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claran

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2016-05-12T22:15:10.714Z

2015-10-12T00:00:00.000Z

6702962

s2

Genome-Wide Identification of Genes Probably Relevant to the Uniqueness of Tea Plant (Camellia sinensis) and Its Cultivars Tea (Camellia sinensis) is a popular beverage all over the world and a number of studies have focused on the genetic uniqueness of tea and its cultivars. However, molecular mechanisms underlying these phenomena are largely undefined. In this report, based on expression data available from public databases, we performed a series of analyses to identify genes probably relevant to the uniqueness of C. sinensis and two of its cultivars (LJ43 and ZH2). Evolutionary analyses showed that the evolutionary rates of genes involved in the pathways were not significantly different among C. sinensis, C. oleifera, and C. azalea. Interestingly, a number of gene families, including genes involved in the pathways synthesizing iconic secondary metabolites of tea plant, were significantly upregulated, expressed in C. sinensis (LJ43) when compared to C. azalea, and this may partially explain its higher content of flavonoid, theanine, and caffeine. Further investigation showed that nonsynonymous mutations may partially contribute to the differences between the two cultivars of C. sinensis, such as the chlorina and higher contents of amino acids in ZH2. Genes identified as candidates are probably relevant to the uniqueness of C. sinensis and its cultivars should be good candidates for subsequent functional analyses and marker-assisted breeding. Introduction Tea (Camellia sinensis) is one of the most popular beverages in the world. It belongs to the genus Camellia (Theaceae: Ericales) and originated from East Asia [1]. The cultivation of C. sinensis probably started more than 2,000 years ago [1]. The extensive secondary metabolites in tea leaves, including polyphenols, theanine, and volatile oils, are good for people's health [2]. Nowadays, many cultivars of C. sinensis, such as Longjing 43 (LJ43), Zhonghuang 1 (ZH1), and Zhonghuang 2 (ZH2), are cultivated extensively in China. In addition to C. sinensis, the genus Camellia includes many other species of great value, such as C. oleifera and C. azalea. C. oleifera has been cultivated for thousands of years in China. It is a kind of small tree with multiple trunks and branches. Its seeds can be pressed to yield edible tea oil, more than 80% of which is monounsaturated fat [3]. Unlike the aforementioned two species, C. azalea is very precious and was discovered in Guangdong Province decades ago. This is a very beautiful plant with great ornamental value. A transcriptome is all the transcripts expressed in one or a population of cells at a certain time. With the advent of next-generation sequencing technology, a great number of transcriptomes, especially those from nonmodel species, have been reported. Given their important economic and ornamental values, transcriptomes of the above three Camellia species have been reported and some of their unique characteristics were identified. Specifically, Shi et al. described the transcriptome of C. sinensis and identified candidate genes related to natural product pathways that are important to tea quality, such as genes involved in flavonoid, theanine, and caffeine biosynthesis pathways [4]; Wang et al. compared the biochemical and transcriptomic differences between LJ43 and ZH2 to uncover mechanisms underlying their phenotypic differences [5]; and Xia et al. characterized the transcriptome from tender shoots, young leaves, flower buds, and flowers of C. oleifera and detected many genes potentially related to lipid metabolism [6]. Despite this body of work, the molecular events underlying the uniqueness of C. sinensis and its cultivars remain largely undefined. Identifying coding regions harboring mutations probably relevant to the uniqueness of a species or a cultivar has attracted the interests of many biologists for decades. Generally, nonsynonymous substitutions are harmful for their carriers and will be eliminated rapidly. However, a small number of nonsynonymous substitutions will benefit their carriers and genes harboring those mutations are termed positively selected genes. When a population extends its range or is moved by human activity to a new environment with environmental factors that are different to the original one, a series of genes may be subject to positive selection and may ultimately result in a new species. Comparing the numbers of nonsynonymous ( ) and synonymous ( ) substitutions