world-wide. Approximately 41 million hectares of GM crops planted are herbicide-resistant crops, which includes an estimated 33.3 million hectares of herbicide-resistant soybean. Herbicide-resistant maize, canola, cotton and soybean accounted for 77% of the GM crop hectares in 2001. However, sugarbeet, wheat and as many as 14 other crops have transgenic herbicide-resistant cultivars that may be commercially available in the near future. There are many risks associated with the production of GM and herbicide-resistant crops, including problems with grain contamination, segregation and introgression of herbicide-resistant traits, market place acceptance and an increased reliance on herbicides for weed control. Integrated weed management: One of the definitions of Integrated Weed Management (IWM) implies methods of controlling weed that require no herbicide or rational use of herbicides 55 . IWM includes more than one method of control viz., seed purity, crop varieties, spacing and methods of planting, cultivations, soil solarization, intercropping, crop rotation, water management, manure application, biological control and herbicides. According to FAO, " the integrated campaign against pests is a method whereby all economically, ecologically and toxicologically justifiable methods are employed to keep the harmful organisms below the threshold level of economic damage, keeping in the foreground the conscious employment of natural limiting factors. Integrating fish culture and dual culture of azolla in transplanted rice is observed to compliment weed control in transplanted rice 56 . Off-season ploughing and mulching the inter row space enhanced the weed control in combination with herbicide in cotton 57 . BENEFITS OF SUSTAINABLE WEED MANAGEMENT The benefits are reviewed in the context of the environment, society and economics, (1) Improved soil and water conservation, (2) Mitigation of global warming, (3) Enhanced biodiversity, (4) Reduction of persistent pollution, (5) Increased food nutrient density, (6) Reduced toxic load in adults and children who eat organic, (7) Better conditions for farm workers, (8) Competitive yields, (9) Price premiums, (10) Direct-to-consumer marketing channels, (11) Lower input costs, (12) Higher per farm income, (13) Improved resilience or lower volatility, (14) Energy savings and (15) Income from carbon markets. CONCLUSION As we know the sustainable farming has the ability to save the natural resources for the future and develop the farm in the little expense, a transition to sustainable weed control is required for environmental, social and economic reasons. Fortunately, sustainable farming is a robust business model, delivering superior economics over conventional farming on a wide variety of metrics such as crop yields, gross and net income per acre, cost of inputs, per farm income and more. As society provides the financial and organizational capital to re-create agriculture, the living soils, plants and animals will respond, over time, to support us. Each acre converted to organic, sustainable methods is one acre closer to a societal tipping point for sustainability-or at least one less acre as a source of harm.

v3-fos

2016-03-14T22:51:50.573Z

2015-05-01T00:00:00.000Z

13737097

s2

Response of Different Genotypes of Faba Bean Plant to Drought Stress Drought stress is one of the major abiotic stresses that are a threat to crop production worldwide. Drought stress impairs the plants growth and yield. Therefore, the aim of the present experiment was to select the tolerant genotype/s on the basis of moprpho-physiological and biochemical characteristics of 10 Vicia faba genotypes (Zafar 1, Zafar 2, Shebam, Makamora, Espan, Giza Blanka, Giza 3, C4, C5 and G853) under drought stress. We studied the effect of different levels of drought stress i.e., (i) normal irrigation (ii) mild stress (iii) moderate stress, and (iv) severe stress on plant height (PH) plant−1, fresh weight (FW) and dry weight (DW) plant−1, area leaf−1, leaf relative water content (RWC), proline (Pro) content, total chlorophyll (Total Chl) content, electrolyte leakage (EL), malondialdehyde (MDA), hydrogen peroxide (H2O2) content, and activities of catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) of genotypes of faba bean. Drought stress reduced all growth parameters and Total Chl content of all genotypes. However, the deteriorating effect of drought stress on the growth performance of genotypes “C5” and “Zafar 1” were relatively low due to its better antioxidant enzymes activities (CAT, POD and SOD), and accumulation of Pro and Total Chl, and leaf RWC. In the study, genotype “C5” and “Zafar 1” were found to be relatively tolerant to drought stress and genotypes “G853” and “C4” were sensitive to drought stress. Results and Discussion In this study, the performance of different genotypes was evaluated in terms of plant height (PH) plant −1 , shoot fresh (SF) and shoot dry (SD) weight plant −1 , area leaf −1 , relative water content (RWC), total chlorophyll (Total Chl), proline (Pro) content, electrolyte leakage (EL), and content of malondialdehyde (MDA) and H2O2, and activity of catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD). The effect of DS treatments on these parameters of genotypes was found to be significant (Tables 1-4). The data reveal that growth performance of faba bean genotypes were affected significantly, depending on the level of water deficit (Table 1). In general, drought stress affected all growth parameters (PH, SF weight and SD weight plant, leaf area) of plants of all genotypes. Genotype "C5" exhibited the highest values for PH and leaf area as compared to the other genotypes under severe drought stress. At severe drought stress, genotype "G853" showed the lowest value for all growth parameters. However, genotype "C5" proved to be the best by giving highest values for all growth characteristics among nine genotypes, and genotype "G853", being at par with genotype "C4" for leaf area, had the lowest values for these parameters. A decrease in growth parameters may be due to the impairment of cell division, cell enlargement caused by loss of turgor, and inhibition of various growth metabolisms [18,19]. These results strengthen the findings of Ouzounidou et al. [20] on faba bean, Ali et al. [21] on faba bean; Farooq et al. [8] on rice and Asrar and Elhindi [22] on marigold, who reported that DS reduced plant growth characteristics. Among the cultivars, "C5" was found to be more tolerant by giving highest values for all growth characteristics in comparison to nine genotypes, and genotype "G853" was found to be sensitive to drought stress. Under drought stress, leaf RWC plays an important role in tolerance of plants to stress by inducing osmotic adjustment due to the accumulation of osmoprotectants [12,23,24]. The maintenance of a high plant water status during stress is an important defensive mechanism to retain enough water by minimizing water loss (e.g., caused by stomatal closure, trichomes, reduced leaf area, senescence of older leaves, etc.) and maximizing water uptake (e.g., by increased root growth) [12]. In the present experiment, Tables 2 and 3 depict that leaf RWC, Pro accumulation, Total Chl content, electrolyte leakage, and content of MDA and H2O2 of all genotypes were significantly affected by water stress. At increasing levels of drought stress, leaf RWC and Total Chl content decreased inversely. The differences in RWC in all genotypes could be associated with their ability of water absorption from soil. Thus, we conclude that genotype "C5" could have better ability to resist drought stress. According to Khanna-Chopra & Selote [25], under stress, the drought-resistant wheat plants exhibited better leaf water relations in terms of turgor potential and RWC as compared to sensitive genotypes. Genotype "C5", being at par with genotype "Giza 3", had the highest RWC under severe water stress conditions. Pro accumulation increased with increasing levels of water stress ( Table 2). The accumulation of Pro in plants reduces the toxic effects of ions on enzymes activity and also lowers the generation of free radicals formed by drought stress. Also, Pro associated with recovery resistance by serving a source of respiratory energy to the plants under stress [26]. Under severe DS, genotype "C5" gave the maximum value for Pro content, and genotype "G853", being at par with genotype "Giza 3", exhibited lower value for content of Pro. Also, genotype "C5", followed by genotypes "Zafar 1" and "Zafar 2", had the maximum value for Pro content (Table 2). Under severe drought stress, genotypes "Zafar 1", followed by genotypes "C5", "Giza 3" and "Makamora", gave the maximum value for Total Chl content (Table 2). This result strongly supports the findings of Ali et al. [21] in faba bean, and Mafakheri et al. [27] in chickpea genotypes. A decrease in Total Chl content may be due to the activity of chlorophyllase, a chlorophyll degrading enzyme [28]. Under drought stress, a low inhibition of Total Chl synthesis in genotypes Zafar 1', "C5" and "Giza 3" could be associated with better light harvesting efficiency, thereby improving dry matter production (Table 1). We observed that electrolyte leakage, and accumulation of MDA and H2O2 were found to be dependent on the severity of drought stress (Table 3). Genotype "C5", being at par with genotype "Zafar 1" for electrolyte leakage, genotypes "Zafar 1", "Giza Blanka" and Zafar 2 for MDA accumulation, gave the lowest values under severe condition of drought stress. Also, genotype "C5" showed the lowest content of H2O2 under water stress condition. All three oxidative stress indicators (electrolyte leakage, and accumulation of MDA and H2O2) were found to be almost lower in genotype "C5" and the highest in cultivars "G853", "C4" and "Makmora". These results agree with the findings of Ouzounidou et al. (20), Terzi and Kadioglu [29]; Ali et al. [21] and Quan et al. [30]. According to Jiang and Huang [31], accumulation of MDA affects the RWC and photosynthetic pigment of plants. Among the cultivars, "C5" had the lowest values for theses parameters. These results reveal that tolerance of genotype "C5" to drought stress could be positively related to leaf RWC and synthesis of Total Chl (Table 2). In general, activity of antioxidant enzymes (CAT, POD, and SOD) were significantly increased with increasing levels of drought stress in plants of all genotypes, (Table 4). Under severe water stress conditions, genotype "Shebam 1", being at par with genotype "C5", gave a higher value for CAT activity. However, the highest enzymes activities were noted in genotypes "C5" for POD and SOD at severe level of water stress. Genotype "Zafar 1" followed by genotype "C5" for the activity of POD and SOD. Moreover, the magnitude of increase in these enzymes' activity in genotypes "C5" was higher than other genotypes of faba bean under DS, except genotypes "Shebam 1" for CAT. Genotype "G853", followed by genotype "C4", exhibited the lowest enzyme activity under water stress. As we know, abiotic stress leads to the generation of reactive oxygen species (ROS: Superoxide anion radicals, hydroxyl radicals, H2O2, alkoxy radicals and singlet oxygen) that may react with a large variety of biomolecules-such as deoxyribonucleic acid, protein, lipids and carbohydrates-Causing lipid peroxidation linked membrane deterioration [32,33]. To overcome oxidative damage, plants develop an antioxidant system to scavenge ROS. In the present experiment, activity of antioxidant enzymes (POD, CAT and SOD) in plants of all genotypes increased under drought stress (Table 4). However, under DS, the highest enzymes activities were noted in genotypes "C5", "Zafar 1", and genotypes "G853" and "C4" exhibited the lowest value. Thus, it could be possible and reasonable to suggest that genotypes "C5" and "Zafar 1" were more tolerant than the other genotypes, because the maximum values for these enzymes' activity were recorded (Table 4). Plant and Treatment Seeds of 10 improved genotypes of Vicia faba L. were obtained from different geographical origins. Seeds of genotypes Zafar 1, Zafar 2 and Shebam from the General organization for Agriculture Research, Yemen, genotypes Makamora and Espan from the local market of Riyadh and genotypes Giza Blanka, Giza 3, C4, C5 and G853 from Agriculture Research Center, Egypt. The experiment was conducted in a growth chamber (temperature 25 ± 3 °C, relative humidity 50%-60%, light 250 μmol of photons m −2 ·s −1 on a 16/8-h light/dark cycle). Seeds were grown in pots containing a mixture of sand and peat (1:1). Drought stress was initiated when seedlings attained 2-3 true leaves. Drought stress treatments were imposed by withholding water. The details of the drought stress treatments were as follows: The leaf area was measured directly using Leaf Area Meter (Model LI-3050A, LI-COR Inc, Lincon, NE, USA). The area of three leaves (upper, middle, and lower) of each plant of the sample (consisting of five plants) was determined. Leaf Relative Water Content Leaf RWC (%) was determined using the methods of Gulen and Eris [35]. Leaf discs of 1.5 cm diameter were taken from the fully expanded and uniform leaves of each of the three plants (replicates) per treatment. First, the FW was recorded, and then samples were placed in a petri dish having distilled water for 4 h. Turgid weight (TW) was then recorded, and the leaf samples were placed in an incubator at 70 °C for 24 h, to determine the dry weight. Leaf RWC % was calculated by: Total Chlorophyll Concentration The youngest fully expanded leaves were subjected to extraction using 80% acetone, and the absorbance was measured using UV-vis Spectrophotometer (SPEKOL 1500; Analytik Jena AG, Jena, Germany) at 663 and 645 nm. The total chlorophyll content was determined by using Arnonʼs formula [36]. Proline Concentration The proline concentration was determined spectrophotometrically using the ninhydrin method of Bates et al. [37]. First, fresh leaf samples were homogenized in 3% sulfosalicylic acid, followed by the addition of 2 mL each of ninhydrin and glacial acetic acid, after which the samples were heated to 100 °C. The mixture was then extracted with toluene, and the free toluene was quantified at 520 nm. MDA Concentration The MDA content was determined according to the method of Heath and Packer [38]. Leaf samples were weighed, and homogenates containing 10% trichloroacetic acid and 0.65% 2-thiobarbituric acid were heated at 95 °C for 60 min, then cooled to room temperature, and centrifuged at 10,000× g for 10 min. The absorbance of the supernatant was read at 532 and 600 nm against a reagent blank. Electrolyte Leakage Electrolyte leakage was used to assess membrane permeability in accordance with Lutts et al. [39]. Samples were washed 3 times with double-distilled water to remove surface contamination, and leaf discs were cut from young leaves and placed in sealed vials containing 10 mL of DDW, followed by incubation on a rotary shaker for 24 h, after which the electrical conductivity of the solution (EC1) was determined. Then, the samples were autoclaved at 120 °C for 20 min, and the electrical conductivity was measured again (EC2) after the solution was cooled to room temperature. The electrolyte leakage was defined as EC1/EC2 × 100 and expressed as percentage. 3.2.6. Hydrogen Peroxide (H2O2) It was measured as described by Velikova et al. [40]. Fresh leaf samples (0.5 g) were homogenized in 5 mL of 0.1% (w/v) TCA. The homogenate was centrifuged at 12,000 rpm for 15 min and the supernatant was added to 10 mM potassium phosphate buffer (pH 7.0) and 1 M potassium iodide. The absorbance of the supernatant was recorded at 390 nm. The content of H2O2 was calculated by comparison with a standard calibration curve plotted using known concentrations of H2O2. Determination of Antioxidant Enzymes' Activity To determine the activities of antioxidant enzymes, a crude enzyme extract was prepared by homogenizing 500 mg of leaf tissue in extraction buffer (0.5% Triton X-100 and 1% polyvinylpyrrolidone in 100 mM potassium phosphate buffer, pH 7.0) using a chilled mortar and pestle. The homogenate was then centrifuged at 15,000× g for 20 min at 4 °C, and the supernatant was used for the enzymatic assays described below. All enzyme activities were expressed as milligram of protein per minute. We applied the method of Chance and Maehly [41] to determine POD (EC 1.11.1.7) activity using 5 mL of enzyme reaction solution containing phosphate buffer (pH 6.8), 50 M pyrogallol, 50 mM H2O2, and 1 mL of the enzyme extract diluted 20 times. The assay mixture was incubated for 5 min at 25 °C, and the reaction was terminated by the addition of 0.5 mL of 5% (v/v) H2SO4. Purpurogallin production was measured spectrophotometrically at 420 nm. One unit of POD activity was considered the amount of purpurogallin formed per milligram of protein per minute. The method of Aebi [42] was used to measure CAT (EC 1.11.1.6) activity. The decomposition of H2O2 was measured as the decrease in absorbance at 240 nm. In this assay, 50 mM phosphate buffer (pH 7.8) and 10 mM H2O2 were used in the reaction solution. Activity of SOD (EC 1.15.1.1) was determined based on the inhibition of nitro blue tetrazolium (NBT) photoreduction according to the method of Giannopolitis and Ries [43]. The reaction solution (3 mL) contained 50 mM NBT, 1.3 mM riboflavin, 13 mM methionine, 75 µM ethylenediamine tetraacetic acid (EDTA), 50 mM phosphate buffer (pH 7.8), and 20 to 50 mL of enzyme extract. The reaction solution was irradiated under fluorescent light at 75 µM·m −2 ·s −1 for 15 min. The absorbance at 560 nm was read against a blank (non-irradiated reaction solution). One unit of SOD activity was defined as the amount of enzyme that inhibited 50% of NBT photoreduction. Statistical Analysis The data were expressed as the mean ± standard error and were analyzed statistically using IBM SPSS Ver.22 statistical software (IBM Corporation and Others, Armonk, NY, USA). The means were compared statistically using Duncan's multiple-range test at the level of p < 0.05. Conclusions All morphological, physiological and biochemical characteristics of 10 genotypes of faba bean reduced under drought stress. We observed all genotypes of faba bean behaved differently under water stress. In the present study, we found some of the genotypes were tolerant ("C5" and "Zafar 1"), mild tolerant ("Giza 3", "Zafar 2", "Giza Blanka", "Espan", "Shebam 1" and "Makamora") and sensitive ("G853" and "C4") to water stress. However, genotypes "C5" and "Zafar 1" performed better by improving RWC and accumulation of Pro. Also, tolerant genotypes had a better ability to reduce oxidative damage by increasing activity of CAT, POD and SOD. For further study, theses genotypes can be used to uncover molecular mechanism(s) involved in building the tolerance of faba bean plants to drought stress.

v3-fos

2016-05-12T22:15:10.714Z

2015-12-14T00:00:00.000Z

7166627

s2

Community Structure of Arbuscular Mycorrhizal Fungi in Rhizospheric Soil of a Transgenic High-Methionine Soybean and a Near Isogenic Variety The use of transgenic plants in agriculture provides many economic benefits, but it also raises concerns over the potential impact of transgenic plants on the environment. We here examined the impact of transgenic high-methionine soybean ZD91 on the arbuscular mycorrhizal (AM) fungal community structure in rhizosphere soil. Our investigations based on clone libraries were conducted in field trials at four growth stages of the crops each year from 2012 to 2013. A total of 155 operational taxonomic units (OTUs) of AM fungi were identified based on the sequences of small subunit ribosomal RNA (SSU rRNA) genes. There were no significant differences found in AM fungal diversity in rhizosphere soil during the same growth stage between transgenic soybean ZD91 and its non-transgenic parental soybean ZD. In addition, plant growth stage and year had the strongest effect on the AM fungal community structure while the genetically modified (GM) trait studied was the least explanatory factor. In conclusion, we found no indication that transgenic soybean ZD91 cultivation poses a risk for AM fungal communities in agricultural soils. Introduction Modern agricultural biotechnology and genetic engineering have allowed the development of crops with improved properties, and global biotech crop hectarage has increased from 1.7 million hectares in 1996 to 181.5 million hectares in 2014 [1]. The field application of genetically modified plants (GMPs), however, might have undesirable consequences on the surrounding ecosystem, such as plant-beneficial soil microorganisms. Non-target organisms could be affected (a) by the products of the transgene itself; (b) by metabolites of the transgene products; or (c) by interaction with an altered plant phenotype [2,3]. Arbuscular mycorrhizal fungi (AMF) represent a potential key non-target organism to be monitored in studies on the ecological effects of GM crops [4]. Several previous studies on the impact of GMPs monitoring changes in the diversity of whole rhizosphere-associated fungal and bacterial communities have revealed either minor or no effect [3,5,6]. However, the total number of studies about the impacts of transgenic crops on AMF is rather low [7], especially about certain traits such as enhanced nutrition (e.g., increased methionine content). Plant-beneficial microorganisms are widely recognized as a crucial natural component of the fertility for agricultural soils. AMF are known to be involved in promotion of plant growth and health, and they are more sensitive to changes in the host plant than free-living soil fungi [7]. AMF, which belong to phylum Glomeromycota, can form mutualistic symbioses with the majority of land plants, including many crops (e.g., soybean) [8]. Verbruggen et al. [9] found that AMF comprise a large portion of soil fungal communities, and it should not be ignored in environmental risk assessment of transgenic crops. Previous studies have shown that GM plants can alter AM fungal development (e.g., delay the colonization, reduce pre-symbiotic hyphal growth, and affect the regular development of appressoria), suggesting that there is a potential for adverse transgenic plant-induced impacts on AMF [10,11]. Furthermore, Liu suggested more work is needed to be done to elucidate the impacts of GM crops on AM fungi [12]. As the methionine (Met) content limits the nutritional value of soybean, GM soybean ZD91 was engineered to enhance its Met content and increase its nutritional value [13]. Transgenic soybean ZD91 was reported to have no significant impact on rhizosphere bacterial community structure [5], but so far, the effect of transgenic soybean ZD91 on plant-beneficial AMF (which is a relevant indicator of microbe-plant symbioses in nutrient acquisition) is unknown [3]. Transient effects of GM crops (e.g., Bt maize, starch modified potatoes, and herbicide tolerant soybean) on soil AM fungal community structure or root colonization have been reported [10,[14][15][16]. As plants vary naturally in their AMF-hosting ability, the GM trait in plants might, in some cases, alter their relationship with AMF [7]. So whether transgenic soybean plants with altered content of methionine will affect AMF? Filion [17] suggested that risk assessment studies on the effect of GMPs should be conducted under field conditions over at least two years. Hannula et al. [16] suggested that it is important to consider the phenological growth stages of plants when assessing the effect of GM crops on the environment. Previous studies usually focused on one year or one growth stage, so the question remains: is there any effect of GM variety on diversity of the soil microbes over multiple years [18]? In our study, potential effects of transgenes introduced into soybean on AMF assessed by comparing communities found in the rhizosphere of transgenic and nontransgenic soybeans at various stages of crop growth and various planting years. Four growth stages of transgenic high-methionine soybean line ZD91 and its parental isoline ZD each year from 2012 to 2013 were included in this study allowing us to determine the long-term (years) and short-term (within growth season) effects of the transgenic high-methionine soybean on AMF community structure dynamics. Ethics statement This study was approved by the Ministry of Agriculture of the People's Republic of China and the genetically modified organisms safety team of Nanjing Agricultural University, China. The land was not privately owned or protected in any ways, and the field studies did not involve any endangered or protected species. Soybean Cultivars Transgenic soybean cultivar (ZD91) contains the Arabidopsis cystathionine γ-synthase (AtD-CGS) gene which has been introduced artificially into the soybean cultivar Zigongdongdou (ZD) using Agrobacterium-mediated transformation, and exhibits a high content of methionine in the seeds. This gene was expressed under the control of a seed-specific promoter legumin B4. Briefly, ZD is a soybean cultivar developed via the conventional breeding process, and it is an unmodified original plant. There are no significant differences on other agronomic traits (e.g., plant height, pod number per plant, seed number per plant, 100-seed weight, and yield) between transgenic plant (ZD91) and wild type line (ZD) [13]. Field setup and sampling This study was performed at Nanchong (30°48 0 N, 106°04 0 E), Sichuan Province, China, in which a completely randomized block design was set out in two consecutive growing seasons (2012-2013, i.e., the third and fourth years of the experiment) of soybean in the same field. The mean monthly temperature and rainfall during the experiment were provided by Nanchong Meteorological Bureau (Table 1). Each cultivar had four randomly distributed blocks, and the field was under standard agricultural practice. Irrigation, fertilization and field management were implemented according to the conventional methods in the area. Rhizosphere soil samples were collected in both years at four different growth stages [seedling stage (SS), flowering stage (FS), pod-setting stage (PS), and maturity-setting stage (MS)]. Collection of soil samples using sterile techniques, the samples were collected as described previously [5]. In brief, five soybean roots per block were removed from the soil by hand and taken to the laboratory. Loosely adhering soil on the roots was shaken off and discarded, and the more tightly adhering soil was then brushed off and collected. Rhizosphere soils from the five plants per block were mixed and used as a composite sample [5]. The rhizosphere soil samples were sieved using a 20-mesh sieve (aperture size is 830 μm) and then stored at -20°C until further use [19]. The main physical and chemical properties of the soil are provided in Table 2. Soil pH was measured with a pH meter (PB-10, Sartorius) using a 1:2.5 soil-to-water solution [19]. The concentrations of total carbon and nitrogen in rhizosphere soil were determined by Vario MICRO cube (Elementar, Germany) [5]. Root Colonization Root colonization is often used as an indicator of symbioses development [20]. 30 randomly chosen roots were carefully washed, cut into 1 cm long segments, cleaned in 10% KOH at 90°C for 20 min, acidified in 2% HCl for 5 min, and stained with 0.01% acid fuchsin [21]. The extent of mycorrhizal colonization was calculated according to the grid-line intersection method [22]. Soil DNA extraction and PCR The soil DNA was extracted from the rhizosphere soil samples by employing the MoBio PowerSoil DNA Isolation Kit (MoBio Laboratories, Inc., USA) with the method recommended by the manufacturer. DNA concentrations were quantified by using a NanoDrop 1000 Spectrophotometer (Thermo Scientific, USA) according to the manufacturer's protocol. Partial SSU rRNA gene fragments were amplified using nested PCR with the universal eukaryotic primers NS1 and NS4 [23]. PCR was carried out in a final volume of 25 μL [each reaction for PCR consisted of 2.5 μL 10 × PCR buffer (Mg 2+ Plus), 2 μL 2.5 mM dNTP, 0.25 μL each of 20 μM primers, and 0.125 μL rTaq DNA polymerase (5 U μL -1 ), to which were added 0.25 μL template DNA and then sterile distilled water to a final volume of 25 μL] and the cycling conditions were as follows: initial denaturation at 94°C for 5 min, then 30 cycles with denaturation at 94°C for 30 s, annealing at 56°C for 30 s, followed by elongation at 72°C for 90 s. The cycle was finalized by elongation at 72°C for 10 min. The PCR products were further amplified in the second step with the AM fungi general primer pair AML1 and AML2 (PCR system: see above; PCR conditions: 94°C for 5 min, then 30 cycles of 30 s denaturation at 94°C, 30 s primer annealing at 64°C and 1 min extension at 72°C, followed by a final extension period of 10 min at 72°C) [23]. Reaction yields were estimated by using a 1% agarose gel containing ethidium bromide. The PCR products obtained from DNA extracted from ZD and ZD91 soil samples were used to construct SSU rRNA gene libraries. Cloning and sequencing The PCR products of the expected size (~800 bp) were purified using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit (TaKaRa, Japan) and were cloned into pMD19-T Vector (TaKaRa, Japan). The vector was transformed into E. coli strain DH5α, which were spread on agar plates and incubated overnight at 37°C. Inserts from 80 randomly selected clones in each resulting SSU rRNA gene library (4 blocks × 4 growth stages × 2 years × 2 cultivars = 64 libraries) were sequenced using M13-f47 and M13-r48 primers on ABI 3730 genetic analyzer. The obtained sequences were trimmed to remove the vector sequence, and sequence similarities were determined using the Basic Local Alignment Search Tool for nucleotides (BLASTn) sequence similarity search tool provided by GenBank [24]. Sequences were clustered at 97% sequence similarity with QIIME [25] and a representative sequence from each OTU was picked for downstream analysis. Taxonomic assignments of OTUs were performed by using QIIME in accordance with SILVA 115 [26]. Representative sequences of the clones generated in this study were deposited at the National Centre for Biotechnology Information (NCBI) GenBank database under the accession numbers KJ740816 to KJ740970. Statistical analysis One-way analysis of variance and Duncan pair-wise comparisons (P < 0.01) were used to determine the minimum significant differences between soybean cultivars by employing SPSS version 17.0 for Windows [27]. In order to assess the efficiency of the clone library, the rarefaction curve was analyzed by the freeware program Analytic Rarefaction 1.3 of the Stratigraphy Lab, University of Georgia [28]. The percentage of coverage was calculated using Good's coverage equation [29], this calculation estimates how well the clones accounted for the biodiversity. Principal component analysis (PCA) was performed in R to compare AMF community structure across all samples [30]. ADONIS differences were calculated by using the vegan package in R [31,32]. ADONIS implements a multivariate analysis of variances using distance matrices and function ADONIS can handle both continuous and factor predictors. We used Jaccard as a distance index and 10,000 permutations. ADONIS analysis between cultivar, growth stage and year and the interactions among them were provided. Variance partitioning analysis (VPA) was also used to determine the contributions of cultivar, growth stage and year, as well as interactions between them on the variation in a AMF community structure with Hellinger-transformed data [31,32]. AMF diversity was calculated using the Shannon (H) index [24] using clone numbers of each OTU in each sample as fungal abundances. Richness of a given sample is all OTU numbers in that sample. Main physical and chemical properties of the soil, mycorrhizal colonization and AMF diversity Through the four different growth stages of each year, the soil pH fluctuated around 8.00, and there were no significant differences between soybean lines ZD and ZD91 ( Table 2). The effects of the transgenic soybean on C and N concentrations in rhizosphere soil were examined, and there were no significant differences between ZD and ZD91 (Table 2). We found that there were no significant differences in the intensity of root colonization by AMF between ZD and ZD91 within the same growth stage in 2012 and 2013, except PS stage in 2013 (Table 2). In general, AMF colonization increased over time, and the highest colonization rate was evident at the maturity-setting stage. The AMF diversity, expressed by the Shannon (H) index, was similar for ZD and ZD91 within the same growth stage in 2012 and 2013 (Table 2). PCR amplification of AMF SSU rRNA gene sequences from soil and diversity analyses In addition to AMF root colonization levels, it is crucial to check whether the composition of AMF communities is altered by GM plants [9]. A total of 5,120 clones from 64 libraries were sequenced and screened for the presence of AMF DNA (80 clones per library). GenBank BLAST search indicated that 2,838 clones were from Glomeromycota. These 2,838 AMF sequences were grouped into 155 OTUs at a 97% similarity threshold (S1 Table). Based on the result of rarefaction curves (Fig 1) and library coverage value calculations (Table 2), the number of clones analyzed was able to identify the major AM fungi in rhizosphere soil. Of the 155 AMF OTUs present in rhizosphere soil, 96 (47.04% of total clone sequences) were found in ZD, 103 (52.96% of total clone sequences) in ZD91, and 44 OTUs were shared between the two. Of the 155 OTUs, 58 were detected in 2012, 138 were detected in 2013, and 41 OTUs were observed in both years. There were no significant differences between AM fungal OTU richness in ZD and ZD91 within the same growth stage in 2012 and 2013 (Fig 2). However, the growth stage had a significant effect on AM fungal OTU richness in 2013, but not in 2012. In 2013, the OTU richness in the seedling and flowering stages of ZD was significantly lower than that in the other two stages; in contrast, the OTU richness in the flowering stage was significantly lower than that in the other three stages in ZD91. AMF community structure We applied PCA to compare AMF community structure between cultivar, growth stage and year (Fig 3). When the cultivar was examined as an explanatory variable, there were no significant differences found in the AMF community structure of ZD and ZD91 in the same year ( Fig 3A), but the community structure were clearly distinct for different years (Fig 3C). In 2013, an effect of the growth stage was observed as the flowering stage was separated from the other stages, however, this effect was not seen in 2012 (Fig 3B). We also calculated ADONIS differences between cultivar, growth stage and year and the interactions among them (Table 3). According to ADONIS, the AMF community structure was most strongly influenced by yearto-year variation and growth stage. Linking the AMF community structure to cultivar, growth stage, and year To quantify the relative contributions of cultivar, growth stage, and year to the total AMF community structure based on OTU composition, VPA was performed and the variation in AMF community structure was partitioned among cultivar, growth stage and year, as well as the interactions between them. These variables explained 12.80% of the observed variation, leaving 87.20% of the variation unexplained (Fig 4). Growth stage explained small portions of the observed variation, which accounted for 2.00% (P = 0.22), while the year accounted for 10.32% (P = 0.04) of the total variation. The variation was mostly explained by interactions between year and growth stage, which accounted for 12.75%. The interactions between year and cultivar, and growth stage and cultivar, accounted for 10.32% and 1.75% of the variation, respectively. The VPA analysis combined with ADONIS analysis and PCA analyses revealed that the year was the most important factor in explaining the shifts in the AMF community structure, while the cultivar was not. Furthermore, the AMF community structure was marginally related to the growth stage. Discussion The present study aims to assess the effect of transgenic soybean ZD91 on AM fungal community structure during two consecutive years (2012-2013). The results demonstrated that both plant growth stage and planting year impacted AMF community structure while the GM trait was the weakest explanatory factor. Our findings were consistent with those of others that evaluated the potential impacts of GM plants on AMF, indicating a transient or no impact of transgenic plants on the AMF [3,9,10,[14][15][16]18,[33][34][35][36][37][38][39][40][41], while three other studies have reported a significant effect of Bt maize on AMF [42][43][44]. Factors such as GM-parent, growth stage, field site, season, and cultivar were used to study the effects of GM crops on AMF [7]. Many studies revealed that the field site, the field season, the plant growth stage and year are important factors affecting AMF while GM-trait have transient or no effect [3,9,14,18,35,36,40,43]. Cotton et al. [45] found that the AM fungal community changes dramatically between years, and they were also suggesting that studies carried out during a single year may be difficult to interpret. In our study, the planting year was the largest explanatory factor, followed by plant growth stage (Table 3, Figs 2-4). Factors such as annual variation and plant growth stage are more influential on AMF community structure than the GM trait [7]. Growth stage-related differences of AMF communities in rhizosphere soil can be explained by the interaction between the abiotic environment and the host phenology [18,46]. Although we have not measured root exudates in this research, there is already evidence that plant growth stage can affect root exudate fluxes which in turn affect soil microorganisms [47,48]. We also detected interesting differences between the planting years. There were no significant differences in average temperature (F = 0.07, P = 0.80) and rainfall (F = 1.21, P = 0.31) between those two years. However, there were significant differences in total carbon content of soil (F = 41.40, P < 0.01) and soil pH (F = 59.04, P < 0.01) between those two years. AMF are strongly affected by changes in soil characteristics [18]. Meyer et al. [3] found that different environmental conditions (e.g., soil properties) occurring in different field seasons considerably affected soil microbial community structure. These factors mentioned above might explain the differences in AMF community structure we observed between 2012 and 2013. Of the 155 AMF OTUs, 58 were detected in 2012, 138 were detected in 2013. It may explain why only 41 were shared between 2012 and 2013. Wu et al. [19] found that the diversity of fungi might be affected by various environmental factors, such as temperature, humidity, and light. And it was showed that the AM fungal community changes dramatically between years [45]. A generalized AMF life-history begins with colonization of a root [49]. Previous study has demonstrated the importance of AM fungal colonization to plant growth and survival [50]. And recent survey of the relationship between soybean and AMF has focused on evaluating AMF root colonization [51]. One common method to quantify AMF abundance is root colonization measurements [52]. Arbuscular mycorrhizal fungi colonize plant roots and deliver many essential nutrients to the plant [53]. Previous studies have suggested that the plant age is an important factor influencing the AMF colonization percentage [3,54,55]. Our present results also indicated that the plant growth stage clearly had the strong effect on AMF colonization, while GM status did not significantly affect AM root colonization, except PS stage in 2013 (Table 2). Wu et al. [19] showed that the inconsistencies in the soil at seeding time may lead to the differences between transgenic and non-transgenic plant. Similarly, Gosling et al. [55] found that sampling time had a significant influence on colonization in soybean. Meyer et al. [3] found that the proportion of roots colonized by AM fungi increased from young to mature plants. The lack of significant yearly changes in root colonization in our study (F = 0.01, P = 0.94) is similar to that of White and Weil [56] and Naghashzadeh et al. [57]. This is similar to the finding by Meyer et al. [3] that AM root colonization was not significantly affected by GM wheat introduced with the Pm3b mildew resistance transgene. Likewise, Powell et al. [14] did not observe significant effect of the transgenic glyphosate resistant soybeans on AMF root colonization. However, Cheeke et al. [43] found a reduction in AMF colonization in multiple Bt maize lines. There is general agreement that GM crops should be assessed on a case-bycase basis [2,58,59]. In this study, the Shannon (H) index showed no significant differences in AMF species diversity between ZD and ZD91 within the same growth stage in 2012 and 2013 ( Table 2). In general, the most frequently detected AMF species in soybean fields belong to the Glomeraceae, Gigasporaceae, Acaulosporaceae, Diversisporaceae, and Paraglomeraceae families [28,55,60,61]. Glomeraceae, Claroideoglomeraceae, Diversisporaceae, Paraglomeraceae, Acaulosporaceae, and Archaeosporaceae were found in the present study. The interaction between host phenology, soil characteristics and habitat can lead to the changes of AM [46]. We postulate that soybean variety, field site, and climate are the underlying factor differentiating AMF phylotypes in these studies. In conclusion, our study examined the effects of a transgenic high-methionine soybean ZD91 on soil AMF community structure. The AMF community structure was strongly affected by natural variations in the environment related to planting years and soybean growth stages. Our results demonstrated that no significant differences in AMF community structure exist between high-methionine soybean ZD91 and its isoline ZD. Supporting Information S1 Table. Different OTUs for each sample. The samples were named in the following form: year-growth stage-cultivar-number of replicate. SS: seedling stage; FS: flowering stage; PS: podsetting stage; MS: maturity-setting stage. (XLS)