per site is often used for diagnosing the extent and direction of selection on sequence evolution, with the ratio / >1, =1, and <1 denoting positive evolution, neutral evolution, and purifying evolution, respectively [7,8]. Church et al. demonstrated that using likelihood-based variable selection models is feasible for comparing sequence pairs [9]. In fact, many studies have reported analyses identifying positively selected genes for several different species [10][11][12]. It seems highly likely that a number of genes may be subject to positive selection during the speciation of C. sinensis. Moreover, during the cultivation of C. sinensis, many important agronomical genes may be subject to artificial selection and may therefore result in a new cultivar. In this report, based on the transcriptomes reported for the three species of the genus Camellia, we identified genes potentially relevant to the uniqueness of C. sinensis. Furthermore, candidate genes probably relevant to the divergence of LJ43 and ZH2 were also detected. Our results should be important for understanding the uniqueness of C. sinensis and its cultivars and provide hints for subsequent breeding. Data Acquisition and Filtering. Paired-end Illumina short reads generated for the floral bud transcriptome of C. azalea (PRJNA257896) [13] and 454 reads for transcriptomes of tender shoots, leaves, flowers, and flower buds of C. oleifera [14], were downloaded from the Sequence Read Archive at the National Center for Biotechnology Information (SRA, http://www.ncbi.nlm.nih .gov/sra/). In addition, paired-end Illumina short reads of the transcriptome of the two cultivars of C. sinensis, LJ43 (PRJNA261659, PRJNA240661, and PRJNA79643) and ZH2 (PRJNA261659), were downloaded from SRA. Quality controls were implemented using NGS QC Toolkit v2.3.3 with default settings [15]. Low quality bases that reside in short reads generated using Illumina and Roche 454 platforms were filtered by scripts included in this package. Single-end 454 reads were further processed using Seqclean (http://sourceforge.net/projects/seqclean/files/latest/download) to trim the vector sequences included in UniVec (ftp://ftp.ncbi.nih.gov/pub/UniVec/). De Novo Assembly. A de novo assembly based on pairedend Illumina short reads was performed using Trinity [16] with default settings for C. azalea. For the assembly of 454 reads of the other two species, the iAssembler package was used, which employs MIRA (http://sourceforge.net/projects/ mira-assembler) and CAP3 [17] and can assemble large-scale ESTs into consensus sequences with significantly higher accuracy [18]. For each species, contigs shorter than 200 bp were discarded in the ensuing analyses. For the de novo transcriptome assembly for each species, TransDecoder (http://sourceforge.net/projects/transdecoder/) was used for predicting the probable open reading frames. Identification of Orthologous Genes and Alignment. Tentative orthologs among the three species were predicted using a transitive Reciprocal Best Hits (RBH) approach implemented in the Ortholuge pipeline [19] with default settings except the -value for blastn being set to 1 − 9. For each ortholog group, we compared each nucleotide sequence with the corresponding protein sequence predicted for C. oleifera using Genewise [20] and used a customized Perl script to extract the matched coding regions and generate the proper alignment format for the subsequent PAML [8] analyses. We excluded alignments with premature stop codons or those shorter than 30 codons after deleting the gaps. Evolutionary Analyses. The evolutionary rates ( , , and / ) for each ortholog group and each separate species in the genus Camellia were calculated using CODEML under the branch-free model (model = ). To test for selection acting on C. sinensis, we used CODEML's branch-site models (model = 2, NSsites = 2), which allowed / to vary among codon sites and across branches of the phylogeny. By setting the branch leading to C. sinensis as the foreground branch, we compared a selection model that allowed a class of codons on that branch to have / > 1 (Model A2, fix omega = 0, omega = 1.5) with a neutral model that constrained this additional class of sites to have / = 1 (Model A1, fix omega = 1, omega = 1). A likelihood ratio test (LRT) with 2 approximation was used to determine the significance of difference between the nested models. All these analyses were implemented twice with different starting omega values to check for convergence. Alignments of positive results were checked and adjusted manually and subject to analyses once again to reduce the possibility of false