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2019-04-25T13:05:21.393Z

2015-01-01T00:00:00.000Z

131362237

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CONTRIBUTION OF INTEGRATED NUTRIENT MANAGEMENT PRACTICES FOR SUSTAINABLE CROP PRODUCTIVITY, NUTRIENT UPTAKE AND SOIL NUTRIENT STATUS IN MAIZE BASED CROPPING SYSTEMS Over applications of inorganic fertilizers lead nutrient imbalances, inefficiency and environmental contamination while insufficient application of nutrients causes soil fertility depletion. This problem drives the use of organic manures, which supply balanced micro and macro nutrients to the current crop and also leave a substantial residual effect on the succeeding crops in different cropping systems. But it is required in bulk as it contains nutrients in small proportion. Hence its availability is scarce for large farms. Therefore, to eliminate both excessive and inadequate applications, judicious use of integrated nutrient management is best alternative for sustainable crop productivity while maintaining soil fertility status in maize and other cereal based cropping systems. Such integrated applications have complementary and synergistic effects. Various research results have confirmed that INM improves sustainable crop productivity and soil fertility status rather than organic or mineral fertilizers alone. Most of research findings reviewed in this paper indicated that among the alternative integrated nutrient management combinations, application of chemical fertilizers integrated with organic manures in equal proportion significantly improved sustainable crop productivity, nutrient uptake and soil nutrient status in maize based cropping systems. In general, combined application of inorganic fertilizers with different sources of organic manures in different proportions has significant role to boost crop productivity, improve nutrient uptake by plants and maintain soil nutrient status in maize based cropping systems. INTRODUCTION Increasing the inputs of nutrients has played a major role in increasing the supply of food to a continually growing world population. However, over application of inorganic fertilizers causes inefficient use, large losses and imbalances of nutrients. It also leads to environmental contamination in a number of areas in developed world. On the other hand, insufficient application of nutrients and poor soil management, along with harsh climatic conditions and other factors, have contributed to the degradation of soils including soil fertility depletion in developing countries like Sub-Saharan Africa [1]. To replenish the soil nutrient depletion, application of chemical fertilizers is essential. However, high cost of chemical fertilizers coupled with the low affordability of small holder farmers is the biggest obstacle for chemical fertilizer use. Moreover, the current energy crisis prevailing higher prices and lack of proper supply system of inorganic fertilizers calls for more efficient use of organic manure, green manure, crop residues and other organic sources along with the inorganic fertilizers to sustain the yield levels [2]. Organic manures supply nutrients to the current crop and also leave a substantial residual effect on the succeeding crops in different sequential cropping systems. The efficiency of applied chemical fertilizers is also increased when applied along with organic manures. Therefore, better management of soil nutrients is required that delivers sustainable agriculture and maintains the necessary increases in food production while minimizing waste, economic loss and environmental impacts [1]. Various long term research results have shown that neither organic nor mineral fertilizers alone can achieve sustainability in crop production. Rather, integrated use of organic and mineral fertilizers has become more effective in maintaining higher productivity and stability through correction of deficiencies of primary, secondary and micronutrients [3]. Therefore, judicious use of integrated nutrient management is best alternative to supply nutrient to crop needs and improve soil conditions [4]. Integrated plant nutrient management (INM) is the combined use of mineral fertilizers with organic resources such as cattle manures, crop residues, urban/rural wastes, composts, green manures and bio-fertilizers [5]. Its basic concept is sustaining soil and crop productivity through optimization of all possible sources of plant nutrients in an integrated manner. In this system, all aspects of mineral and organic plant nutrient sources are integrated into the crop production system FAO (Food and Agriculture Organization of the United Nations) [6] and are utilized in an efficient and judicious manner for sustainable crop production [7]. It contributes in attaining agronomically feasible, economically viable, environmentally sound and sustainable high crop yields in cropping systems by enhancing nutrient use efficiency and soil fertility, increasing carbon sequestration, reducing nitrogen losses due to nitrate leaching and emission of greenhouse gases [3, 6]. Since cropping system serves as a component of INM for sustaining the productivity of the system through efficient nutrient cycling, balanced fertilization must be based on the concept of the cropping system to sustain productivity of a system as a whole rather than a single crop [5]. Intensified and multiple cropping systems require judicious application of chemical, organic and bio-fertilizers for yield sustainability and improved soil health [8]. Such integrated applications are not only complementary but also has synergistic effects. Therefore, the nutrient needs of crop production systems can best be met through integrated nutrient management [9]. The main objective of this paper is to review the contribution of integrated nutrient management practices on sustainable crop productivity, nutrient uptake and soil nutrient status in maize based cropping systems. ROLE OF INM ON CROP PRODUCTIVITY IN MAIZE BASED CROPPING SYSTEM Various studies revealed that sustainable yield and yield related parameters of maize are significantly improved by integrated nutrient management (INM) practices. Balanced application of NPK fertilizers with FYM and lime improved sustainable crop productivity and growth of maize [10,11]. Twenty years of experimental study, at Kathalagere, India, revealed that higher maize yields were observed with application 50% N through FYM and 50% NPK through inorganic fertilizers [2]. Study in Islamabad revealed that substitution of 25 or 50% N with FYM + 4 kg Zn/ha performed better grain and straw yield than 100% N (120kg/ha) from chemical fertilizer alone. Maximum maize grain yield (5.18 t/ha) was obtained with 75% chemical fertilizer (CF) + 25% Farm Yard Manure (FYM) and 4 kg Zn/ha, which was statistically at par with application of 50% CF + 50%FYM or 4 kg Zn/ha or 75% CF + 25% FYM and 8 kg Zn/ha [12]. Another study revealed that application of 50% organic manure (poultry and farm yard manure) along with 50% nitrogen from urea resulted in higher yield and yield components compared to either organic or mineral nitrogen alone. Application of mineral N and 50% poultry manure produced higher ear length, grain per ear, grain and biological yields of maize [13]. The results of Ahmad, et al. [14] showed that combining FYM with 50% of recommended NPK fertilizers produced the highest grain and biological yields of maize over the 50% NPK treatment and were statistically at par with those receiving 100% NPK fertilizers. Moreover, the net return was greatest when organic sources were combined with 50% of recommended NPK fertilizers. According to Mugwe, et al. [15], sole application of cattle manure at 60 kg N ha -1 and combined application of cattle manure (30 kg N ha -1 ) with inorganic fertilizer (30 kg N ha -1 ) gave significantly higher yields than the recommended rate of inorganic fertilizer. The highest grain and Stover yields (8.0 tons ha -1 and 8.9 tons ha, -1 respectively) of maize were recorded by the combined applications of 60 kg N ha -1 from poultry manure and mineral fertilizer at 60-40-40 kg ha -1 NPK compared to the unfertilized treatment which recorded the lowest grain and Stover yields of 2.10 tons ha -1 and 4.30 tons ha -1 respectively [16]. The research result of Ali, et al. [13] also showed that poultry and farm yard manure along with urea at equal proportion resulted in higher yield and yield components of maize than sole organic or mineral nitrogen ( [17]. Similarly, Cheema, et al. [18] found that applying 50% N from poultry manure and remaining from urea fertilizer produced maximum grain yield of maize (5.6 t ha -1 ), harvest index (24.91%) and grain weight per cob (68.98 g) compared to unfertilized treatment which gave the lowest harvest index (15.71%), grain yield (2.40 t ha -1 ) and weight per cob (44.53 g). Agricultural wastes alone or in combination with reduced rates of NPK fertilizer increased seed weight per plant, 100-seed weight, number of seeds per cob and grain yield of maize compared with un-amended treatment [19]. Combination of industrial by-products in 2:1 P ratio produced 14 to 27% more dry matter yield of 40-days old maize plants compared to the chemical fertilizer alone. Its residual effect on wheat following maize was also higher in straw and grain yield [20]. INM including vermicompost showed best results in yield parameters of maize like number of grains per cob, weight of the cob, 100 seed weight and yield [21]. Fanuel and Gifole [22] recommended to apply combination of compost at 5 ton ha -1 along with inorganic fertilizer (50 kg urea ha -1 + 100kg DAP ha -1 ) to obtain better yield of maize. Tithoniaapplied solely or in combination with inorganic fertilizer at 60 kg N ha -1 can be used as nutrient sources to meet N requirement for maize in smallholder farming systems maintaining maize yields at 4 to 6 t ha -1 . Though herbaceous legumes yielded the lowest maize yields, the increase in maize yield with application of herbaceous legumes compared with the control demonstrate that legumes make a significant contribution to maize crop production. Farmers can therefore be benefited by incorporating these legumes in the farming systems as an option to subsistence farming where farmers currently crop their farms without any inputs. Use of Tithoniacombined with chemical fertilizer was most effective in increasing maize yields [15]. (c) Potato yield after maize harvest (t ha -1 ) Figure-1. Productivity of maize-wheat and maize-potato cropping system under different nutrient management in North West India (the figure is drawn by the data taken from Naresh, et al. [4]. ROLE OF INM ON NUTRIENT UPTAKE OF CROPS IN MAIZE BASED CROPPING SYSTEMS Study in Islamabad showed that substitution of 25 or 50% N with FYM + 4 kg Zn/ha performed better nutrient uptake than 100% N (120kg/ha) from chemical fertilizer alone. The highest N uptake (98.7 kg/ha) was observed with 50% CF + 50% FYM and 8 kg Zn/ha application, while maximum Zn uptake (250.7 g/ha) was observed with 75% CF + 25 % FYM and 4 kg Zn/ha application [12]. Combined application of NPK mineral fertilizer and poultry manure has significantly higher NPK uptake values of maize than the sole organic and inorganic fertilizers. Integrated applications of 60 kg ha -1 N as poultry manure and mineral fertilizer at 60-40-40 kg ha -1 NPK resulted in higher NPK uptake values than either organic or inorganic fertilizers alone [16]. Integrated use of P sources not only increased crop yield but also increased nutrient uptake, protein content and P recovery efficiency in maize. The P recovery efficiency and NP uptake by maize following the application of poultry manure with inorganic P source showed higher values than those recorded by applying inorganic P sources alone indicating that integrated use of poultry manure with chemical P sources can save 30 to 40 kg mineral P fertilizer [25]. Integration of poultry waste and di-calcium phosphate in 2:1 P ratio significantly increased total P-uptake and P fertilizer use efficiency of maize by 30 to 66% over single supper phosphate alone. Its residual effect showed that P-uptake of wheat was higher by straw and was lower by grain in single supper phosphate alone but it was higher in grain and lower in straw with application of integrated use of industry byproducts which indicates that transportation of assimilates from source to sink was relatively higher and better utilized for grain production by integrated management. It was also observed that integrated use of nutrients increased Pfertilizer use efficiency from 2.8 to 59.7% over chemical fertilizer alone [20]. The results of Wakene Negassa, et al. [24] showed that the uptake of N and P was significantly increased from Mucuna as improved fallow and supplemented with low dose of NP fertilizers and FYM. The uptake of K was significantly low only in treatment received the recommended NP fertilizers (Table 2). ROLE OF INM ON SOIL NUTRIENT STATUS IN MAIZE BASED CROPPING SYSTEMS Different results reported that integrated nutrient management practices significantly improved macro and micronutrient status of soils in maize cropping system. Balanced application of NPK fertilizers with FYM or agricultural wastes improved the soil fertility status in addition to increase in maize yield [11,19]. As depicted in figure 3, organic matter content in INM superior than farmers' practice and initial soil organic matter content in maize-wheat and maizepotato cropping system [4]. Substitution of 25 or 50% N with FYM + 4 kg Zn ha -1 increased soil organic matter content [12]. Application of compost at 5 ton ha -1 along with inorganic fertilizers (50 kg urea ha -1 + 100 kg DAP ha -1 ) improved physico-chemical properties of the soil on sustainable basis rather than using inorganic fertilizer alone [22]. Similarly, twenty years of experimental study showed that application of 50% N through FYM and 50% NPK through inorganic fertilizers improved soil fertility status [2]. The soil analysis after maize crop harvest revealed that soil organic matter, total N, extractable P and K, were greatest from plots receiving organic sources with 50% of recommended NPK fertilizers, suggesting integrating organic sources with 50% of recommended NPK fertilizers are appropriate for sustainable crop production on a low fertility soil [14]. The availability of nutrients in soil were significantly high in organic and integrated nutrient management practices compared to chemical nutrient management practices at harvest of both kharifand rabi crops in maize based cropping system. At the end of the fourth year in the study, there was increase in available N, P2O5, K2O and S by 19.0, 46.3, 9.6 and 54.1%, respectively due to integrated nutrient management over its initial values. There was also significant build up of the micronutrient at the end of fourth year due to integrated nutrient management practices as