positives. Mutations between LJ43 and ZH2 and Experimental Verification. All Illumina paired-end reads of LJ43 and ZH2 were used for identifying mutation sites potentially relevant to their divergence. For these two cultivars, all reads were mapped back to the nonredundant transcriptome using BWA (-n 0.005 -k 5) [21], and all duplicate reads were removed using the MarkDuplicates program from the software package Picard (http://broadinstitute.github.io/picard/). Reads with minimum mapping quality < 20 were removed using SAMtools [22], and then a synchronized file was generated using the program mpileup2sync in the software package PoPoolation2 [23]. The synchronized file listed allele frequencies for every population at every base in the nonredundant transcriptome in a concise format. Sites with base frequencies more than five in LJ43 but absent from ZH2 were identified as unique mutations and were compared with the predicted transcript structure to decide if they were located in the coding regions and changed the encoding amino acids. Due to the fact that RNA-Seq may produce false positives, mutation sites residing in some genes and probably related to the phenotype differentiation were selected and subject to experimental verification. Briefly, total RNAs from the leaves of LJ43 and ZH2 were extracted with Trizol Reagent Kit (Invitrogen, Madison, USA) and were reverse transcribed into cDNA using the M-MLV RTase cDNA synthesis kit (Takara, Dalian, China); sequences involved in the pathway "Porphyrin and chlorophyll metabolism" harboring nonsynonymous mutations were selected and amplified using gene-specific primers ( Table 1). Sequences of the amplified productions were bidirectionally sequenced using an ABI 3730 Model DNA sequencer (Shanghai Sangon, China) and submitted to Genbank. 2.6. Pathway Analysis. KEGG pathways were assigned to all the ortholog groups using the KOBAS software [24]. After that, the values for each pathway with more than fifteen genes assigned were compared between C. sinensis and the other two species separately using the binomial test. Contigs with mutation sites that encoded different amino acids between LJ43 and ZH2 or were identified as positively selected genes in C. sinensis were individually subject to pathway enrichment analysis using the KOBAS software. Differentially Expressed Gene Families between C. sinensis and C. azalea. The identification of differentially expressed genes between species is blocked by the accurate assignment of ortholog relationships, especially for nonmodel species for which whole genome sequences are unavailable. To understand the pivotal genes probably relevant to the uniqueness of C. sinensis such as the high content of flavonoid, theanine, and caffeine, we employed a strategy used for the identification of differentially expressed gene families cross species [25]. Briefly, the proteome of Arabidopsis thaliana was downloaded from Ensembl Plants (http://plants.ensembl.org/) and clustered as gene families using CD-HIT [26]. The sequence identity threshold was set to 0.6 and a representative sequence for each gene family was selected. Then, short Illumina reads for LJ43 and C. azalea were mapped back to the de novo assembled transcriptomes of C. sinensis and C. azalea separately, using bowtie [27] with default settings. After that, each sequence of the transcriptomes of the two species was uniquely mapped to the representative of each gene family, and the number of mapped short reads was accumulated if two or more sequences were mapped to the same gene family for each species. Finally, edgeR [28] was used to identify the differentially expressed gene families between the two species, and the method of Benjamini and Hochberg was used to adjust values for multiple comparisons [29]. Changes in expression patterns of gene families including genes involved in the pathways synthesizing "flavonoid," "theanine," and "caffeine" were scrutinized [4]. Results and Discussion With de novo assembly methods, we obtained 246,972, 103,002, and 141,099 sequences for C. azalea, C. sinensis, and C. oleifera, respectively. The number of sequences obtained for C. azalea was similar to that reported previously [13]. However, using the Newbler software, other researchers obtained 60,479 and 120,425 sequences for C. sinensis and C. oleifera, respectively [6,14]. The significantly greater number of sequences we obtained for these two species may be because Newbler performs best for restoring full-length transcripts [30,31], but iAssembler can identify incorrectly assembled contigs and should be more conservative [18]. Moreover, the