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Novel candidate genes AuxRP and Hsp90 influence the chip color of potato tubers Potato (Solanum tuberosum L.) tubers exhibit significant variation in reducing sugar content directly after harvest, cold storage and reconditioning. Here, we performed QTL analysis for chip color, which is strongly influenced by reducing sugar content, in a diploid potato mapping population. Two QTL on chromosomes I and VI were detected for chip color after harvest and reconditioning. Only one region on chromosome VI was linked with cold-induced sweetening. Using the RT-PCR technique, we showed differential expression of the auxin-regulated protein (AuxRP) gene. The AuxRP transcript was presented in light chip color parental clone DG 97-952 and the RNA progeny of the bulk sample consisting of light chip color phenotypes after cold storage. This amplicon was absent in dark chip parental clone DG 08-26/39 and the RNA bulk sample of dark chip progeny. Genetic variation of AuxRP explained up to 16.6 and 15.2 % of the phenotypic variance after harvest and 3 months of storage at 4 °C, respectively. Using an alternative approach, the RDA-cDNA method was used to recognize 25 gene sequences, of which 11 could be assigned to potato chromosome VI. One of these genes, Heat-shock protein 90 (Hsp90), demonstrated higher mRNA and protein expression in RT-qPCR and western blotting assays in the dark chip color progeny bulk sample compared with the light chip color progeny bulk sample. Our study, for the first time, suggests that the AuxRP and Hsp90 genes are novel candidate genes capable of influencing the chip color of potato tubers. Electronic supplementary material The online version of this article (doi:10.1007/s11032-015-0415-1) contains supplementary material, which is available to authorized users. Introduction Glucose and fructose are osmotically active substances that protect plants during low temperature stress or act as cryoprotectants during frosts (Stitt and Hurry 2002). In potato (Solanum tuberosum L.) tubers, Electronic supplementary material The online version of this article (doi:10.1007/s11032-015-0415-1) contains supplementary material, which is available to authorized users. the content of the reducing sugars immediately after harvest is generally low. During tuber dormancy, sugar accumulates as a result of starch breakdown (Sonnewald and Kossmann 2013). This process depends on the genotype and reflects the environmental conditions of plant growth, the harvest date and the storage regimen (Tai and Coleman 1999;Jakuczun and Zimnoch-Guzowska 2004). Temperatures of 4-6°C are the most favorable for tuber storage; however, these conditions stimulate the activity of amylolytic enzymes and the degradation of starch to sucrose. Sucrose can be transported into vacuoles or broken down into glucose and fructose. This phenomenon is known as cold-induced sweetening (CIS) (Isherwood 1973) and protects the plants from cold stress but is unacceptable to consumers. The content of reducing sugars in tubers for the processing industry should not exceed 0.07 % of fresh weight (FW) or 3.5 % of dry weight (DW) (McCann et al. 2010), because during processing reducing sugars participate in the Maillard reaction. The effect of the reaction can be seen as an undesirable brown coloration with an intensity that is proportional to the reducing sugar content. As a result, French fries and chips have an undesirable, bitter flavor. During CIS, the reducing sugar content can reach 2 % of the FW in tubers and significantly reduce their processing quality (Isherwood 1973). Therefore, low level of reducing sugars in potato tubers is one of the most important requirements in their processing worldwide. Several enzymes involved in starch synthesis and breakdown, glycolysis and hexogenesis have been identified and characterized at the biochemical and molecular levels in potato tubers (Sowokinos 2001;Solomos and Mattoo 2005;Lin et al. 2015). These data, together with knowledge about the intracellular partitioning of metabolites (Malone et al. 2006;Farre et al. 2008), has led to the conclusion that the biochemistry of carbohydrate metabolism in potato tubers is well studied. However, the genetic and metabolic regulation of this process is still unclear. Reducing sugars content in potato tubers is a quantitative trait, with heritabilities ranging from 0.47 to 0.98 (Cunningham and Stevenson 1963;Pereira et al. 1994;Jakuczun and Zimnoch-Guzowska 2004;Hamernik et al. 2009). Quantitative trait loci (QTL) analyses (Douches and Freyre 1994;Menendez et al. 2002;Werij et al. 2012) as well as the construction of the potato molecular function map (Chen et al. 2001) represent milestones in the application of a candidate gene approach to assess DNA variation of genes important for carbohydrate metabolism and transport in potato tubers. These findings revealed that different sets of genes may control the synthesis of reducing sugars in tubers at harvest, after cold storage and after reconditioning (controlled tuber warming). Recently, natural DNA variation in several genes that function in the starchsugar interconversion was found to be associated with tuber quality traits (Schreiber et al. 2014). The loci on chromosomes IX and X encoding apoplastic, cell-wall-bound isoforms of the acid invertase (Li et al. 2005(Li et al. , 2008Draffehn et al. 2010) and the locus on chromosome III encoding an intra-cellular, soluble acid invertase (Draffehn et al. 2010;Li et al. 2010Li et al. , 2013 were described as the most promising genetic factors associated with chip quality in tetraploid potatoes. The high flexibility of carbon metabolism depends on the physiological state, environmental conditions and sequence diversity. The DNA polymorphisms in the candidate genes (single nucleotide polymorphisms, insertion or deletion polymorphisms) with potential influence on their functions additionally complicate the identification of key genetic factors involved in cold sweetening. Comparative proteomics (Fischer et al. 2013) and mapping of expression QTL (e-QTL) (Kloosterman et al. 2012) have created new possibilities for the recognition of factors involved in the accumulation of reducing sugars in potato tubers. The objective of this study was to identify candidate genes that affect the chip color of potato tubers in interspecific Solanum diploid hybrids. We applied two alternative approaches to identify novel genetic factors. The first method was based on QTL mapping, followed by a reverse transcription polymerase chain reaction (RT-PCR). The second method was based on transcriptome investigation by representational difference analysis of cDNA (RDA-cDNA), followed by RT-quantitative PCR (RT-qPCR) and western blotting analysis. We have successfully used the RDA-cDNA technique for the identification of genes specifically expressed in a liverwort Pellia endiviifolia male and female thalli producing antheridia and archegonia, respectively (Sierocka et al. 2011(Sierocka et al. , 2014. Here, combining information from genetic studies and gene/protein expression data may yield a more accurate picture of genetic processes underlying complex traits than that obtained by using them separately (Pérez-Enciso et al. 2003). Plant material The diploid potato (S. tuberosum) parental clones DG 97-952 and DG 08-26/39 were crossed in 2010. Both parents were interspecific, multigenerational Solanum hybrids originating from S. tuberosum, S. acaule, S. chacoense, S. demissum, S. gourlayi, S. microdontum, S. phureja, S. stenotomum, S. verrucosum and S. yungasense. The theoretical input of the Solanum spp. to the genetic composition of the parents was calculated based on the pedigree data. The percentage of S. tuberosum in the origin of DG 97-952 and DG 08-26/ 39 were 67 and 68 %, respectively. Values for chip color after harvest of the parental clones DG 97-952 and DG 08-26/39 were 8.5 and 4.5, respectively. The parental clones and 92 F1 individuals (population 11-40) were used for DArT map construction and QTL analysis. Plants were first sprouted for 2 weeks in the sprouting chamber and then planted in tents where they were grown from May to October (average: 18 weeks). Three replications of the parents and each of the progeny were grown in the tents in 2011 (seedlings) and in 2012-2013 (first and second tuber generations). Chip color assessment The chip color for tubers of the parental clones and F1 individuals was evaluated in three subsequent years (2011, 2012 and 2013). Tubers harvested in 2011 were assessed in one batch 2 weeks after harvest (AH). Tubers harvested in 2012-2013 were fried in three batches each in three replications per genotype. The first batch was fried 2 weeks after harvest, the second batch 3 months after cold storage at 4°C (CS) and the third batch 2 weeks after reconditioning at 19°C (RC). For each replication, four slices of each of two potato tubers were fried. The frying color was visually assessed on a scale from 1 (dark) to 9 (light) as described by Jakuczun et al. (1995). Cultivars Pasat and Saturna were used as dark-and light-colored standards, respectively. Genetic mapping and QTL analysis Genomic DNA of parental clones and F1 individuals was isolated from leaves using the DNeasy Plant Maxi kit (Qiagen GmbH, Hilden, Germany) according to manufacturer's protocol. DArT analysis was performed using the Diversity Array Pty Ltd. Canberra, Australia, as described for S. michoacanum and S. ruiz-ceballosii by Ś liwka et al. (2012a, b), following the protocols previously developed for other plant species (Jaccoud et al. 2001;Wenzl et al. 2004). The genetic map was enriched in sequence-specific CAPS (cleaved amplified polymorphic sequence) and SCAR (sequence characterized amplified regions) markers (Supplementary Table 1). The PCR mixture (20 ll) contained 19 DreamTaq buffer, 0.25 mM of each dNTP, primers (0.25 lM), DreamTaq Polymerase (0.01 U, Fermentas) and DNA. The PCR parameters were initial denaturation at 94°C for 60 s, followed by 40 cycles of denaturation at 93°C for 25 s, annealing at 48-62°C (Supplementary Table 1) for 35 s and extension at 72°C for 90 s with a final extension at 72°C for 5 min. The PCR products were digested and visualized in a 1.2 % agarose/TBE (100 mM Tris-HCl, 83 mM boric acid, 1 mM EDTA, pH 8.4) gel containing 0.5 lg ml -1 ethidium bromide. Linkage analysis was performed as described previously using JoinMap Ò 4 (Van Ooijen, 2006) with the following settings: CP population type (first creating maternal and paternal linkage maps and then creating a common population map), independence LOD as a grouping parameter (linkages with LOD [ 3 were considered significant), regression mapping algorithm and Haldane's mapping function (Ś liwka et al. 2012a). The obtained linkage groups were oriented and named (chromosomes: I-XII) by comparison with earlier DArT maps of related species (Sharma et al. 2013;Ś liwka et al. 2012a, b). Interval mapping of the QTL was performed using MapQTL Ò 6 (Van Ooijen 2009). QTL with LODs equal or exceeding 3 were treated as significant. RNA isolation and construction of RNA and cDNA bulk samples RNA was isolated from tubers harvested in 2012 (first tuber generation) after 3 months of cold storage at 4°C. Parental clones DG 97-952 and DG 08-26/39 and ten F1 individuals, each in two replications, characterized by different chip color (5 with chip color 1-3 and 5 with chip color 8-9) were used for RNA isolation according to the protocol of Chomczyński and Sacchi (1987) using the TRIZOL reagent. Briefly, 1 g of frozen tubers were ground in liquid nitrogen prior to the addition of 4 ml of TRIZOL reagent. After incubation at room temperature and centrifugation, the supernatants were transferred to fresh tubes. The extraction was performed twice in 3 ml of chloroform. The RNA was precipitated 15 min after the addition 0.6 ml of salt solution (0.8 M sodium citrate and 1.2 M sodium chloride) and 0.6 ml of isopropanol. The RNA concentration and quality were determined using a biophotometer (Eppendorf) at 260, 280 and 230 nm. Two types of bulk samples were prepared. For bulk samples L I and D I , reverse transcription of RNA (2 lg) from each genotype was performed using the RevertAid kit (Fermentas) according to the manufacturer's protocol. The cDNA from five genotypes (in two replications) that were characterized by light chip color (color 8-9) and five that were characterized by dark chip color (color 1-3) were mixed together at equal amounts to form the bulk samples L I and D I , respectively. For the bulk samples L II and D II , equal amounts (60 lg) of the RNA progeny samples were mixed together to prepare the bulk samples. Expression of the AuxRP gene The cDNA of the bulk samples L I and D I was used for PCR amplification (Supplementary Table 1). The PCR mixture (20 ll) contained 19 DreamTaq buffer, 0.25 mM of each dNTP, primers (0.25 lM), Dream-Taq Polymerase (1 U, Fermentas) and cDNA. Water was used as a negative control. The PCR products were confirmed using electrophoresis in 1 % agarose/ TBE gels with ethidium bromide (Midi Horizontal Electrophoresis Unit Set, Thermo Scientific). All reactions were performed using the G-STORM thermal cycler (Gene Technologies, UK) and visualized using transilluminator UV (Vilber Lourmat, Germany). The PCR products for AuxRP (PGSC0003DMT400077929) when cDNA was used as a template were cloned in pGEM-T vector in the Laboratory of DNA Sequencing and Oligonucleotides Synthesis IBB PAS, Warsaw. After cloning, 96 clones were sequenced. Consensus sequences were obtained using blastn algorithm available at the Potato Genomics Resource platform (http://solanaceae.plantbiology. msu.edu). Sequence-specific primers were constructed based on the cloned sequences. Specific primers were developed as follows: AuxRP forward: 5 0 -AAGGCG GACGGAAAAGTAATCT-3 0 and AuxRP reverse: 5 0 -CAAGTTCAAGCAAGTCCATC-3 0 . The expression levels of AuxRP were measured on cDNA from the bulk samples L I and D I using the RT-PCR method. Experiment was repeated at least five times, and data obtained from representative, individual experiments were presented. Representational difference analysis of cDNA (RDA-cDNA) procedure The poly(A) ? RNA was isolated from 600 lg of total RNA from bulk samples L II and D II using the PolyATract mRNA Isolation System (Promega, No. Z5300) according to manufacturer's protocol. Doublestranded cDNA (ds-cDNA) was synthesized using the cDNA Synthesis System (ROCHE, No. 11117831001) according to manufacturer's protocol. The oligonucleotides utilized in RDA-cDNA were published by Hubank and Schatz (1994). The ds-cDNAs (1.5 lg) of the TESTER and the DRIVER were used to generate amplicons. RDA-cDNA was conducted in two directions, each in independent experiments: ds-cDNA from genotypes with a light chip color was used once as the TESTER and the second time as the DRIVER. Four rounds of subtractive hybridization (SH I -SH IV ) were performed using the quantitative TESTER to DRIVER ratios as follows: 1:100 for the first round, 1:800 for the second round, 1:400,000 for the third round and 1:1,000,000 for the final round. Hybridization was performed at 67°C for 48 h. After each round of hybridization, the RDA-cDNA products were separated by electrophoresis in 1.5 % agarose gels (Midi Horizontal Electrophoresis Unit Set, Thermo Scientific). The final product of the fourth hybridization round was sent to the Laboratory of DNA Sequencing and Oligonucleotides Synthesis IBB PAS, Warsaw, for cloning and sequencing. Sequences of the SH IV products were analyzed using the blastn algorithm available at the Potato Genomics Resource platform (http://solanaceae.plantbiology.msu.edu/). RT-qPCR analysis of the Hsp90 gene selected by RDA-cDNA Validation of gene expression derived after subtractive hybridization was performed using the RT-qPCR method. The same RNA isolated during the RDA-cDNA experiment (tuber after CS) was used. Two micrograms of each RNA sample from the parents and F1 individuals were reverse transcribed into cDNA using the RevertAid kit (Thermo Scientific, no. K1691) with Random Hexamer primers. Equal amounts of the cDNA of ten F1 individuals were mixed (two replications each; five characterized by light chip color and five characterized by dark chip color). For RT-qPCR, 50 ng of each bulk and parental genotype was collected. Analysis was performed using the LightCycler Ò 480 SYBR Green I Master (Roche), and DDC t values were calculated. Specific primer pairs were designed on the basis of the cloned amplicon Hsp90 (PGSC0003DMT400074377) derived from the RDA-cDNA experiment: forward primer Hsp90a 5 0 -GTTCCCTTGCTTTTTGAGAC CG-3 0 and reverse primer Hsp90a 5 0 -GGGAACTC CAATGCAGGCGTG-3 0 . a-tubulin was used as the reference gene with the following primers pairs: 57a-tubulin forward 5 0 -AATTTGTCGACTGGTGTC CT-3 0 and 57-a-tubulin reverse 5 0 -GTCAATGCGA GAGAAGACCT-3 0 (Śliwka et al. 2013). The following program was applied: denaturation at 95°C for 5 min; 40 amplification cycles of 95°C for 10 s, 65°C for 20 s and 72°C for 30 s. Then, PCR product melting was performed in a temperature range of 65-97°C, and the melting curve was analyzed to confirm the amplification of gene-specific products. A single peak on the melt curve analysis indicated the presence of the single PCR product. The results were expressed as relative expression levels from three independent biological experiments, with four replications in each set. Western blot analysis of Hsp90 Total proteins were isolated from tubers of the parental clones and ten genotypes differing in chip color (chip color 1-3 and 8-9) each in two replications (the same genotypes used in the RDA-cDNA and gene expression experiments) after storage at 4°C in 2012. Protein isolation was performed separately for each clone according to the protocol of Urbany et al. (2012). Briefly, 250 mg of potato tubers were ground in liquid nitrogen and extracted in extraction buffer (2 % SDS; 0.1 % Triton X-100; 10 mM EDTA; 25 mM DTT; 30 % sucrose; and 0.05 M TRIS/HCl, pH 8.0). After incubation on ice, the extract was treated with basic phenol. The upper phase was transferred to fresh tubes and washed with extraction buffer. Proteins were precipitated in 0.1 M ammonium acetate in methanol at -20°C. After centrifugation, the proteins were washed first with acetone, second with methanol, and then solubilized in a 2 M thiourea/7 M urea solution. Equal amounts of proteins from genotypes with light or dark chip colors were mixed and grouped into the protein bulk samples L II and D II . A total of 5 lg of total proteins from each sample were resolved by electrophoresis on a 10 % SDS-PAGE gel and subjected to electrotransfer onto a nitrocellulose membrane (GE Healthcare, 0.45 lm). Then, the nitrocellulose was incubated in 5 % non-fat dry milk powder in T-TBS buffer (0.05 M Tris-HCl, 0.15 M NaCl, and 1 % Tween, pH 7.6) for 1 h. After blockage of nonspecific protein binding, the nitrocellulose was incubated with the specific antibody anti-Hsp90-1 (Agrisera, No. AS08 346) or anti-Hsp90-2 (Agrisera, AS11 1629) in T-TBS with 2 % dry milk overnight (both diluted 1:3,000). Next, the membrane was incubated with a secondary antibody (goat anti-rabbit IgG, Agrisera, AS09 602) conjugated with horseradish peroxidase in T-TBS with 2 % dry milk diluted 1:25,000 for 2 h. For color development, DAB (3,3 0diaminobenzidine solution, Sigma Aldrich, No. D3939) was added to the membrane and incubated for 2 min. The Western analysis was performed for four independent experiments. Chip color after harvest, cold storage and reconditioning Chip color measured after harvest (AH), cold storage (CS) and reconditioning (RC) in the parents DG 97-952 and DG 08-26/39 was 8.5 and 4.5; 7.0 and 4.0; 6.7 and 5.3, respectively. The corresponding values for F1 individuals from mapping population 11-40 are presented in Supplementary Table 2. After harvest, 66 genotypes had chip color ranging from 4 to 6. Cold storage resulted in higher concentrations of reducing sugars in 79 % of genotypes. The most frequent genotypes (45 %) had chip color 3-4, with the simultaneous appearance of genotypes with chip color 1-2 (17 %). The number of genotypes with chip color 1-3 increased during RC (8 % of genotypes became darker after RC compared with CS). After reconditioning process genotypes with chip color scores 2-4 were the most frequent (71 %). The distribution of data was normal for chip color after AH and deviated from normality for CS and RC ( Supplementary Fig. 1). Major QTL for chip color are localized on chromosomes I and VI Among the 3331 DArT markers scored in population 11-40, 2089 markers were segregated (i.e., were present in more than 10 % and less than 90 % of progeny individuals). We excluded from analyses markers with more than 10 % missing data (172 markers) and those with unknown origins (parental clone data missing, 130 markers). Further markers were removed on the basis of the DArT quality parameter call rate\85 (3 markers), resulting in 1784 remaining markers. Markers with identical patterns of segregation were excluded by the JoinMap Ò 4 program. The final genetic map consisted of 1420 markers, including 1410 DArT markers and 8 CAPS and 2 SCAR markers. A total of 370 markers originated from parent DG 97-952 and 490 from DG 08-26/39, while 560 markers descending from both parents. The total length of the map reached 1000.2 cM. The numbers of markers located on particular chromosomes varied from 29 on chromosome IV to 226 on chromosome IX. The lengths of the chromosomes ranged from 55 (for VI) to 108 cM (for II) (Supplementary Fig. 2). The QTL for chip color were detected on chromosomes I and VI (Table 1, Supplementary Fig. 3). The QTL on chromosome I was significant in the AH and RC datasets, while the QTL on chromosome VI was significant for all three datasets (AH, CS and RC). The QTL for chip color after CS was detected only on chromosome VI. The effect of the QTL on the mean chip color after AH reached 17.5 % (LOD 3.84) of the variance explained by the QTL on chromosome I and 18.3 % (LOD 4.03) by the QTL on chromosome VI. Marker alleles on chromosomes I and VI affecting the trait descended from DG 08-26/39. The major QTL for mean chip color after CS was mapped onto chromosome VI between 71.1 and 83.7 cM and explained up to 23.5 % of the variation. The most significant QTL for chip color after RC was detected on chromosome VI RC12 and explained 24.0 % of the variance (LOD 5.47); this QTL descended from DG 97-952 and DG 08-26/39. The impacts of the QTL on mean chip color after RC on chromosome I and VI was 17.5 % at LOD 3.83 and 3.84, respectively. The QTL effect varied between data sets over the time course of the study, indicating the significance of the environment during growing season and genotype 9 environment interaction on the sugar metabolism later in the storage. Most of the QTL for chip color after CS on chromosome VI were significant in all data sets (CS12-CS13 and mean CS) (Table 1), indicating that this trait was stable in different vegetation seasons. The QTL on chromosome I was significant in three out of four datasets obtained after harvest (AH12, AH13 and mean AH), while the QTL on chromosome VI was detected in a different combination of three AH datasets (AH11, AH12 and mean AH). The least stable trait was chip color after RC; the significant QTL were detected only for the mean RC on chromosome I and RC12 and mean RC on chromosome VI (Table 1). CAPS and SCAR markers on chromosome VI The genetic map of the DArT markers was enriched with eight CAPS and two SCAR markers which were selected from the QTL region on chromosome VI (Supplementary Table 1), of which nine were developed based on DArT sequences or gene sequences available at the Potato Genomics Resource (PGSC) platform within chromosome VI. One DNA marker for the locus Hsp90 was developed using the sequences identified in the RDA-cDNA experiment. Eight and two markers originated from DG 97-952 and DG 08-26/39, respectively. Five of the markers showed linkage to chip color in at least two datasets. The DNA markers AuxRP, Chaperone DnaJ, Myb48d and Zinc were linked with chip color in the AH and CS samples, while the marker Nod was linked with chip color in the CS and RC samples. The markers 965p2, Myb48 g and pPt874a were significantly linked only to CS. The most significant CAPS markers for chip color AH were derived from the loci AuxRP and Myb48d; the same phenotype effects explained 16.6 % of the variance (LOD 3.62). For CS, Nod explained 20.7 % of the variance (LOD 4.64); moreover, it was the only marker allele with significant effect on RC, where it explained 14.8 % of the phenotypic variance (LOD 3.20) (Table 2). AuxRP expression The expression of four genes mapped onto chromosome VI. Chaperone DnaJ, Myb transcription factor (Myb48), zinc finger protein (Zfp) and auxin-regulated protein (AuxRP) was assessed using the RT-PCR technique. Only one gene (AuxRP) was shown to be expressed differentially in the light and dark chip samples after CS. The specific amplicon was 660 bp long and was expressed only in DG 97-952 and bulk L I with light chip color phenotypes after cold storage (Fig. 1). Linkage to a QTL for chip color and differential expression indicated that AuxRP may be a candidate gene influencing the chip color of potato tubers. Evaluation of differentially expressed genes The RDA-cDNA technique was employed to identify candidate genes responsible for reducing sugar accumulation in potato tubers stored at 4°C. The cDNA obtained from mRNA isolated from genotypes with light chip color after CS was used once as the TESTER (L II ) and once as the DRIVER (D II ). After the fourth round of hybridization (SH IV ), two distinct bands at 250 bp and 300 bp were obtained from the L II and D II samples, respectively (Fig. 2). The cDNA fragments were cloned and sequenced. Of the 109 clones from L II and D II , 41 different sequences were obtained, in which 24 came from bulk L II and 17 from bulk D II (Supplementary Table 3). Among them, 16 and 9 sequences were assigned to protein coding genes in the bulk samples L II and D II , respectively. Sequences that could be located to chromosome VI represented 27 % of the gene sequences obtained from the RDA-cDNA (Supplementary Table 3). To confirm which of these genes were characteristic for bulk L II and D II , RT-PCR Table 1 QTL detected for mean (all years of testing) chip color after harvest (AH), cold storage (CS) and re-conditioning (RC) as well as in particular seasons 2011-2013 (e.g., AH11-AH13) in the diploid potato population 11-40 Interval mapping of QTL was performed using MapQTL Ò 6 (Van Ooijen 2009) a P1-inherited from DG 97-952; P2-inherited from DG 08-26/39 was performed with the same RNA template used for the RDA-cDNA experiment. Hsp90 gene expression and Hsp90 protein expression Among the candidate genes selected in the RDA-cDNA experiment, we found that only Hsp90 was informative and differentially expressed in bulk samples L II and D II . The cDNA sequences of Hsp90 found in bulk samples L II and D II were 240 bp and 154 bp To confirm this finding and determine which of these alleles were characteristic for bulk L II and D II , specific primer pairs for both sequences were designed. Expression of Hsp90 (originated from bulk D II ), determined by RT-qPCR, was not significantly different in the parents DG 97-952 and DG 08-26/39. However, the expression level of Hsp90 in bulk D II was significantly higher than in bulk L II (Fig. 3a). The protein expression levels of the Hsp90-1 and Hsp90-2 isoforms were detected by western blotting. Hsp90-2 is the constitutive isoform, and its expression did not vary between the parental clones and the bulk samples (Fig. 3b). Hsp90-1 is the isoform involved in responses to biotic and abiotic stresses (Kadota and Shirasu 2012;Xu et al. 2012). In this case, the amounts of the protein in the parental clones were similar. The amount of Hsp90-1 protein in bulk D II was significantly higher compared with bulk L II (Fig. 3b). Bulks L II and bulk D II were constructed for tubers consisting very low (chip color 8-9) and very high (chip color 1-3) content of reducing sugars, respectively (Fig. 3a). Thus, the higher expression level of stress-induced Hsp90-1 isoform as well as the higher expression of Hsp90 gene in bulk D II indicates that this gene may have an effect on reducing sugar content in potato tubers. Discussion Between two and six QTL for chip color have been detected in previous studies. Six QTL for chip color (two on chromosome II, one on IV, two on V, and one on X) were identified in a population of the diploid hybrid of S. tuberosum and S. chacoense after storage at 10°C for 45 days (Douches and Freyre 1994). Menendez et al. (2002) revealed six QTL explaining more than 10 % of the variability in reducing sugars that were located on chromosomes I, III, VII, VIII, IX, and XI in diploid S. tuberosum tubers stored at 4°C for 3 months. Recently, two (on linkage groups IX and X), four (on linkage groups III, V, VIII and X) and two (on linkage groups V and X) QTL for chip color were detected in diploid potato clones of S. phureja, S. vernei and S. tuberosum origin after harvest, cold storage and reconditioning, respectively (Werij et al. 2012). In our study, two QTL on chromosomes I and VI that were linked with chip color were detected both at harvest and after reconditioning. We identified only one QTL for chip color after cold storage, located at the end of the long arm of chromosome VI. From three previous chip color/reducing sugar QTL linkage mapping studies (Douches and Freyre 1994;Menendez et al. 2002;Werij et al. 2012), one (Menendez et al. 2002) has reported 3 sugar QTL on chromosome VI: Sug6a, Sug6b and Sug6c. In the recent paper (Schreiber et al. 2014), invertases and hexokinase have been proposed to contribute to the effect of chromosome VI on chip color. The genes Zfp, Myb48, Nodulin 26 (Nod) and Chaperone DnaJ that were mapped to chromosome VI showed QTL effects at different significance levels in population 11-40. They accounted for 14.7-20.7 % of the phenotypic variance. Myb48 is a member of the Myb superfamily of sequence-specific transcription factors; the members of this family are recognized as Fig. 3 a Expression level of the Hsp90 gene in tubers after cold storage (CS); ns non-significant differences between parental clones (Student's t test); a and b homogenous groups between bulk samples L II and D II (Tukey's test). b Amounts of Hsp90-1 and Hsp90-2 protein isoforms in tubers after cold storage (CS); Marker-Prosieve Ò QuadColor TM Protein Marker. Parental clones: DG 97-952 (chip color 7) and DG 08-26/39 (chip color 4); bulk samples: L II (chip color 8-9) and D II (chip color 1-3) genetic factors associated with increasing tolerance to abiotic stresses in plants (Dubos et al. 2010;Shin et al. 2011). Nod encodes an aquaporin involved in selective water transport that allows the regulation of osmotic pressure and adjustment of osmotic potential. Eight aquaporins have been localized on potato chromosome VI, including the gene St-TIP1;2 (Venkatesh et al. 2013). This gene corresponds with the map position of Nod detected in our study. The gene Zfp belongs to a large family of transcription factors that can play various regulatory roles in plants, including abiotic stress responses (Giri et al. 2011). We used two alternative approaches (QTL studies and transcriptome analysis) to identify the new candidate genes AuxRP and Hsp90. Auxin-regulated proteins are involved in the regulation of the auxin transduction pathway and take part in the response to abiotic stresses. Genes encoding these proteins have also been recognized as stress-responsive (Ghanashyam and Jain 2009; Wu et al. 2012). In our study, the AuxRP gene explained up to 16.6 and 15.2 % of the phenotypic variance in chip color AH and after CS, respectively. Moreover, AuxRP was expressed only in tubers with light chip color phenotypes. Heat-shock proteins (Hsp) are a large family of molecular factors involved in many chaperoning functions in plants (Xu et al. 2012). In potatoes, significant differences in protein expression of the chloroplast small heat-shock protein class I, heatshock protein 70 (Hsp70) and 101-kDa heat-shock protein were detected in tubers between CIS-tolerant and CIS-sensitive cultivars before and after cold storage (Fischer et al. 2013). In microarray experiments, a group of small Hsp genes was recognized as cold-linked genes in potato tubers (Bagnaresi et al. 2008). These data are consistent with our finding that a marker derived from the Chaperone DnaJ gene encoding the small Hsp40 factor accounted for up to 16.1 and 17.8 % of the chip color after harvest and cold storage, respectively (Table 2). Hsp90 is a highly conserved and essential molecular chaperone that is also involved in abiotic stress responses (Kadota and Shirasu, 2012;Xu et al. 2012). In the present study, we show for the first time increased Hsp90 mRNA expression and Hsp90 protein expression in the dark chip color progeny bulk samples in comparison with the bulk samples of light chip color phenotypes. Hsp90 was mapped to potato chromosome VI at 30.0 cM. We did not find a significant association between allelic variation in Hsp90 and the phenotype (Table 2). However, the gene expression may be regulated by transcription factors or other regulatory elements/ proteins located next to or far from the gene that act in cis or trans configurations (Rockman and Kruglyak 2006). In studies on the response to iron deficiency in soybeans (Glycine max L.), only 58 of the 835 (7 %) candidate genes identified in the microarray experiment were mapped within known QTL regions (O'Rourke et al. 2009). Therefore, we suggest that both AuxRP and Hsp90 genes are novel candidate genes capable of influencing the chip color of potato tubers.