greater number of sequences for C. azalea was much more than the other two species and may result from the different sequencing platforms and assemblers, since short reads for C. azalea were generated by using Illumina sequencing technology and the long reads for the other two species were generated by 454 pyrosequencing. Using the transitive RBH method, 8,787 one-one-one ortholog groups were identified and 4,617 groups were selected for subsequent evolutionary analyses. Distribution of alignment length for each ortholog group reveals that most groups are shorter than 600 bp. This may be because sequences used in the current analyses were generated by RNA-Seq and have a bias for shorter sequence reads (Figure 1). Using a likelihood method and the binomial test, we found that the evolutionary rates of genes involved in metabolic pathways were not significantly different among the three species (data not shown). The likelihood method identified a total of 97 sequences as positively selected genes in C. sinensis, and pathways that these sequences may participate in are shown in Supplemental Table S1 (in the Supplementary Material available online at http://dx.doi.org/ 10.1155/2015/527054). The pathways are mainly related to the metabolism of carbohydrates, lipids, amino acids, and some secondary metabolites, such as "glycolysis/gluconeogenesis," "steroid biosynthesis," "fatty acid degradation," "arginine and proline metabolism," "histidine metabolism," and "butanoate metabolism." However, genes participating in pathways synthesizing important secondary metabolites of the species, such as "flavonoid," "theanine," and "caffeine," were not identified in the current analyses. These results may be because positively selected genes involved in these pathways were not included in the original dataset and the conservatism of the method in the condition of few sequences [32]. Further investigations employing the transcriptomes of more species may address this issue. Changes of expression patterns of some pivotal genes may also contribute to the uniqueness of C. sinensis. To test this hypothesis, differentially expressed gene families between 4 International Journal of Genomics C. sinensis (LJ43) and C. azalea were identified. Specifically, profiles of the genes that participate in the synthesis of the three important secondary metabolites in C. sinensis are shown in Table 2. We found that most gene families including the genes that participate in the pathways synthesizing "flavonoid," "theanine," and "caffeine" were significantly upregulated and expressed in C. sinensis. In contrast, the significance of gene families including those genes that were downregulated in C. sinensis was not so notable, except the family including the gene "leucoanthocyanidin reductase." Thus, we suggest that the uniqueness of C. sinensis may result from the upregulation of some pivotal genes. However, the reliability of the results needs further investigation since these results are based on the comparison of expression patterns of gene families and employing the combined expression data from different tissues. Mutation analyses found polymorphisms present between LJ43 and ZH2 for more than 10,000 sites. Further investigations showed that 3,655 mutations were located in the coding regions and 2,021 of them were nonsynonymous mutations. Pathway analysis showed that genes harboring these nonsynonymous mutations were involved in a number of pathways. In particular, the pathways "alanine, aspartate, and glutamate metabolism," "Porphyrin and chlorophyll metabolism," "glycine, serine, and threonine metabolism," "valine, leucine, and isoleucine degradation," and "flavonoid biosynthesis" were well represented (Supplemental Table S2). Gene ontology analysis showed that genes with those nonsynonymous mutations were significantly enriched in GO terms "chloroplast part" and "chloroplast stroma" (Supplemental Table S2). To validate the mutations identified using high-throughput analyses, the sequences involved in the pathway "Porphyrin and chlorophyll metabolism" were selected and subjected to experimental verification. Bidirectional sequencing confirmed seven of the eleven nonsynonymous sites residing in these genes (Table 1). Biochemical analyses revealed that contents of free amino acids and flavonoid are different in the yellow-leaf tea cultivar ZH2 and normal cultivar LJ43 [5]. Our study suggests that nonsynonymous mutations residing in the coding regions of some genes may also take part in the formation of differences between the two cultivars, in addition to the differential expression of some other genes [5].