v3-fos

2017-06-29T13:19:49.388Z

2015-07-22T00:00:00.000Z

337727

s2

Development of transgenic wheat (Triticum aestivum L.) expressing avidin gene conferring resistance to stored product insects Background Wheat is considered the most important cereal crop all over the world. The wheat weevil Sitophilus granarius is a serious insect pests in much of the wheat growing area worldwide and is responsible for significant loss of yield. Avidin proteins has been proposed to function as plant defense agents against insect pests. Results A synthetic avidin gene was introduced into spring wheat (Triticum aestivum L.) cv. Giza 168 using a biolistic bombardment protocol. The presence and expression of the transgene in six selected T0 transgenic wheat lines were confirmed at the molecular level. Accumulation of avidin protein was detected in transgenic plants compared to non-transgenic plants. Avidin transgene was stably integrated, transcribed and translated as indicated by Southern blot, ELISA, and dot blot analyses, with a high level of expression in transgenic wheat seeds. However, no expression was detected in untransformed wheat seeds. Functional integrity of avidin was confirmed by insect bioassay. The results of bioassay using transgenic wheat plants challenged with wheat weevil revealed 100 % mortality of the insects reared on transgenic plants after 21 days. Conclusion Transgenic wheat plants had improved resistance to Sitophilus granarius. Background Wheat is the first most important field crop worldwide in terms of crop value and total production. Wheat production is limited by both biotic and abiotic factors [1]. Insect infestations are major factors for post harvest loss of grain quantity and quality. Preventing or at least slowing the stored product infestation is important in maintaining wheat's quality and marketable volume [2]. Insect pests contaminate stored cereals and cause damage by their feeding, producing highly toxic and carcinogenic compounds, and creation of allergens. Additionally, their metabolic products change the smell and taste of the contaminated baking products [3]. There is an urgent need to eliminate wheat insect infestation in stores, during transportation and processing, which ensures a supply of wholesome food to the consumers [4]. Chemical insecticides are being used to reduce the negative impact of the transmission of the insect infestations [5]. Complete dependency on the heavy use of chemicals has created numerous unacceptable agricultural, environmental and human health problems. Another concern is the development of resistance in target organisms [6,7]. These factors have encouraged the scientific community to discover alternative, bio-friendly, economically acceptable strategies for insect control [8][9][10]. Considerable progress has been made over the past two decades in manipulating genes from diverse sources and inserting them into crop plants to confer resistance to insect pests, diseases, tolerance to herbicides, drought, improved nutritional quality, increased effectiveness of bio-control agents, and a better understanding of the nature of gene action and metabolic pathways [11][12][13][14]. Transgenic biotechnology can be utilized as an alternative choice for protection of crops from attack by insect pests using insect growth-inhibiting proteins e.g., insect chitinase [15], cry protein [16], vip3A protein [17] and avidin protein [18]. If genes coding for these proteins are introduced into wheat with adequate and stable expression, they can provide resistance against stored product pests over several generations. Avidin is a glycoprotein that binds biotin strongly and prevents acquisition of biotin by many organisms [19]. Biotin is a cofactor needed during important carboxylation reactions. Insects have no biosynthetic pathway for biotin and thus, must obtain it from other sources. Therefore, diets containing avidin are toxic to a wide range of insects [20]. The molecular weight of avidin is about 67 kDa. The insecticidal effect of chicken avidin has been known since 1959 when it was first reported that the protein is toxic to the housefly, Musca domestica [21], this effect is due to the strong binding of avidin to biotin [22]. Due to its insecticidal properties, avidin has been expressed in a variety of agriculturally important plant species, for example, tobacco, maize and rice [18,19]. Avidin differs from other transgenic insecticidal toxins because it is not directly damaging to tissues, rather it merely withholds an essential nutrient from the insects [23]. The biotin binding activity of avidin is greatly destroyed by cooking, rendering the avidin harmless to humans following cooking. In the same way that cooked eggs (or precisely egg white) are not harmful to humans and are considered as a normal component of many people's diet [24]. Ninety seven percent of avidin's functional activity is lost by heat denaturation (i.e. cooking) at 95°C for 5 min. In addition, avidin has the considerable advantage over conventional insecticides that it is not washed away during processing and continues to act as an insecticide during storage [20]. In this study, we introduced a modified avidin gene into wheat in order to protect it from stored product insects, and we investigated the insecticidal activity of transgenic wheat against wheat weevil. Insect Wheat weevil Sitophilus granarius were obtained from the insectary at the Agricultural Genetic Engineering Research Institute (AGERI) -ARC-Egypt. Construction of recombinant genes The synthetic avidin gene including Barley alpha amylase signal peptides was designed as outlined by [20]. Codon bias was checked through the codon usage table of wheat (NCBI-GenBank, http://www.kazusa.or.jp/codon). An 496 bp EcoRI fragment containing the full-length synthetic avidin (synthesized by Bioneer co., http:// eng.bioneer.com) was filled using Klenow fragment and blunt-end ligated to the previously digested and filled BamHI site of pAHC17 [25].The 2100 bp HindIII fragment containing bar gene cassette with the ubiquitin promoter was at the HindIII site of pAHC17 ( Fig. 1). Wheat transformation Immature embryos were isolated from field grown bread wheat (Triticum aestivum L.) cv. Giza 168(G168).Tissue culture and transformation systems were performed [26]. Primary transformants were transferred to the biocontainment facility and tested using leaf painting assay with a 0.1 % aqueous solution of Basta™ (Bayer Crop Science PVT Ltd) containing 20 % glufosinate ammonium. Polymerase chain reaction (PCR) Genomic DNAs were extracted from putative transgenics plants, resistant to the herbicide Basta, as well as the wild type control, using the CTAB method [27]. PCR was performed by the amplification of avidin gene (496 bp) as well as for the bar gene (400 bp) using specific primers ( Table 1). Southern blot analysis Genomic Southern blot analysis [28] was carried out using DIG High Prime DNA Labeling and Detection Starter Kit II (Roche cat. No. 11 585 614 910) for the six selected T 0 transgenics. Genomic Southern analysis of the six avidin transgenic plants AVD1, AVD2, AVD3, AVD4, AVD5 and AVD6 . Genomic DNA of each line was digested with NcoI and SpeI and fragmented by 0.8 % agarose gel electrophoresis. The blot was probed with a SpeI and HindIII fragment involving a maize ubi intron1, maize ubi promoter. Ubi is 2.08 kb. RT-PCR Total RNA was extracted from transgenic as well as control non-transgenic plants using total RNA isolation system (Trizol reagent -Sigma-Aldrich, USA). Expression of the integrated transgene was tested on the RNA extracted from T 1 plants that showed positive results after being sprayed with the Basta herbicide using a semiquantitative RT-PCR according to the protocol described by Eltayeb et al., [29]. RT-PCR was performed with RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific TM , cat. No. K1631) using avidin and actin reverse primers, separately. The reactions were followed with PCR reaction using GoTaq Flexi DNA polymerase (Promega, USA) identical to those used for the PCR analysis to generate an expected product size of 496 bp. was used as a house keeping gene to normalize the initial variations in sample concentration. Actin was amplified using forward primer: 5' TGA CGT GGA TAT CAG GAA GG 3' and reverse primer 5' GCT GAG TGA GGC TAG GAT GG 3' to generate expected product at196 bp, and the avidin primer was used as mentioned above. PCR conditions were: initial denaturation at 94°C for 3 min; 40 cycles of 94°C for 10 s, 58°C for 30 s and 72°C for 15 s; elongating at 72°C for 10 min. ELISA and dot blot analysis ELISA performed with average of four replicates per samples according to the procedure of Clark et al., [30] and Dot blot analysis was performed as outlined in Gil et al., [31] to confirm the presence of the avidin protein in the grains of the T 2 transgenic plants using antiavidin rabbit whole serum (Sigma, USA, Cat NO. A5170). Two μg of avidin protein (Sigma-Aldrich cat. A9275-1MG) was used as a positive control. Insects Bioassay An insect feeding bioassay experiment was conducted according to the method described earlier [19,20] using three replicates of crushed seeds of transgenics and nontransgenic plants. Every replicate contained 100 mature insects of Sitophilus granarius for 21 days at 25-30°C. Bioassay was repeated 3 times and mortality was scored daily until death or pupation. Results This study aimed to introduce the synthetic avidin gene into immature embryo-derived calli of an Egyptian hexaploid bread wheat (spring wheat cv. Giza 168), and to investigate its ability to confer insecticidal effect on wheat plants via microprojectile bombardment using a eukaryotic expression vector containing the bar gene as the selectable marker. The modified avidin gene sequence that was used in our transformation experiments was synthesized according to a report [20] where the same sequence in the transformation of rice (Oryza sativa) plants was used. Molecular Analysis Construction of the plant expression vector pAHC17/avidin/ bar harboring the avidin gene The plant expression vector used in this study was pAHC17/avidin/bar harboring the modified avidin gene [20] under the control of Zea mays ubiquitin promoter and bar gene under the control of CaMV 35S promoter. PCR was performed using specific primers of the two transgenes (bar and avidin) using DNA extracted from the T 0 transgenic plants that were scored for resistance to the Basta herbicide, in order to confirm the presence of both genes in their genomic DNA. From the results reported in Fig. 2a and b, both transgenes are present in the genomic DNA of the six putative transgenic plants. Figure 2a shows the PCR product corresponding to the expected size of the partial-length bar gene amplification product (400 bp) and Fig. 1b shows the PCR product corresponding to the expected size of the full-length avidin gene (496 bp). Southern blot analysis Total genomic DNA isolated from leaf tissue of plants that tested positive in the leaf painting assay with the herbicide and these that tested positive in PCR analyses of putative transgenics as well as the non-transgenic wheat cultivar Giza 168 as negative control were digested with the restriction enzyme mixture of SpeI and NcoI. The digestion products were subjected to electrophoresis followed by southern blot analyses and hybridized with the HindIII/SpeI restriction fragment (2.08 kb) of the pAHC17/avidin/bar plasmid, which was used as a probe to confirm the integration of pAHC17/ avidin/bar construct in the wheat genome. Figure 3 shows the hybridization patterns of the probe with genomic DNA of the six putative transgenics (AVD1, AVD2, AVD3, AVD4, AVD5 and AVD6) and the negative control (G 168). As expected, a fragment with expected size (~1 kb) was detected from genomic DNA of the six transgenics and was completely absent from the non-transgenic wheat cultivar (G 168). Different insertion sites patterns (variable bands sizes per well) confirms that the six transgenic events were independently originated from multiple independent embryogenic calli representing different integration events. In addition, the numbers and sizes of bands indicated different copy numbers of the transgene avidin. For example AVD1 has four copies and AVD5 has two copies and AVD2 has one copy. Semi-quantitative Reverse transcriptase-polymerase chain reaction (SqRT-PCR) analysis: Expression of the integrated gene was tested on the RNA extracts of the T1 plants that showed positive results after being sprayed with the Basta herbicide. The results of avidin gene expression indicated the presence of the expected cDNA band size 496 bp (Fig. 4). SqRT-PCR detected differential expression levels between the six transgenic lines. The transcription level of avidin in the AVD1, AVD4, AVD5 and AVD6 is shown to be relatively higher than those of AVD 2 and AVD3 lines. Protein Analysis Protein dot blot assay Avidin protein in the grains of transgenic plants, which were resistant to the Basta herbicide and Giza 168 nontransgenic cultivar was used as negative control. The six transgenic lines AVD1, AVD2, AVD3, AVD4, AVD5 and AVD6 (Fig. 5) expressed the avidin protein as well as the positive signal of the authentic avidin, while the nontransgenic cultivar, Giza 168, showed negative signal. The ELISA was performed on the grains from T 2 plants to quantify the relative amounts of the avidin protein in each of the six transgenic events (Table 2). AVD1, AVD5 and AVD6 had the highest absorbance values which refer to the highest avidin protein concentration (1.5 and 1.4), then AVD4 (1.2) and finally AVD2 and AVD3 giving the lowest absorbance indicating low avidin concentration (0.8). Insect Toxicity Assay All insects of the S. granarius died after 21 days of feeding on a diet consisting of crushed seeds of transgenic avidin wheat, whereas, none of the tested insects died when they were fed on a diet of ground seeds from nontransgenic wheat (Table 3). Means within rows and/ or columns followed by the same letter (s) are not significantly different by Duncan's new multiple range test (P < 0.05). Discussion Wheat is an important staple crop in many countries. Depending on the climatic and storage conditions, it can become infested by a wide variety of storedproduct insect pests [1]. Avidin-containing wheat and its processed products would be resistant to infestations caused by all of the species. Detection and control methods for stored product insects have to be through integrated pest management program (IPM). Major efforts involving sanitation practices, exclusion techniques, habitat modifications, fumigation, and insecticide applications are usually required to prevent damage. The conventional insect control method is mainly dependent on the intensive and extensive use of chemical pesticides, which have drawbacks such as doing harm to the ecological system, producing residual poisons to human beings and animals, and high cost. Moreover, some insects have developed resistance to some of the available insecticides [32,33]. Therefore, it is desirable to develop insect-resistant plants through the introduction of insect-resistant genes (e.g. avidin) through genetic transformation. In present study, synthetic avidin coding DNA was transferred to wheat plants. Avidin accumulation was detected in transgenic plants by ELISA and western dot blot. We found that avidin wheat has excellent resistance to storage insects. Bioassay experiments proved that insect mortality in the first week was about 30 %, in second week it was about 70 % and in the third week the mortality was 100 %. Similar results were obtained with the red flour beetle Tribolium confusum, and flat grain beetle Cryptolestes pusillus [19]. Avidin transgenic tobacco halted growth and it caused mortality in larvae of two lepidopterans, Helicoverpa armigera and Spodoptera litura. The insects showed very poor growth over their first 8 days on a diet consisting of the leaves from transgenic plants and significant mortality were reported after 11 or 12 days and all insects were dead after 22 days [34]. In conclusion, the stable avidin transgenic wheat showed high level of resistance to the stored product insect (Sitophilus granarius). This study will hopefully decrease the loss of wheat seeds in warehouses significantly. In addition, the avidin-transgenic wheat powder can be used as a bioinsecticide. Avidin may interfere with enzymes that depend on enzyme bound biotin, such as those involved in carboxylation, decarboxylation, and transcarboxylation reactions [35]. Biotin deficiency in the blowfly, Aldrichina grahami, caused decreases in several fatty acids [36]. Presumably, a similar biochemical effect led to the stunted growth and mortality of the stored-product insects studied here. The public acceptability of avidin wheat as a food or feed is difficult to predict. Careful examination of its safety, however, is needed before consumption by humans and livestock can be considered. Kramer et al., [19] reported that at least there is no acute toxicity of avidin when feed to mice. Long-term ingestion of high levels of avidin maize may be a problem, because a biotin deficiency can decrease the growth rate of mice and affect reproduction [36,37]. However, avidin is a food protein that is consumed in the form of egg at a concentration of >400 part per million (ppm) by dry weight, which is four times higher than its concentration in most of the wheat used in the present study. Moreover, avidin has an antidote (biotin), which can be used to prevent toxicity or to rescue potential victims from adverse effects. Food and feed uses of avidin wheat might involve processing that includes supplementation with the vitamins. Another method that would help to prevent potential toxicity of the avidin wheat is the heat treatment, which would denature most of the avidin as well as the avidin-biotin complex and release most of the vitamins [38][39][40]. Currently development of wheat expressing transgenic avidin as a food or feed grain could be considered after thorough risk assessment. In addition to its efficacy against postharvest insect pests, avidin also is effective against preharvest pests such as the beet armyworm, black cutworm, bollworm, and other species for which biotin is an essential growth factor [41,42]. Transferring the avidin gene to other crops will be important in determining its potential usefulness in a variety of other commercial protein production and pest control situations. Risk assessment of these transgenics can be done following the National Biosafety Committee guidelines for the most efficient avidin-transgenic line. In addition, different species of stored cereals insects can be challenged with the avidin transgenic wheat grains and flour. Conclusion A synthetic avidin gene was introduced into spring wheat (Triticum aestivum L.) cv. Giza 168 using a biolistic bombardment protocol. Functional integrity of avidin was confirmed by insect bioassay. The results of bioassay using transgenic wheat plants challenged with wheat weevil revealed 100 % mortality of the insects reared on transgenic plants after 21 days. In conclusion, the stable avidin transgenic wheat showed high level of resistance to the stored product insect (Sitophilus granarius). This study will hopefully decrease the loss of wheat seeds in warehouses significantly. Competing interests The authors declare that they have no competing interests.

v3-fos

2017-05-29T19:19:00.474Z

2015-04-02T00:00:00.000Z

13956437

s2

Transcriptome Analysis and Gene Expression Profiling of Abortive and Developing Ovules during Fruit Development in Hazelnut Background A high ratio of blank fruit in hazelnut (Corylus heterophylla Fisch) is a very common phenomenon that causes serious yield losses in northeast China. The development of blank fruit in the Corylus genus is known to be associated with embryo abortion. However, little is known about the molecular mechanisms responsible for embryo abortion during the nut development stage. Genomic information for C. heterophylla Fisch is not available; therefore, data related to transcriptome and gene expression profiling of developing and abortive ovules are needed. Methodology/Principal Findings In this study, de novo transcriptome sequencing and RNA-seq analysis were conducted using short-read sequencing technology (Illumina HiSeq 2000). The results of the transcriptome assembly analysis revealed genetic information that was associated with the fruit development stage. Two digital gene expression libraries were constructed, one for a full (normally developing) ovule and one for an empty (abortive) ovule. Transcriptome sequencing and assembly results revealed 55,353 unigenes, including 18,751 clusters and 36,602 singletons. These results were annotated using the public databases NR, NT, Swiss-Prot, KEGG, COG, and GO. Using digital gene expression profiling, gene expression differences in developing and abortive ovules were identified. A total of 1,637 and 715 unigenes were significantly upregulated and downregulated, respectively, in abortive ovules, compared with developing ovules. Quantitative real-time polymerase chain reaction analysis was used in order to verify the differential expression of some genes. Conclusions/Significance The transcriptome and digital gene expression profiling data of normally developing and abortive ovules in hazelnut provide exhaustive information that will improve our understanding of the molecular mechanisms of abortive ovule formation in hazelnut. Introduction Hazelnut (Corylus L. spp.) is an edible nut crop that has great economic value. Its kernel is an important ingredient that is widely used in the production of dairy products, baked goods, chocolates, and other confectionery products. The unsaturated fatty acids in the kernel are reported to provide human health benefits by lowering cholesterol levels in blood and controlling the adverse effects of hypertension [1,2]. Due to the economic and ecological importance of hazelnut cultivation in northeast China, it is an important option for farmers who wish to increase their incomes in mountainous areas [3,4]. C. heterophylla Fisch. ex Besser and its hybrids with European hazelnut (C. heterophylla × C. avellana) are the most important Corylus varieties that are cultivated in northeast China, and their excellent resistance to cold will ensure their continued dominance of hazelnut production in China in the short term. However, excessive empty fruit formation of C. heterophylla causes serious yield losses [3]. A number of studies have suggested that selfincompatibility is associated with a higher frequency of blanks, nuts that contain no edible kernel [5,6]. In a previous study, empty fruit formation of C. heterophylla Fisch was verified to be closely associated with embryo abortion, using comparisons of morphological anatomy in normally developing nuts with empty nuts [3]. However, the molecular regulation mechanisms of embryo abortion in hazelnut remain unknown. In recent years, effective next-generation genome-wide gene profiling techniques have provided fascinating opportunities to survey the coding sequences of a total genome [7]. For example, Illumina sequencing technology provides millions of sequence reads from a single instrument run, and the assembled unigenes may be mapped to a reference transcriptome profile to obtain their molecular annotations. Despite the rapidly increasing amount of sequencing data, expressed sequence tags (ESTs) of C. avellana remain the only available large-scale sequencing data for the Corylus genus [8]. Therefore, data from transcriptome and expression profiling of developing and abortive ovules are needed in order to understand the molecular mechanisms regulating ovule abortion in hazelnut. In this study, we constructed a total pooled RNA library of developing and abortive ovules. De novo transcriptome Illumina sequencing of the pooled RNA library generated more than 4 billion nucleotides of high-quality RNA sequences. Due to the lack of genome information for hazelnut, 55,353 of the unigenes acquired provided necessary and useful reference sequences after transcriptome assembly and annotation analysis. Further transcriptome RNA-seq (quantification) analyses were carried out to identify differentially expressed genes (DEGs) that were associated with ovule and embryo development and to determine their possible functions. The assembled, annotated transcriptome sequences and gene expression profiles provide useful information that can help identify genes involved in embryo abortion during the fruit development stage. Materials and Methods Materials Fruit from young hazelnut trees was sampled from a hazelnut orchard (43°07 0 06@N, 124°2 8 0 39@E) near Siping, Jilin province, China. No specific permission was required in order to work in this location; also, the experiment did not involve endangered or protected species in China. In early spring, pollen from the cultivar 'Dawei' was collected from the orchard and airdried using the method described by Liu et al. [3]. At the same time, more than 600 pistillate inflorescences of C. heterophylla Fisch were randomly selected and bagged. On April 20, hand pollination was performed. Starting on June 20, fertilized ovules began to grow rapidly, resulting in an obvious size difference between developing ovules and abortive ovules. Young fruits were sampled three times at 15-day intervals after June 20. The samples were stored on ice until dissection, at which time the ovules from each fruit were carefully dissected. To obtain complete gene-expression data from ovules during the stages of fruit development, the sampled fruits were further dissected to determine whether the ovules were abortive. Developing ovules had diameters ranging from about 10 mm to more than 100 mm, and about 50 developing ovules in 50 fruits were collected. Abortive ovules had diameters of less than 3 mm during the fruit development stage and about 400 ovules were collected in 200 fruits. The developing and abortive ovules were immediately frozen in liquid nitrogen and stored at −80°C for RNA extraction. cDNA library preparation and Illumina sequencing for transcriptome analysis Total RNA of the ovule samples was extracted using the RNA Easyspin Isolation System (Aidlab Biotech, Beijing, China), and RNase-free DNase I was used to eliminate the residual genomic DNA in the raw RNA extract, according to the manufacturer's protocol (Promega, Beijing, China), with minor revisions. The concentration and integrity of total RNA were measured according to spectrophotometer analysis and using denaturing agarose gel electrophoresis. To obtain complete gene expression information, data from developing and abortive ovules at three equivalent stages were pooled, and these samples were used for transcriptome analysis. After the pooled RNA samples were treated with DNase I, magnetic beads with Oligo (dT) were used to isolate mRNA. The mRNA was fragmented into short segments (about 200 bp) by mixing with the fragmentation buffer. Next, the first strand of cDNA was synthesized by using a random hexamer primer. Buffer, dNTPs, RNAase H, and DNA polymerase I were added to synthesize the second strand. After end repair was performed and a 3'-end single nucleotide A was added, the short fragments were connected using adapters. The fragments were enriched by polymerase chain reaction (PCR) amplification and purified to create cDNA libraries. The Agilent 2100 Bioanalyzer and the ABI StepOnePlus Real-Time PCR System were used respectively to quantify and qualify the sample libraries. Finally, the cDNA libraries were sequenced using HiSeq 2000 Sequencing System (Illumina, Inc., USA). All raw transcriptome data was deposited in SRA (NCBI BioSample Accessions Number: SAMN03152274, SAMN03152275). Transcriptome de novo assembly was carried out with the short-read assembly program Trinity [9]. The resulting sequences were considered unigenes. The removal of redundant sequences and further splicing of the assembled unigenes from each sample were carried out using the software program TGICL [10], after which sequence clustering software was used to acquire non-redundant unigenes for as long as possible. BLASTX alignments (E-value < 10 −5 ) between unigenes and protein databases, including NR, Swiss-Prot, the Kyoto Encyclopedia of Genes and Genomes (KEGG), and COG, were performed, and the best aligned results were used to determine the sequence direction of unigenes. If the results of different databases conflicted with one another, a priority order of NR, Swiss-Prot, KEGG, and COG was followed in order to decide the sequence direction of unigenes. When a unigene did not align with sequences in any of the databases, ESTScan software [11] was used to deduce its sequence direction. GO and KEGG Orthology annotations of the unigenes were determined using Blast2Go, a universal tool for annotation, visualization, and analysis in functional genomics research [12], and InterProScan software. Differential gene expression (DGE) library preparation and sequencing Equal amounts of RNA from three time points for developing ovules or abortive ovules were separately pooled for DGE profiling analysis. The total RNA extraction was carried out following the protocol previously mentioned. First, the two pooled total RNA samples were treated with DNase I to degrade any DNA from accidental contamination. Then the mRNA was enriched by using oligo (dT) magnetic beads. After it was mixed with fragmentation buffer, the mRNA was fragmented into short segments (about 200 bp). Then the first strand of cDNA was synthesized by using a random hexamer primer. Buffer, dNTPs, RNase H, and DNA polymerase I were added to synthesize the second strand. The double-stranded cDNA was purified with magnetic beads. End reparation was then performed and 3'-end single nucleotide A (adenine) was added. Finally, sequencing adaptors were ligated to the fragments. The fragments were enriched by PCR amplification. The Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System were used to quantify and qualify the two sample libraries. The library products were then sequenced using the HiSeq 2000 Sequencing System (Illumina, Inc., USA). All raw data from the DGE library sequencing has been deposited in SRA (NCBI BioSample Accessions Number: SAMN03196408). Screening of DEGs Genes that were differentially expressed among samples were screened, and GO functional enrichment analysis and KEGG pathway enrichment analysis were performed for these DEGs [13]. A strict algorithm identifying DEGs between two samples was developed to distinguish the significance of DGE. If the number of unambiguous clean tags from gene A is denoted x, given that every gene's expression occupies only a small part of the library, p(x) will closely follow the Poisson distribution. pðxÞ ¼ e Àl l x x! ðλ is the real transcript of the geneÞ The total clean tag number of the sample 1 is N1, and the total clean tag number of sample 2 is N2. Gene A holds x tags in sample1 and y tags in sample2. The probability that gene A will be expressed equally between two samples may be calculated using the following equation: pði j xÞ > 0:5Þ The P-value corresponds to the DGE test. False Discovery Rate (FDR) is a method to determine the threshold of the P-value in multiple tests [14]. FDR 0.001 and the absolute value of log 2 Ratio ! 1 were the thresholds to judge the significance of gene expression difference. Quantitative reverse transcription (qRT)-PCR validation Total RNA from abortive and developing ovules was extracted and pooled as described for the DGE library previously mentioned, and three replicate experiments were prepared in each sample. First, reverse transcription to cDNA was performed on 100 ng total RNA using the PrimeScript RT reagent Kit (TaKaRa, Tokyo, Japan), following the manufacturer's instructions. qRT-PCR was performed with 1μL cDNA template, 1× reaction buffer, 0.2 mM dNTP mixture, 0.4 μM each primer, 0.6× SYBR Green I dye (Generay Biotech), and 0.3 units of rTaqDNA polymerase (TaKaRa) in a Rotor-Gene 2000 thermocycler (Corbett Research, Sydney, Australia) using the SYBR Premix Ex Taq Kit (TaKaRa), according to the manufacturer's protocol. Three technical replicates were run for each experiment. The cycle threshold (CT) values for each gene were normalized to the housekeeping gene β-actin using the formula (Unigene-ACTIN), where ACTIN is the mean CT of triplicate β-actin genes runs; Unigene is the mean CT of triplicate runs of the unigene of interest. Transcript fold-changes describing the change in expression of the target gene in samples from abortive ovules relative to that of the developing ovules transcript were calculated using the 2 −ΔΔCt method described by Schmittgen and Livak [15]. Results of qPCR were analyzed using one-way ANOVA followed by Dunett's multiple comparison tests in SAS version 8.01 (SAS Institute, Inc., Cary, NC, USA). P values lower than 0.05 were considered significant. The error bars indicate the standard deviation from three different experiments. The primers used in qRT-PCR analysis are listed in S1 Table. Results Illumina sequencing and sequence assembly A total of 54,000,000 raw reads were generated after Illumina sequencing analysis. Reads with adaptors, unknown nucleotides larger than 5%, and low-quality reads with more than 20% lowquality bases (base quality 10) were removed. A total of 48,585,250 clean reads with 4,372,672,500 nucleotides (nt) was obtained with a Q20 percentage of 98.17%. The final sequence assembly results using Trinity [9] was 55,353 unigenes, including 18,751 clusters and 36,602 singletons, with a mean length of 844 nt. The unigene size distribution is shown in Fig 1, which indicates that unigenes shorter than 2000 nt accounted for 91.45% of the total unigenes. GO and KEGG ontology classification The Blast2GO program [12] was used to obtain GO annotation of the unigenes on the basis of the NR annotation data. Unigenes with GO annotation accounted for 51.74% of all the unigenes and are listed in S3 Table. The GO categories of the unigenes are shown in Fig 3. The terms "cell" and "cell part," "binding" and "catalytic," and "process" and "locomotion" were dominant in the categories of cellular components, molecular functions, and biological processes, respectively. All unigenes were queried against the KEGG pathway database, and 38.63% of the 55,353 unigenes were given pathway annotations that were related to 128 pathways, including metabolism, plant hormone signal transduction, RNA transport, and purine metabolism. Unigenes with KEGG annotation are listed in S4 Table. Sequencing of two DGE libraries The empty library and full cDNA library that were generated from the developing and abortive ovules, respectively, of C. heterophylla Fisch were sequenced and generated approximately 7.4 million raw reads each. Dirty reads, which contained adapters, unknown bases, or low-quality bases, and which constituted fewer than 1% of the total reads, were filtered out. Next, clean reads of each library were mapped to the assembled unigenes on the basis of the results of transcriptome analysis. The number of total mapped reads was about 6.4 to 6.5 million base pairs, accounting for approximately 87% to 88% of total reads ( Table 1). The percentage of total unmapped reads was about 12% in the two libraries. In total, 45,641 unigenes in the empty library and 44,863 unigenes in the full library were mapped to our transcriptome reference database. The average length of mapped unigenes was about 800 bp, and their average coverage was about 55% ( Table 2). The gene expression level was calculated using the RPKM method [16]. The RPKM method eliminates the influence of different gene lengths and sequencing discrepancies on the calculation of gene expression level. The calculated values of the gene expression level may be directly used to compare the differences in gene expression among samples. The reads per kb per million reads of mapped unigenes was about 19 (Table 2). Detailed information about unigenes in the empty and full libraries that could be mapped to our transcriptome reference database is listed in S5 and S6 Tables, respectively. Coverage represents the percentage of a gene covered by reads, and the genes' distribution coverage of the empty and full libraries is shown in Variations in gene expression between developing and abortive ovules In order to determine the variations in gene expression between developing and abortive ovules, the empty and full libraries were compared. FDR 0.001 and the absolute value of log 2 Ratio ! 1 were used as the threshold to judge the significance of gene expression difference. Comparative analysis revealed 2,352 significantly differentially expressed unigenes between the empty and full libraries. Among these unigenes, 1,637 were upregulated and 715 were downregulated in the empty library, compared with the full library (Fig 5). The significantly differentially expressed unigenes (S7 Table) were further analyzed to determine their biological and molecular functions. Their functional categories were assembled according to information in several public databases and related literature. In the GO ontology, the terms "cell" and "cell part" were dominant in the category of cellular components, the term "catalytic activity" was dominant in the category of molecular functions, and the terms "cellular process" and "metabolic process" were dominant in the category of biological processes (S8 Table). According to GO classification, many of the differentially expressed genes were associated with oxidoreductase, peroxidase, and antioxidant activity (S9 Table). Among 2,352 DEGs, 1,209 DEGs with pathway annotation were identified and classified into 116 distinct categories (S10 Table). The top five pathways were metabolic pathways 4 Perfect Match: portion of total mapped reads that can be perfectly mapped to reference. 5 < = 2 bp Mismatch: portion of total mapped reads that can be mapped to reference with < = 2 bp mismatches. 6 Unique Match: portion of total mapped reads that have only one mapped site in reference. 7 Multi-position Match: portion of total mapped reads that have multiple mapped sites in reference. 8 Total Mapped Reads: number of reads that have no similar sequences as any part of reference. 9 Empty: cDNA library generated from abortive ovules. 10 Full: cDNA library generated from developing ovules. However, all the DEGs in the empty library that were involved in the abscisic acid and salicylic acid signal pathways were upregulated. In the abscisic acid signal pathway, these changes included abscisic acid receptor (PYL) (Unigene4733_D2), protein phosphatase 2C (PP2C) (CL2293.Contig2_D2, CL3319.Contig2_D2, CL7150.Contig2_D2, and CL6767.Contig1_D2)), and ABA responsive element binding factor (ABF) (Unigene16189_D2). In the ethylene regulating signal pathway, these upregulation changes included serine/threonine-protein kinase CTR1 (CTR1) (Unigene19967_D2, CL102.Contig7_D2, Unigene16685_D2), ethylene-insensitive protein 3 (EIN3), ethylene-responsive transcription factor 1 (ERF1) (Unigene31203_D2, Unigene15326_D2) and ethylene-responsive transcription factor 2 (ERF2) (Unigene15326_D2, CL3888.Contig2_D2). In this study, we focused on a group of downregulated DEGs involved in embryo development (Table 3). According to GO classification and KEGG annotation, these DEGs execute important biological functions, including the generation of late embryogenesis Confirmation through qRT-PCR To evaluate the validity of Illumina analysis, six upregulated DEGs involved in abscisic acid and ethylene signal pathways, including ABF (ABA-responsive element-binding factor), ERF1 (Ethylene-responsive transcription factor 1), CTR1 (serine/threonine-protein kinase CTR1), ERF2 (Ethylene-responsive transcription factor 2) EIN3 (Ethylene-insensitive protein 3) and PYL (Abscisic acid receptor PYR/PYL family), two downregulated DEGs which involved in embryo development, including TRA2 (transformer-2 protein) and SUPT5H (transcription elongation factor SPT5), were selected for examination by real-time RT-PCR. All the genes mentioned are probably involved in the dormancy and senescence of seed or embryo development. Information about these genes and their gene-specific primers is shown in S1 Table. The DGE results (Fig 6A) for these genes were identical to those obtained by RT-PCR (Fig 6B). In order to better understand the gene expression information in ovules of C. heterophylla Fisch, two cDNA libraries generated from developing ovules and abortive ovules were constructed during the ovule development stage to acquire complete transcriptome information. The empty and full DEG libraries were created to reveal the DEGs related to embryo development. We obtained a total of 55,353 unigenes. The mean length of unigenes was 844 nt and the N50 was 1,347 bp, which was much better than the assembled sequence quality of the EST database (28,255 contigs with an average length of 532 bp and an N50 of 961) [17]. Furthermore, our unigene data assembled from transcriptome sequencing data have far fewer redundant sequences than do the EST data. In addition, RNA samples used in the present study were extracted from the ovules of nuts rather than the leaves of young seedlings, which enrich the current Corylus database and aids future studies of the function of genes involved in hazelnut fruit development. The DEGs were involved in all of the plant hormone signal transduction pathways. In the present study, we specifically focused on the abscisic acid and ethylene signal pathways (Table 4), which were probably involved in the dormancy and senescence of seed. In the abscisic acid signal pathway, all three DEGs were upregulated, including abscisic acid receptor (PYL), protein phosphatase 2C (PP2C), and ABA-responsive element binding factor (ABF). PYLs are ABA receptors functioning at the apex of a negative regulatory pathway that controls ABA signaling by inhibiting type 2C PP2Cs [19]. The upregulation of ABF is believed to enhance abiotic stress signaling in rice [20]. The upregulation of PYL, PP2C, and ABF likely induce embryo dormancy through the ABA signal pathway. In addition, four DEGs were involved in the ethylene-regulating signal pathway, including serine/threonine-protein kinase CTR1 (CTR1), ethylene-insensitive protein 3 (EIN3), ethylene-responsive transcription factor 1 (ERF1), and ethylene-responsive transcription factor 2 (ERF2). Among these, EIN3 is thought to be involved in ubiquitin-mediated proteolysis, and the nuclear proteins ERF1/2 act on downstream components of EIN3 in the ethylene signaling pathway. All four proteins acted sequentially in a cascade of transcriptional regulation initiated by ethylene gas [21]. Although the ovules used in the study were far from mature, the activation of the ethylene signaling pathway is likely to result in the senescence of the young ovule and embryo. In addition to a cascade of DEGs in the plant hormone signal pathway, we also identified some important downregulated DEGs that were involved in the regulation of embryo development according to GO enrichment analysis. First, two important protective proteins in the abortive ovule were downregulated. Late embryogenesis abundant protein (LEA) in plants protects other proteins from aggregation due to desiccation or osmotic stresses associated with low temperature; in particular, it protects mitochondrial membranes against dehydration damage [22][23]. Superoxide dismutase (SOD2) is a protein cofactored with either iron or manganese, which catalyzes the dismutation of superoxide (O 2 − ) into oxygen and hydrogen peroxide. It is an important antioxidant enzyme that functions in the defense of nearly all cells exposed to oxygen. Thus, in the abortive ovule, these protective proteins fail to execute their defensive functions, which reduce the survival or development of the embryo. Second, some important proteins involved in the splicing of mRNA, RNA transcription, DNA synthesis, and protein modification were significantly downregulated. Among them, transformer-2 protein (TRA2) is an essential component of a splicing enhancer complex, and transcription elongation factor SPT5 (SUPT5H) and DNA polymerase epsilon subunit 3 (POLE3) are important components involved in RNA transcription and DNA duplication, respectively, while amidophosphoribosyltransferase (purF) catalyzes the first step of de novo purine nucleotide biosynthesis. Brassinosteroid (BR) insensitive 1-associated receptor kinase 1 (BAK1) positively regulates the BR-dependent plant growth pathway and negatively regulates the BR-independent cell-death pathway, and its downregulation would activate the cell death pathway in young ovules and embryos of hazelnut. Finally, several DEGs coding for important components such as actin-like protein 6B (ACTL6B) and histone deacetylase 1/2 (HDAC1_2) were downregulated, and plants in which transport inhibitor response 1 (TIR1) is disrupted are deficient in a variety of auxin-regulated growth processes, many of which may be involved in the abortive process of ovules and embryos in hazelnut. A large number of DEGs were not identified by KEGG annotation, GO annotation, or BLAST NR results. At the same time, the functions of many DEGs cannot be identified due to mismatched annotations in mammals or microorganisms. These may be identified at some point in the future as more annotations become available in public databases. This study is the first report of the involvement of DEGs in the embryo development pathway of Corylus, and our findings facilitate a better understanding of the developmental process in hazelnut, which may improve our ability to obtain high yields from this economically important crop. Conclusions Two cDNA libraries generated from developing ovules and abortive ovules were constructed during the ovule development stage in order to acquire complete transcriptome information. Sequencing and assembly results revealed 55,353 unigenes, including 18,751 clusters and 36,602 singletons. Transcriptome analysis results allowed gene expression changes between developing and abortive ovules in hazelnut to be compared. The results of the comparison indicated that a total of 1,637 and 715 unigenes were significantly upregulated and downregulated in the empty library compared with the full library. Among 2,352 DEGs, 1,209 DEGs with pathway annotation were identified and classified into 116 distinct categories. The top five pathways were metabolic pathways (33.66%), biosynthesis of secondary metabolites pathways (18.86%), plant-pathogen interaction pathways (10.84%), plant hormone signal transduction pathways (7.69%), and phenylpropanoid biosynthesis pathways (5.46%). We particularly focused on two groups of genes that were probably involved in the dormancy and senescence of seed and embryo development. (1) PYL, PP2C, and ABF in the abscisic acid pathway and CTR1, EIN3, ERF1, and ERF2 in the ethylene signal pathway were upregulated in the abortive ovule, and these changes may trigger the abscisic and ethylene signal pathways to induce the dormancy or senescence of ovules in hazelnut. (2) Some important proteins, including LEA, SOD2, TRA2, SUPT5H, POLE3, purF, BAK1, ACTL6B, and HDAC1_2 was downregulated in the abortive ovule, which probably damaged the protective function, activated the cell death pathway, and blocked development in the abortive ovule. Supporting Information S1