v3-fos

2020-04-30T09:03:18.333Z

2015-10-01T00:00:00.000Z

220452632

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Assessment of the Diversity in Fruit Yield and Fruit Components among Sri Lanka Tall Coconut Accessions Conserved Ex-Situ Characterization of conserved coconut germplasm has been undertaken globally for identification of important features of different accessions for them to be effectively used in coconut breeding. One hundred and fifty seven accessions comprising of local and exotic material have been conserved in ex-situ field genebanks of Coconut Research Institute in Sri Lanka. The objective of this study is to quantitatively characterize nut yield and fruit components by weights among Sri Lanka Tall (Typica) coconut accessions. Twenty local tall coconut accessions were characterized for nut yield and fruit components following Bioversity International descriptors for coconut. Bunch wise nut yield was recorded in all the coconut phenotypes in the six most mature bunches in 25 randomly selected palms from each accession. Sampled nuts were scored for weights of fresh nut, husked nut, split nut and kernel and the weights of husk, water and shell of each nut were derived from the scored data. Analysis of variance by general linear models procedure and mean separation by Duncan’s multiple range test were performed in SAS v8 and principal component analysis and cluster analysis using squared Euclidean distances were performed in Minitab V17. General linear models procedure revealed significant differences for nut yield and all the fruit components at 5% probability level. Walahapitiya recorded the highest average nut yield followed by the Razeena with statistically equal performances. Clovis recorded the highest values for most of the parameters for fruit component analysis followed by the accession Margaret, grouping together in Dendogram and the scatter plot. The highest per nut kernel producer, Clovis, was followed by Margaret with statistically equal performances and this is important because kernel is the main economically important component followed by the husk. Results revealed that there is no significant correlation between nut yield and all the fruit components in tall accessions indicating the importance of taking these two parameters separately to formulate germplasm conservation strategies. Introduction The Coconut palm, Cocos nucifera L. is the most important palm of the wet tropics and extensively grown palm tree worldwide. It is a diploid species with a chromosome number 2n = 32 and belongs to the family Arecaceae under monocotyledons. Every part of the plant is useful and, in many cases, human life in certain parts of the world would be impossible in its absence. Hence, it acts as an important fruit tree in the tropical regions and the fruit can be made into a variety of foods and beverages. Conservation of coconut germplasm has been undertaken globally in view of its economic importance. Coconut genetic resources have been traditionally collected and conserved in major coconut producing countries with the objective of using these to improve the genetic makeup of their existing cultivars. Selected germplasm are generally used as planting material to improve the coconut productivity, test material to determine the phenotypic and genotypic characters of value and population base for breeding superior hybrids/varieties (Baudouin and Santos, 2005). Currently coconut germplasm is conserved as accessions and there are 24 institutes managing ex-situ coconut field genebanks globally, totalling to more than 1500 accessions. These conserved germplasm is used for enhancing breeding strategies, used as national repository, facilitates materials for Germplasm exchange and used for characterizing and evaluating important traits. Characterization of coconut germplasm can be done in several ways; Morphological characterization, Biochemical Characterization and Molecular Characterization. Morphometric characterization can be done by using stem, leaf and inflorescence characters and nut yield and fruit components also can be used for further characterization. Literature revealed the availability of a number of publications for morphometric characterization using stem, leaf and inflorescence characters (Sankaran et al., 2012;) but, only a very few publications deal with nut yield and fruit characters Harries, 1981). Different components of the fruit provide major economically important produce offered by the coconut palm such as kernel, liquid endosperm, husk and shell. Furthermore, producing high quality fruit is a key breeding objective and overall breeding focus on quality traits includes; husk, shell and kernel proportions; kernel oil content and fatty acid composition; and coconut water levels and composition (Johnson et al., 2014). Therefore, the quantitative evaluation of each of these components by weight is a highly important aspect of morphological characterization as it directly deals with the yielding capacity of the coconut palm. Components of the fruit combined with the nut yield of the palm provide the actual yield of the coconut palm in terms of the total kernel, husk, water or shell produced by a given palm . Coconut in Sri Lanka is currently classified into 15 different coconut forms grouped under three varieties, namely Typica, Nana and Aurantiaca. The Coconut Research Institute of Sri Lanka (CRISL) initiated a systematic collection and conservation of coconut germplasm on 1984 (Wickremaratne, 1984). Currently, 157 accessions comprising of local and exotic material collected and have been conserved in the field genebanks of coconut at the CRISL (CGRD database). And also, coconut germplasm has been effectively utilized in breeding programs such as production of Brown Dwarf hybrids (Kumara et al., 2014 andDissanayake et al., 2012) and San Ramon hybrids (Perera et al., 2010). Further, these conserved germplasm display considerable diversity for morphological characters indicating the potential of them to be utilized in coconut breeding and the effectiveness of sampling in the conservation process. The objective of this paper is to quantitatively