v3-fos

2018-04-03T03:52:46.186Z

2015-03-26T00:00:00.000Z

25600027

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Protein freeze concentration and micro-segregation analysed in a temperature-controlled freeze container Highlights • Freeze concentration of bovine serum albumin studied in a laboratory freeze container.• Protein micro-segregation visualized by confocal laser scanning microscopy.• Freezing protocol affected protein freeze concentration.• Spatial heterogeneities created by freeze concentration.• Micro-segregation was affected by freezing parameters. Introduction Proteins are high-value products of biotechnology with a rapidly growing importance on pharmaceutical markets [4,19,27]. The high costs of their production (e.g. Ref. [16]) makes it necessary that proteins are stabilized and stored with only minimum alteration of their biological activity [2,21,26]. Besides freeze drying [6,7,15], freezing is often used for preservation and storage of protein solutions [9,22]. Physically, freezing involves partitioning of the protein solution into an ice phase and a freezeconcentrated liquid [1]. Proteins like other solutes are rejected from the ice [8]. Spatial segregation of the protein is thus induced [5,25]. The consequent change in microenvironment involves various stresses on the protein, including exposure to solid ice surfaces and molecular crowding, which can result in unfolding and aggregation [5,13,20,24,28]. The freezing process should be designed to minimize loss of product quality due to critical stresses incurred. Understanding of how process control variables (e.g. the cooling rate, degree of supercooling) impact the freezing-induced phase separation at different scales would be highly important to assess consequent effects on protein stability [3,[9][10][11]12,14,22]. Studies at microliter scale have quantified spatial heterogeneity in frozen lysozyme solutions prepared by different freezing protocols, showing a protein freeze concentration by up to 7-fold [5]. Denaturation of lysozyme at the interface region of ice and liquid was also demonstrated [5]. However, in view of freezing as a unit operation of industrial biotechnology, investigations of the freezing-induced phase separation and the resulting protein freeze concentration must also be performed at larger scale and in controlled apparatus of defined geometry [9,10,17,22]. Here, evidence from freezing solutions of bovine serum albumin in a temperature-controlled 200 ml freeze container [18] is presented. Materials BSA was from Sigma-Aldrich. Fluorescein isothiocyanate (FITC) and other chemicals were from Roth. FITC labeling 2.3 ml of FITC solution (1 mg ml À1 ) in anhydrous DMSO was added in 20-ml aliquots under gentle stirring to 20 ml of BSA solution (5 mg ml À1 , 0.5 M sodium carbonate buffer, pH 9.5). After incubation for 12 h at 4 C in the dark, 2.2 ml of 0.5 M NH 4 Cl solution was added and incubation continued for 2 h. Unbound FITC was removed and buffer exchanged to 50 mM potassium phosphate (pH 7.5) using Vivaspin ultrafiltration. The molar FITC/ BSA ratio (F/P) was determined with the relationships: [11], and 0.614 is BSA absorption at 280 nm at 1.0 mg ml À1 . Absorbances measured (A) are given with wavelength in subscript. They were recorded on a Beckman Coulter DU 800 spectrophotometer. Freezing, sampling, sample processing and analysis Native BSA and FITC-labeled BSA were used. About 200 ml of protein solution (0.1 mg ml À1 ) were frozen in a freeze container (described in Ref. [18]) Silicone oil (M40.165.10 by Huber, Germany) was used as thermofluid. The different freezing protocols applied are described in Section 3. Temperature inside the freeze container was measured with 8-channel PCE-T 800 Multi-Input Thermometer, with probes at seven positions (A-G) indicated in Fig. 1. Freezing time was defined as the time between nucleation and complete solidification of the bulk, which can be identified as a plateau in the measured temperature profiles [18]. When seeding was performed, frozen buffer droplets were introduced next to the cooled container walls as soon as the bulk temperature was À2 C. After freezing and equilibration of bulk temperatures, the ice block was removed from the container. For native BSA concentration determination, the removed ice block was cut into pieces (1-12) as indicated in Fig. 1. The samples were thawed, and the soluble BSA concentration was measured in each of them. Roti-Nanoquant protein assay was used. The FITC label interfered with protein determination, thus precluding the same protein analysis when using the FITC-BSA conjugate. To perform confocal laser scanning microscopic analysis on frozen material, samples were drilled from the frozen ice block using a hollow drill with an inner diameter of 25 mm. From each run three samples were taken from positions I-III, as shown in Fig. 1. Ice cores were stored in 50-ml Falcon tubes at À70 C until microscopic examination. Images were obtained with a Leica DMI 6000 inverted microscope in a TCS SP5 system. Excitation was at 488 nm, acquisition from 502 to 603 nm. It should be noted that images presented later on were chosen to represent the predominant microscopic appearance of the probed position. However, between 5 and 8 CLSM images with adequate quality were taken from each position. In addition, for every sample at least one acquisition of stacked images in z-direction was performed, each stack containing approx. 50-100 single images. Every single or stacked image pictured an area of 620 Â 620 mm. It was ensured through careful comparison of different sample areas that the images shown are fully representative for the frozen sample as a whole. Images were collected as fast as possible before any sample melting through laser beam exposure could occur. Macroscopic freeze concentration of BSA Using a common thermofluid set temperature of À40 C, solutions of unlabeled BSA were frozen at different freezing rates. The fastest freezing rate involved immediate switch from room to set temperature. Relatively slower freezing rates involved linear temperature ramps from room to set temperature over 4 h and 14 h. Freezing over the 4 h temperature ramp involved an additional seeding step. No seeding was applied during the 14 h temperature ramp in order to maximize the effect of supercooling. The rapid lowering of temperature during fast freezing resulted in immediate nucleation at the container walls, thus making seeding unnecessary. Aim of the experiments was to evaluate the influence of the freezing protocol on freeze concentration at 12 different points in the freeze container (see Fig. 1). Results are summarized in Table 1. Analysis of the protein distribution across the different sampling positions 1-12 is based on a total protein recovery of 95% or greater. Spatial characteristics of BSA freeze concentration were similar in each experiment where protein concentrations were highest in the slowly freezing central region of the container around the longitudinal axis, in particular at positions 6 and 7. In container side regions that froze fastest (positions 1, 4, 9 and 12), by contrast, BSA concentrations were lowest. The freeze concentration can be characterized by the maximum and minimum protein concentrations observed across the container. Slowing down the freezing rate resulted in increased protein concentrations in the maximally freeze-concentrated regions of the container. The minimum protein concentration was lowest for the seeded 4-h freezing run, and also the ratio between maximum and minimum protein concentration was highest under these conditions. Maximum/ minimum protein concentration ratios of between 1.4 and 1.7 indicate macroscopic freeze concentration to have been substantial under all freeze conditions used. Considering the protein distribution in frozen bulk (Table 1) and assuming comparable cryoconcentration patterns of labeled and unlabeled BSA, positions I-III were chosen in regions with low, intermediate and high protein concentrations to obtain samples for microscopic imaging by CLSM analysis. Visualization of the distribution of freeze-concentrated FITC-BSA conjugate The FITC-labeled BSA preparation used had a F/P ratio of 3.4. For CLSM imaging experiments, fast freezing (freezing time = 53 min) and 4-h seeded freezing (freezing time = 87 min) were performed as in Table 1. In addition, a 4-h freezing run was done without seeding (freezing time = 73 min) to induce supercooling. Seeding was however used in the slow-freezing run where set temperature was decreased linearly to À40 C within 12 h (freezing time = 4 h 22 min). Note that the set temperature decreases were comparable but not identical to the freezing experiments described in Table 1. Representative images from CLSM analysis of ice core samples are shown in Fig. 2 where images are arranged by position and freezing time. Note: in the non-seeded 4-h freezing experiment (freezing time = 73 min), supercooling was achieved in the whole bulk whereby just before spontaneous nucleation, temperatures of À4.6 C and À1.0 C were recorded in peripheral (thermocouples B, C, F, E in Fig. 1) and central regions of the freeze container (thermocouples A, G, D in Fig. 1), respectively. In most of the sample images, irregular distributions of labeled protein were found. Protein was accumulated in localized and contained regions, circular or elongated in shape and approxi- Discussion Freezing induced a substantial amount of macroscopic spatial heterogeneity in BSA solutions (Table 1). Maximum BSA concentration in freeze-concentrated regions increased on increase in the freezing time. Ratio between maximum and minimum protein concentration also changed on variation of the freezing rate. Explanations for the macroscopic observations were found through analysis of microscopic effects of the freezing, as follows. Of the sampling positions selected for CLSM analysis, position III was located in the freeze container's very last point to freeze and as expected, it was the one to show the highest protein concentration. The remaining positions (I, II) represented faster freezing points of the container, and they exhibited decreased protein concentration. Fig. 2 shows that freeze-concentrated protein regions were formed in larger number and bigger size at sampling position III, as compared to positions I and II, under conditions of slow freezing (87 min, 4 h 22 min). The effect was weakened (73 min) or even absent (53 min) at faster freezing rates. Fig. 2 also shows that slower freezing times (87 min, 4 h 22 min) resulted in fewer and larger freeze-concentrated regions and also in wider protein-free regions, except for position III (see below). Table 1 Spatially resolved protein distribution in BSA solutions frozen at different freezing rates (fast, 4 h, 14 h) to thermofluid temperature of À40 C. Sampling positions 1-12 refer to Fig. 1. N is the number of replicate freezing runs. Seeding was performed in the 4-h freezing run. Results are given in percent concentration (C) where 100% equals the initial solution concentration of 0.1 mg l À1 . Mean values AE standard deviations are shown. Range = C max À C min ; Ratio = C max /C min . In the highlighted experiment (pink), no seeding was performed. Images were obtained with a Leica DMI 6000 inverted microscope in a TCS SP5 system. Excitation was at 488 nm, acquisition from 502 to 603 nm. Magnification was 630-fold. Samples were placed in a plastic dish with a glass slide bottom while dry ice was used for cooling. Images were collected as fast as possible before any sample melting through laser beam exposure could occur. Multiple images were collected from each position. It was ensured through careful comparison of different sample areas that the images shown were fully representative for the frozen sample as a whole. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Faster freezing, by contrast, caused a higher number of smaller freeze-concentrated regions, as revealed on comparing the 53 min and 4 h 22 min freezing runs at the position I and position II. The effect of seeding is made evident by comparing the 73 min and 87 min freezing runs. In the non-seeded 73 min run, freezeconcentrated protein regions were much smaller and distributed in a way more irregular fashion than in the seeded 87 min run. Microscopic protein distribution at position I is relevant in particular: almost instant solidification of the whole sampling position was noticed after spontaneous nucleation whereas in the seeded experiment, the freezing occurred much slower in this region. The comparably regular (linear) appearance of freezeconcentrated protein regions in the seeded experiment is therefore noted, at position I but also at position II. Linear alignment of the protein domains could be caused by dendritic ice growth, which is often found in bulk-scale freezing [23]. One expects dendritic growth to be pronounced under conditions where solidification occurs relatively slow, as in the seeded experiment with its low degree of supercooling. There are practical ramifications of the evidence presented. Formation of a relatively large number of freeze-concentrated protein domains under conditions of fast freezing implies pronounced exposure of proteins to ice crystal surfaces. High cooling rates could therefore be unfavorable for freezing of surfacesensitive proteins. At slow freezing rates, fewer and larger protein domains are present. It is quite likely that the protein concentration will be higher in the large as compared to the small domains and therefore, slow freezing may create problems with proteins having low solubility limits and tend to native-like aggregation.