characterize nut yield and fruit components in tall coconut accessions at Pottukulama Research Station in Sri Lanka. Materials and methods Twenty coconut accessions belonging to the tall coconut variety (Table 1) conserved in Pottukulama ex-situ field gene bank were characterized for nut yield and fruit components following Bioversity IPGRI Descriptors for coconut (1995). All the accessions are categorized under Sri Lanka Tall variety (Figures 1 and 2) collected randomly covering different geographical locations except Clovis and Margaret were collected bias sampling method. The plants were 25-30 years of age at the time of data recording and were managed with average standard management practices for coconut. Bunch wise nut yield was recorded in all the coconut phenotypes in the 6 most mature bunches in 25 randomly selected palms from each accession. A total of 40 nuts from each coconut phenotype were sampled as 2 nuts from a palm. Sampled nuts were subjected to fruit component analysis and weights of fresh nut (FW), husked nut (HNW), split nut (SNW) and kernel (KW) were recorded and the fruit components husk weight (HW), water weight (WW) and shell weight (SW) of each nut were derived from the scored data. The data were analyzed using analysis of variance and general linear models procedure followed by mean separation procedures Duncan's multiple range test and least squares using statistical software package SAS version 8 and Minitab version 17 used for principal component analysis, distance matrix, correlation matrix, cluster analysis and dendrogram based on squared Euclidean distances. Nut yield General linear models procedure revealed significant differences (P= 0.006) at 5% probability level for scored nut yield. The average number of nut yield of the six most mature bunches in the studied coconut phenotypes varied from 28.6 to 46.2. Walahapitiya (acc no: 22) recorded the highest average nut yield followed by Razeena (acc no: 11) with statistically equal performances for mean separation procedures of Duncan's multiple range test ( Table 2). Further that is proven by recording the highest average number of fruits per bunch in Walahapitiya (acc no: 22) and Razeena (acc no: 11) indicating the possibility of utilization of these two varieties for future coconut breeding programs. Analysis of Variance General linear models procedure revealed statistically significant differences at 5% probability level among all of the quantitative traits scored. Clovis (acc no: 4) revealed the highest values for most of the parameters followed by Margaret (acc no: 7) for mean separation procedures Duncan's multiple range test (Table 3). The edible part of the coconut fruit (coconut meat and coconut water) is the endosperm tissue which gives the economic value to the fruit mainly (Yong et al., 2009). The 6.4 6.9 6.6 6.5 6.4 7.1 5.6 6.6 5.5 7.6 7.0 6.8 6.7 5.9 5.0 5.9 4.8 6.7 7.7 5.2 Means with the same letter are not significantly different ( ≤ 0.05). highest average per nut kernel producer, Clovis, was followed by Margaret with statistically equal performances and this fact is vital as kernel is the main economically important fruit component followed by the husk. Further, the highest average husk weight was recorded in Goyambokka (acc no: 15) followed by Wellawa (acc no: 23), with statistically significant differences between them. Multivariate Discrimination Principal component Analysis was used to describe the variation in the data set. The first three principal components (PC1, PC2 and PC3) accounted for 69.4%, 16.2% and 10.6% of the variation respectively accumulating to a total of 96.1% variability among the coconut accessions evaluated (Table 4) According to the PC scatter plot, the accessions were organized into several distinct groups which were further proved by the Phenetic tree by forming of major three groups of accessions at the 66.6% similarity level. Both plots revealed that Clovis (acc no: 4) and Margaret (acc no: 7) grouped together indicating the relative genetic similarity of the accessions Margaret and Clovis which has a history of an ancestry with the variety San Ramon (SNRT). Distance matrix and Correlation matrix Pair-wise distance matrix (Table: 5) revealed the highest distance between Clovis (acc no: 4) and Pitiyakanda (acc no: 3) and the lowest distance between the accession Goyambokka (acc no: 15) and St. Annes' (acc no: 6) among all accessions. Pearson correlation coefficients among the quantitative traits are presented in Table 6. Highly strong positive correlations (>0.75) were observed between many of the fruit components but, results revealed that there is no significant correlation between Nut Yield (NY) and the other fruit components. Therefore, nut yield cannot be taken as a predictor for fruit components in the studied tall accessions of coconut. Further, Kernel weight (KW) showed strong positive correlation with all the other fruit components except Husk Weight (HW). Conclusion Nut yield and the fruit components of all tall accessions at Pottukulama genebank were quantitatively characterized in this research. Accordingly, Walapitiya and Razeena reported the highest values for nut yield and grouped together in scatter plot and the dendogram. Further, these two accessions reported comparatively high values for fruit components as well. Clovis and Margaret scored the highest values for fruit component analysis and grouped together in scatter plot and the dendogram. Further evidence for this was given in the distance matrix by showing minimum distance (157.6) between these two accessions. Clovis is a San Ramon variety. The results identified Table 5. Pair-wise distance matrix among the accessions based on the scored morphological traits Margaret as the closest accession with Clovis indicating an exotic San Ramon origin, yet within the variety tall. Finally, results revealed diversity of nut yield and fruit components among tall accessions indicating the potential of them to be utilized in breeding programmes. The study also unveiled certain duplications among accessions with respect to fruit components which will help in formulating future conservation strategies.

v3-fos

2016-05-17T18:09:33.915Z

2015-05-31T00:00:00.000Z

14625132

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Pyramiding of three bacterial blight resistance genes for broad-spectrum resistance in deepwater rice variety, Jalmagna Background Jalmagna is a popular deepwater rice variety with farmers of India because of its good yield under waterlogged condition. However, the variety is highly susceptible to bacterial blight (BB) disease. The development of resistant cultivars has been the most effective and economical strategy to control the disease under deepwater situation. Three resistance genes (xa5 + xa13 + Xa21) were transferred from Swarna BB pyramid line, using a marker-assisted backcrossing (MAB) breeding strategy, into the BB-susceptible elite deepwater cultivar, Jalmagna. Results Molecular marker integrated backcross breeding program has been employed to transfer three major BB resistance genes (Xa21, xa13 and xa5) into Jalmagna variety. During backcross generations, markers closely linked to the three genes were used to select plants possessing these resistance genes and markers polymorphic between donor and recurrent parent were used to select plants that have maximum contribution from the recurrent parent genome. A selected BC3F1 plant was selfed to generate homozygous BC3F2 plants with different combinations of BB resistance genes. The three-gene pyramid and two gene pyramid lines exhibited high levels of resistance against the BB pathogen. Under conditions of BB infection, the three-gene pyramided lines exhibited a significant yield advantage over Jalmagna. The selected pyramided lines showed all agro-morphologic traits of Jalmagna without compromising the yield. Conclusion The three major BB resistance genes pyramided lines exhibited high level of resistance and are expected to provide durable resistance under deep water situation where control through chemicals is less effective. High similarity in agro-morphologic traits and absence of antagonistic effects for yield and other characters were observed in the best pyramided lines. Background Rice (Oryza sativa L.) is an important food crop that serves as a major carbohydrate source for nearly half of the world's population. In India, it is grown in 43 million hectares accounting for 42% of food grain production and 55% of cereal production. To sustain self-sufficiency and to meet food grain requirement of future, India has to produce 135-140 million tones of rice by 2030. This has to necessarily meet from less land, less water, less labor and fewer chemicals, constant battle against new emerging pathogens and pests and possible adverse effects from climate change (Khush 2005). This ecosystem covers around 4 million hectares of which 3 million hectares are under deepwater ecology and 1 million hectare under very deepwater ecology (floating rice).Under deepwater ecology, the crop remains waterlogged for a period of more than a month with more than 50 cm water depth while in floating type the water depth remains more than one meter. The average productivity of deepwater ecosystem is around 1 t/ha while floating type is again very low yield. Bacterial leaf blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is the most important disease of deepwater rice in India. In some areas of Asia, it can reduce crop yield by up to 50% (Khush et al. 1989) or even up to 80% (Singh et al. 1977). It also causes poor quality fodder. This affects photosynthetic areas and reduces the yield drastically and produce partial grain filling and low quality fodder yield. Although a large number of rice varieties have been released for different agro-ecosystems in India, only a few are widely grown in deep water situations. Rice varieties, Jalmagna and Dinesh are very widely grown in deep water areas of India. Theses varieties are popular among rice farmers and consumers because of its high yield, medium slender grains and excellent cooking and eating qualities. Despite popularity, these varieties are highly susceptible to many pests and diseases including BB. BB is the most important disease of deepwater rice in India and has become a major production constraint. In absence of effective chemical or other control agents against the pathogen in deepwater situation, host plant resistance has gained enormous importance in controlling this disease (Devadath 1989). Therefore, host plant resistance offers the most effective, economical and environmentally safe option for management of BB pathogen in deepwater situation (Khush et al. 1989). In other ecology also, development of resistant cultivars carrying resistant genes have been the most effective and economical strategy to control BB disease and no environmental pollutions (Huang et al. 1997;Jena and MacKill 2008;Singh et al. 2001;Sundaram et al. 2008;Rajpurohit et al. 2011;Dokku et al. 2013;Suh et al. 2013). Globally, thirty eight BB resistance genes have been identified from diverse