v3-fos

2016-03-22T00:56:01.885Z

2015-06-01T00:00:00.000Z

11455577

s2

Covalently Cross-Linked Arabinoxylans Films for Debaryomyces hansenii Entrapment In the present study, wheat water extractable arabinoxylans (WEAX) were isolated and characterized, and their capability to form covalently cross-linked films in presence of Debaryomyces hansenii was evaluated. WEAX presented an arabinose to xylose ratio of 0.60, a ferulic acid and diferulic acid content of 2.1 and 0.04 µg∙mg−1 WEAX, respectively and a Fourier Transform Infra-Red (FT-IR) spectrum typical of WEAX. The intrinsic viscosity and viscosimetric molecular weight values for WEAX were 3.6 dL∙g−1 and 440 kDa, respectively. The gelation of WEAX (1% w/v) with and without D. hansenii (1 × 107 CFU∙cm−2) was rheologically investigated by small amplitude oscillatory shear. The entrapment of D. hansenii decreased gel elasticity from 1.4 to 0.3 Pa, probably by affecting the physical interactions between WEAX chains. Covalently cross-linked WEAX films containing D. hansenii were prepared by casting. Scanning electron microscopy images show that WEAX films containing D. hansenii were porous and consisted of granular-like and fibre microstructures. Average tensile strength, elongation at break and Young’s modulus values dropped when D. hansenii was present in the film. Covalently cross-lined WEAX containing D. hansenii could be a suitable as a functional entrapping film. Introduction Arabinoxylans are important non-starch cereal polysaccharides constituted by a linear backbone of β-(1→4)-linked D-xylopyranosyl units to which α-L-arabinofuranosyl substituents are attached through O-2 and/or O-3 [1]. Some of the arabinose residues are ester linked on (O)-5 by ferulic acid (FA, 4 hydroxy-3-methoxycinnamic acid) [2]. These polysaccharides are classified as water extractable (WEAX) or water-unextractable (WUAX). WEAX form highly viscous solutions and can gellify through covalent ferulic acid cross-linking upon oxidation by some chemical or enzymatic free-radical-generating agents [3,4]. Laccase (p-diphenol oxygen oxidoreductase, EC 1.10.3.2), a blue multi-copper enzyme of white rot fungi [5] oxidizes FA from WEAX resulting in the formation of five different diFA structures (5-5ʹ-, 8-5ʹ benzo-, 8-O-4ʹ-, 8-5ʹ-and 8-8ʹ di-FA), the 8-5 and the 8-O-4 forms being always preponderant [6][7][8]. The presence of a trimer FA structure (tri-FA) in WEAX gels has been also reported [9]. This covalent WEAX cross-linking has commonly been considered as responsible for the WEAX network development, even if weak interactions also contribute to the final gel properties [10,11]. WEAX gels present interesting functional properties such as stability to temperature and pH changes, as well as neutral taste and odor, which are all desirable properties for industrial applications. As dietary fibers, WEAX and WUAX present potential health benefits for lipid metabolism, colon function, and reduction of heart disease, among others [9,10]. In addition, arabinoxylans' ability to form a continuous and cohesive matrix has led to their use as film formers [12]. In recent years there has been a growing consumer interest in health, nutrition, food safety and environmental issues. This has led to renewed interest in food packaging based on natural macromolecules has been due to concerns about the environment, a decrease in fossil resources, and a need to reduce the amount of disposable packaging materials. All this has led companies and researchers to explore alternatives such as polysaccharide-based film applications in food systems [13]. Nevertheless, one of the major problems in polysaccharide films applications is their stability during thermal processing. Most currently used polysaccharide films are stabilized by physical (hydrogen bonding and/or ionic) interactions, while films based on polysaccharide covalent networks such as gelled cross-linked arabinoxylans are not common. Polysaccharide films can be used to protect perishable food products from deterioration [14]. It has been reported that antimicrobial compounds or biocontrol microorganisms can be incorporated into films to maintain the stability of food storage [15]. Incorporation of biocontrol microorganisms into the films could improve the control of postharvest diseases. In this regard, the capacity of the yeast Debaryomyces hansenii to control blue mold decay of lemon has been demonstrated [16]. Nevertheless, no research has been reported on the incorporation of microorganisms into oxidatively cross-linked WEAX films. The objective of this research was to investigate the incorporation of D. hansenii in laccase-induced WEAX films and to evaluate the morphological and mechanical properties of the material formed. Extraction and Characterization of WEAX Yield of WEAX extracted from wheat flour was 0.45% (w/w) on a dry matter basis (db, w WEAX/w wheat flour). Similar WEAX yield values have been reported for flours of different wheat varieties [17,18]. In the present study, wheat flour WEAX were used as a convenient model, but more complex commercial arabinoxylans will be tested later on in order to make these films viable for foodstuffs as they must have a competitive price. WEAX composition is presented in Table 1. The arabinoxylans content of the extract was estimated from the sum of xylose + arabinose as 65% db, which is close to the value reported for other wheat WEAX [18]. A residual amount of glucose was also quantified. The FA content (2.1 μg•mg −1 WEAX) was in the range reported for other wheat WEAX [3,4,8]. Small levels of di-FA have been detected in WEAX (0.04 μg•mg −1 WEAX), suggesting that some arabinoxylan chains might be cross-linked as previously reported [19][20][21]. The percentages of each one of the different di-FA presents in the WEAX were 80, 16, and 4% for the 8-5ʹ (mainly in the benzofuran form), 8-O-4ʹ, and 5-5ʹ structures, respectively. The 8-8ʹ dehydrodimer was not detected in this study. The predominance of 8-5ʹ and 8-O-4ʹ di-FA structures has also been reported in arabinoxylans from wheat and barley flour [6][7][8]. The tri-FA 4-O-8ʹ, 5ʹ-5ʺ was detected only in trace amounts. The degree of substitution (arabinose to xylose ratio, 0.60) was characteristic of wheat endosperm arabinoxylans (0.53-0.7) [9,10]. The intrinsic viscosity ([η]) and viscosimetric molecular weight (Mv) values were 3.6 dL•mg −1 and 440 kDa, respectively, which are in the range previously reported for other wheat WEAX [4]. The Fourier transform infrared (FTIR) spectrum of WEAX ( Figure 1) shows absorbance bands for polysaccharides at 1200-800 cm −1 , a main band centered at 1035 cm −1 that could be assigned to C-OH bending, with small shoulders at 1158, and 897 cm −1 that have been related to the antisymmetric C-O-C stretching mode of the glycosidic link and β(1→4) linkages [22]. The amide I band (1640 cm −1 ), related to protein content, which is mainly assigned to polypeptide carbonyl stretching, was also detected [23]. The band at 3413 cm −1 corresponds to stretching of the OH groups and the one at 2854 cm −1 corresponds to the CH2 groups [24]. The gel permeation chromatography profile of WEAX is presented in Figure 2. The molecular weight distribution profile shows a major peak in the high molecular weight region (retention time between 10 and 14 min). A shoulder was registered to the right of the major peak indicating the presence of a WEAX population with low molecular weight values. This molecular weight distribution pattern has been previously reported for wheat WEAX [17]. WEAX Gelation The cross-linking process of WEAX was rheologically investigated by small amplitude oscillatory shear. Figure 3A shows the development of G' and G" moduli vs. time of 1% (w/v) WEAX solution undergoing oxidative gelation by laccase. Storage (G') and loss (G") moduli rise to reach a pseudoplateau region. The final G' and G" values of 1% (w/v) for gels were 1.4 and 0.4 Pa, and for gels containing yeast 0.3 and 0.2 Pa, respectively. This decrease in G' could be related to loss, or weakening in WEAX chains physical interactions which may reduce the network connectivity, as previously suggested in WEAX gels containing Bifidobacterium longum [25]. Nevertheless, yeast cells and bacteria are rather different (in surface properties, size, shapes, etc.), which may lead to different interactions with the WEAX network. The gelation time (tg), calculated from the crossover of the G' and G" curves (G' > G") was 31 min and for gels containing yeast 69 min. The tg value indicates the sol/gel transition point and at this point G' = G" or tan δ = G"/G' = 1 [26]. The mechanical spectra of WEAX after 3 h gelation ( Figure 3B) was typical of solid-like materials with a linear G' independent of frequency and G" much smaller than G' and dependent of frequency [27]. This behavior is similar to that previously reported for arabinoxylans gels cross-linked by laccase or peroxidase/H2O2 system [10]. During WEAX gelation, ferulic acid was oxidized leading to the formation of covalent cross-linking structures (0.122 µg of di-FA per milligram of WEAX and traces of tri-FA). The 8-5ʹ benzofuran form, 8-5ʹ, 8-O-4ʹ and 5-5ʹ dimers represented 66%, 15%, 14% and 5% of the total di-FA amounts respectively ( Figure 4). The main increase in di-FA concerned the 8-5ʹ benzofuran form. The predominance of 8-5ʹ benzofuran form and 8-5ʹ dimers and absence of the 8-8ʹ structure was also observed in other WEAX gels [7,9,11]. The amounts of di-FA and tri-FA produced did not counterbalance the lost in FA. Therefore, at the end of gelation, 63% of the initial FA in the WEAX solution disappeared, with only 37% recovered as di-FA. Low ferulate recovery after oxidative treatment of arabinoxylans has been previously reported [7,9,11]. This behavior could be explained by the possible formation of other ferulate structures which are not determined by the method used in the present study. WEAX Films WEAX gelation system with or without D. hansenii formed films when was left to dry on Petri dishes. The films were slightly yellow, but still transparent, flexible, homogeneous, and with smooth surfaces (no pores or cracks). The thickness of WEAX films was 25.7 ± 1.27 and 24.1 ± 3.25 µm for WEAX films containing yeast. Figure 5A,C,E shows WEAX film while images 5(B,D,F) correspond to WEAX film entrapping D. hansenii. Figure 5C-F show stereomicrographs of the WEAX films surface, which are without apparent fractures. Without any oxidative gelation of the WEAX, the material formed presents a discontinuous surface and became far too fragile to handle. Table 2. Significant differences (P < 0.05) in L, a, b values were detected among the films. The main difference in color values among these materials was the increased b value (yellowish) in presence of yeast cells. Color changes due to incorporation of D. hansenii can be more fully described using other color function such as YI which indicates degree of yellowness [28]. The entrapment of D. hansenii in WEAX film resulted in a significantly increase (P < 0.05) in YI. In the present study, FT-IR spectra of WEAX film and WEAX film containing yeast cells are shown in Figure 6. In general, film spectra were similar to those of non-cross-linked WEAX (Figure 1). However, in cross-linked WEAX a small absorption band at 1720 cm −1 was observed, which represents the carbonyl stretching vibrations of esters [23,24]. It has been reported that the intensity of the absorption band at 1414 cm −1 for CO asymmetric stretching increases after WEAX cross-linking in lyophilized microspheres [29]. However, no significant increase in this band was found in WEAX films, which could be attributed to the lower amount of polysaccharide used for the sample preparation (WEAX solutions at 1%, w/v) in comparison to that used for WEAX microspheres (WEAX solution at 4%, w/v). The FTIR spectrum of WEAX film containing D. hansenii was also investigated because the protein from yeast cell could favor the formation of protein-WEAX adducts which have been reported and seen by FTIR [30]. Nevertheless, these adducts were not observed in WEAX film containing yeast cell, which could be explained by the relatively low amount of entrapped cells. SEM images of the WEAX film containing D. hansenii at different scales are presented in Figure 7. The surface morphology reflects the complexity of interactions within the WEAX film. The film surface presents some unevenness on the microscopic level which could be attributed to the high viscosity of WEAX solution in the final stages of gel formation and/or to the presence of yeast cells suspended in the WEAX gelation system. It is possible to observe the presence of granular-like and fibre microstructures that may be attributed to WEAX chain aggregates. A higher magnification clearly shows that the WEAX film contains pseudo-circular structures of 1-3 µm size, which can be attributed to entrapped yeast cells. SEM images show that WEAX films containing D. hansenii are porous. This morphological microstructure is similar to the microstructure of films made from corn hull arabinoxylans [31], xylans [32] and glucans [33]. From a general point of view, the porosity of WEAX films could be a relevant parameter because this characteristic may prolong D. hansenii viability and thus improve the inhibitory efficiency of the films on the control of postharvest diseases. Average tensile strength, elongation at break and Young's modulus values dropped when D. hansenii was present in the WEAX film, although it should be noted that the standard deviation for the test samples was high (Table 3). It is possible that the presence of D. hansenii could act as defects in the WEAX films and contribute to the early breaking of the samples during tensile testing. These findings are in good agreement with the decrease in G' of the WEAX gels entrapping yeast (Figure 3). WEAX and WEAX containing D. hansenii mechanical properties were in the range reported for non-oxidative coupled arabinoxylan films, however such films were prepared by using polysaccharide solutions at higher concentrations (7.5% w/v) [31] while in the present study a WEAX solution at 1% (w/v) was used. Another study reported non oxidative coupled arabinoxylan films presenting higher tensile strength values but lower elongation at break (4.7%) than the films formed in the present study [34]. It has been reported that the polysaccharide covalent cross-linking improves the mechanical properties of the films formed [35]. Our results suggest that oxidative coupling of WEAX enhance the mechanical properties of the films formed. Materials Water extractable arabinoxylans (WEAX) were extracted from wheat flour which was kindly provided by a wheat milling industry in Northern Mexico (Molino La Fama). D. hansenii was obtained from seawater samples collected at a depth of 100 m at the Cortez Sea (Baja California, Mexico) and belonging to the Yeast Collection of the CIBNOR S.C. Commercial laccase (benzenediol:oxygen oxidoreductase, E.C.1.10.3.2) was from Trametes versicolor. All chemical reagents were purchased from Sigma Chemical Co. (St Louis, MO, USA). WEAX Extraction and Characterization WEAX were extracted as described previously [17]. Laccase activity was measured at 25 °C from a laccase solution at 0.125 mg•mL −1 dissolved in 0.05 M citrate-phosphate buffer pH 5.5 as previously reported [9]. Neutral sugar content in WEAX was determined by hydrolysis of the polysaccharides with 2 N trifluoroacetic acid at 120 °C for 2 h as reported before [17]. Samples were filtered through 0.2 µm (Whatman) and analysed by HPLC using a Supelcogel Pb column (300 × 7.8 mm; Supelco, Inc., Bellefonte, PA, USA) eluted with 5 mM H2SO4 (filtered 0.2 µm, Whatman) at 0.6 mL•min −1 and 50 °C. A Varian 9012 HPLC (Varian, St. Helens, Australia) equipped with q Varian 9040 refractive index detector and a Star Chromatography Workstation system control version 5.50 were used. The protein content in the WEAX powder was determined according to the Dumas method [36], using a Leco-FP 528 nitrogen analyzer (Leco, St. Joseph, MI, USA). Viscosity measurements were made by determination of the flow times of WEAX solutions in water (from 0.06 to 0.1% w/v). An Ubbelohde capillary viscometer at 25 ± 0.1 °С immersed in a temperature controlled water bath was used. The intrinsic viscosity ([η]) was estimated from relative viscosity measurements (η rel) of WEAX solutions by extrapolation of Kraemer and Mead and Fouss curves to "zero" concentration [9]. The viscosimetric molecular weight (Mν) was calculated from the Mark-Houwink relationship, Mν = ([η]/k)1/α. Molecular weight distribution of WEAX was determined by Size Exclusion-High Performance Liquid Chromatography (SE-HPLC) at 38 °C using a TSKgel (Polymer Laboratories, Shropshire, UK) G500 PMWX column (7.8 × 300 mm). A Water 2414 refractive index detector was used for detection. Isocratic elution was performed at 0.6 mL•min −1 with 0.1 M LiNO3 filtered through 0.2 μm [17]. FT-IR spectra of dry WEAX and WEAX film powder were recorded on a Nicolet FT-IR spectrophotometer (Nicolet Instrument Corp., Madison, WI, USA). The samples were pressed into KBr pellets (2 mg sample/200 mg KBr). A blank KBr disk was used as background. Spectra were recorded between 400 and 4000 cm −1 [22]. WEAX Gelation A WEAX solution (1% w/v) was prepared in distilled water pH 5. Laccase (1.675 nkat per mg WEAX) was added to WEAX solution as cross-linking agent. Gels were allowed to develop for 3 h at 25 °C [9]. WEAX solution concentration and laccase amount used were based on previous reports [4,9,11]. The advantage of using laccase as cross-link agent is that this enzyme is generally recognized as safe (GRAS Notice 000122). Small amplitude oscillatory shear was used to follow the gelation process of WEAX solution. Cold (4 °C) WEAX solution (1% w/v) in distilled water pH 5 was mixed with laccase and immediately poured on plate-plate geometry (4.0 cm in diameter) of a strain controlled rheometer (Discovery Hybrid Rheometer, TA Instruments, New Castle, DE, USA). Exposed edges were recovered with silicone to prevent evaporation. WEAX gelation was started by a sudden increase of temperature from 4 to 25 °C and monitored at 25 °C for 2 h by recording the storage (G') and loss (G") moduli. Measurements were carried out at 1.0 Hz frequency and 10% strain. From strain sweep tests, WEAX gels showed a linear behavior from 0.02 to 100% strain. 10% strain was used in all the rheological measurements. The mechanical spectra of gels were obtained by frequency sweep from 0.01 to 10.0 Hz with a 10% strain at 25 °C. The same conditions were applied to WEAX solution containing D. hansenii [25]. WEAX Films Preparation and Characterization A WEAX solution (1% w/v) and a WEAX solution (1% w/v) containing D. hansenii (1 × 10 8 CFU•mL −1 ) were prepared in distilled water (pH 5) as reported previously [25]. Laccase (1.675 nkat per mg WEAX) was used as cross-linking agent. The amount of yeast cells used was based on a previous report about WEAX gels containing Bifidobacterium longum [25]. Glycerol (0.05% w/w WEAX) was used as plasticizer. Dispersions were poured over polyethylene Petri dishes (90 mm in diameter) covered with a 1.5 mm thickness Teflon film to facilitate the recovering of the dried films. The dispersions were left to dry approximately 36 h at room temperature and low relative humidity in a desiccator. WEAX films formed presented a thickness value of 0.02 ± 0.001 mm, measured with a micrometer (Code No. 293-230, Mitutoyo Corp., Kawasaki, Kanagawa, Japan). WEAX film color was evaluated with a colorimeter (Minolta CR400, Ramsey, NJ, USA) and standardized with respect to white calibration plate. Colorimeter provided CIE L, a, and b values. L is lightness, and a (−greenness to +redness) and b (−blueness to +yellowness) are the chromaticity coordinates. Yellowness index (YI) was calculated [28]. Four readings were taken at different locations on each film. Measurements were done in triplicate. FT-IR spectra of WEAX films were recorded on a Nicolet FT-IR spectrophotometer (Nicolet Instrument Corp.). The films were placed in a sample holder. Air (blank) was used as background. Spectra were recorded between 400 and 4000 cm −1 . WEAX films were frozen at −20 °C and lyophilized at −37 °C/0.133 mbar overnight in a Freezone 6 freeze drier (Labconco, Kansas, MO, USA). The structure of the WEAX films was analyzed with a Motic BA300Pol microscope (Motic Incorporation Ltd., Hong Kong, China) at low magnifications (4× and 40×). The structure of freeze-dried WEAX films was studied by scanning electron microscopy (JEOL 5410LV, Peabody, MA, USA) at low voltage (20 kV). SEM image was obtained in secondary electrons image mode. Tensile tests were carried out using a TA-XT2 texture analyser (Stable Micro Systems, Godalming, England). Film strips (14 mm × 50 mm) were attached on tensile grips and stretched at 0.5 mm•s −1 in tension mode [37]. Conclusions Oxidative cross-linked WEAX film was achieved for the first time. In addition, in the present study, it was possible to entrap D. hansenii in the WEAX film. The presence of yeast results in a slight reduction of WEAX gel elasticity, probably by affecting the physical interactions taking place between WEAX chains. Average tensile strength, elongation at break and Young's modulus values drop when D. hansenii was present in the WEAX film. WEAX films are porous and consist of granular-like and fibre microstructures. The results suggest that oxidative cross-linked WEAX films can be potential candidates for the entrapment of yeast as a functional entrapping film. WEAX films barrier properties, water stability and yeast viability will be investigated during the second part of the project, where the WEAX film entrapping D. hansenii will be tested as a biological control of blue mold decay in Mexican lemon.

v3-fos

2016-05-16T23:41:15.414Z

2015-12-01T00:00:00.000Z

34791927

s2

Loop-mediated Isothermal Amplification Assay to Rapidly Detect Wheat Streak Mosaic Virus in Quarantined Plants We developed a loop-mediated isothermal amplification (LAMP) method to rapidly diagnose Wheat streak mosaic virus (WSMV) during quarantine inspections of imported wheat, corn, oats, and millet. The LAMP method was developed as a plant quarantine inspection method for the first time, and its simplicity, quickness, specificity and sensitivity were verified compared to current reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR quarantine methods. We were able to quickly screen for WSMV at quarantine sites with many test samples; thus, this method is expected to contribute to plant quarantine inspections. Wheat streak mosaic virus (WSMV) is an unreported virus in and quarantine has raised the possibility that WSMV can cause serious food loss and economic damage (French and Robertson, 1994) by infecting wheat, corn, oats, and millet (Lee et al., 2013a). Quarantine inspections have used reverse transcriptionpolymerase chain reaction (RT-PCR) and nested PCR to detect pathogens since 2012 in Korea (Lee, 2013;Lee et al., 2013b;Lee et al., 2013c). However, these methods require 10 hrs for results, so they are inconvenient for field use where rapid inspection and subsequent quarantine are needed. Therefore, in this study, we developed a LAMP assay as an inspection method to detect WSMV in a one-step quick screen during quarantine inspection. Samples of WSMV and reference viruses [Cereal chlorotic mottle virus (CCMV) and Cucumber mosaic virus (CMV)] were collected through import approval of prohibited goods. Nucleic acids were extracted and cDNA was synthesized using a method reported previously Lee et al., 2015;Shin and Rho, 2014). RNA of 170-200 ng/µl was extracted from the samples, and 100 µl cDNA was synthesized from the RNA. The WSMA complete genome (NC_001886), 15 WSMV strains and their base sequences, and the base sequences of 20 reference virus strains that could infect the same host were collected from the National Center for Biotechnology Information. Two sets of WSMV-specific LAMP primers were designed based on the base sequences collected using the LAMP primer designing software PrimerExplorer (Table 1). Three WSMV template cDNAs were chosen and reacted for 1 hr at three different temperatures (60, 62, and 65 o C) to select the best LAMP conditions for detecting WSMV after 10 min at 95 o C and 1 min at 4 o C. As a result, condition 3 using an 11 µl total volume and no distilled water showed the most specific reaction. The analysis indicated that the specific reaction occurred at all temperatures, so the most stable conditions at 62 o C were selected (Fig. 1). In addition, set 1 of the cDNAs for the CCMV and CMV reference viruses indicated nonspecific amplification for both the reference virus and the negative control, so it finally selected as the LAMP primer combination for detecting WSMV (Supplementary Fig. 1). The LAMP method developed here can detect WSMV in imported samples within 1.5 hrs (30 min for cDNA synthesis and 60 min for LAMP) after RNA extraction. Thus, Development of LAMP Assay for Detecting Wheat streak mosaic virus 439 this method reduces the time to detect WSMV from the previous 10 hrs using the RT-PCR, electrophoresis, nested PCR, and additional electrophoresis steps. Furthermore, it is much simpler and easier to use as the standard quarantine method. Additionally, specificity improved compared to that of the PCR method. The WSMV cDNA template for LAMP was diluted up to 10 −9 . The RT-PCR primer combination [forward: 5′-TGG CGA TGA AGA TGT CAG-3′, reverse: 5′-CCA TTT CTG TGA AGG CTT T-3′ (834 bp)] was used previously to diagnose WSMV. The PCR template result was used for the nested PCR reaction to amplify the specific 299 bp gene (Lee et al., 2013a). The detection sensitivities of the two methods were compared. As a result, a specific band was formed at up to a 10 −2 dilution in the nested PCR but the specific ladder from the LAMP assay was analyzed at up to a 10 −3 dilution (Fig. 2). After LAMP assay, the prepared 10 µl of LAMP products were digested with 10 U of restriction enzyme BfaI (New England Biolabs, US) at 37 o C for 2 hrs, then they were subjected to electrophoresis in a 1.5% agarose gel and visualized (digestion fragments [194 + 156 bp]) ( Supplementary Fig. 2). The LAMP assay was applied to quickly screen for WSMV during quarantine inspection of imported wheat, corn, oats, and millet. Instead of using Taq DNA polymerase, LAMP amplifies denatures, anneals, and extends at the same temperature by using Bst polymerase, resulting in quicker detection than that of the existing PCR method. In addition, the 5' → 3' exonuclease feature provides high detection sensitivity and a quick result (Ahn et al., 2010;Cho et al., 2013). The LAMP developed in this study is simple and more specific rather than the current two-step quarantine methods using RT-PCR and nested PCR and reduced processing time by approximately 8 hrs. Furthermore, the LAMP method had 10 fold higher detection sensitivity than the current two-step PCR method. We expect that this LAMP method will be applied for use during quarantine inspections in the future.

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