sources (Bhasin et al. 2012). A number of these resistance genes have been tagged by closely linked molecular markers (Yoshimura et al. 1995;Sonti 1998;Rao et al. 2002;Gu et al. 2008). A few of these genes like Xa4 have been incorporated widely in many high yielding varieties through conventional breeding (Khush et al. 1989). However, widespread cultivation of varieties with Xa4 has led to predominance of Xoo races that can overcome this gene (Mew et al. 1992). The deployment of rice cultivars that have multiple BB resistance genes is expected to lead to more durable resistance. Pyramiding multiple R genes in a single line confers wide-spectrum and durable resistance. Tightly linked DNA markers have been developed for several BB resistance genes. The BB resistance genes, Xa1, xa5, xa13, Xa21, Xa26 and Xa27 have been cloned and used for breeding program. With the exception of xa5 and xa13, the BB resistance genes are dominant in nature and the markers developed from the sequencing information of these genes are widely used in MAS (Song et al. 1995;Yoshimura et al. 1998;Gu et al. 2005;Chu et al. 2006a). Using the gene pyramid approach, improved indica rice cultivars with broad spectrum durable BB resistance have been developed by combining different genes (Huang et al. 1997;Sanchez et al. 2000;Shanti et al. 2001;Singh et al. 2001;Joseph et al. 2004;Pha and Lang 2004;Bharatkumar et al. 2008;Hu et al. 2008;Perez et al. 2008;Sundaram et al. 2008;Rajpurohit et al. 2011;Dokku et al. 2013;Suh et al. 2013). A three-gene combination appeared to be the most effective; with Xa21 contributing the largest component of resistance. Therefore, incorporation of three BB resistant genes combination was taken up in the popular variety Jalmagna background by integrating markerassisted backcrossing with phenotypic selection for development of pyramiding lines for the handicapped ecology. Pyramiding of bacterial blight resistance genes The parent polymorphism was detected for the donor (CRMAS 2232-85) and recurrent parent (Jalmagna) with the markers pTA 248, RG 136 and xa5S, R (multiplex) for the genes Xa 21, xa13 and xa 5 respectively (Table 1). The parents were polymorphic with respect to these genes. In addition, the parents were screened with 236 rice microsatellite markers (Table 2) of which 120 were polymorphic and 60 were used for background selection. Molecular markers were integrated in the backcross breeding programme upto BC 3 F 2 generation. During the breeding procedure, foreground selection was practiced from F 1 generation till BC 3 F 3 generation at each stage to select the plants having resistance alleles of the three target genes and only progenies having the resistance alleles were advanced for the next generation (Figures 1, 2 and 3). Background selection was started from BC 1 F 1 to BC 3 F 1 generation and in each step genotype possessing highest genome content of the recipient parent was selected to hybridize for next backcross. A total of 650 F 1 plants were produced and 150 F 1 plants were tested for the hybridity and confirmed by their heterozygosity for the resistance gene linked markers of which 143 plants were observed to be true F 1 s. The true F 1 s were backcrossed using Jalmagna as a recurrent parent. These crossed seeds were raised (360 BC 1 F 1 seeds) for further backcrossing with Jalmagna. Ninety three BC 1 F 1 plants showed the presence of Xa21 resistance gene specific bands (1000 bp) while 91 plants showed the presence of xa13 resistance gene specific bands (490 bp and 530 bp). One hundred sixteen BC 1 F 1 plants showed the presence of xa5 resistance gene specific bands (160 bp). Based on the amplification of resistance specific bands, 31 BC 1 F 1 plants showed the presence of Xa21 and xa13 resistance genes while 42 BC 1 F 1 plants showed the presence of Xa21 and xa5 resistance genes. Forty six BC 1 F 1 plants showed the presence of xa13 and xa5 resistance genes. Only fourteen plants showed the presence of three BB resistance genes Xa21, xa13 and xa5. Out of these 14 BC 1 F 1 progenies, plant showing 77.5% of recurrent genome (Plant No.53) was backcrossed with recurrent parent Jalmagna (Table 3). Total of 122 BC 2 F 1 progenies were produced of which, twenty one, thirty three and thirty six BC 2 F 1 plants showed presence of resistance genes, Xa21, xa13 and xa5, respectively. Based on the amplification pattern, 11 BC 2 F 1 plants showed the presence of Xa21 and xa13 resistance genes while 13 BC 2 F 1 plants showed the presence of Xa21 and xa5 resistance genes. Twenty three BC 2 F 1 plants showed the presence of xa13 and xa5 resistance genes. Only nine plants exhibited the amplification of three resistance genes Xa21, xa13 and xa5. The background selection of these nine BC 2 F 1 plants with sixty polymorphic SSR markers exhibited the presence of 88.13 % to 91.82 % with an average of 90.95% of recurrent genome content. The plant containing 91.82% genome content of Jalmagna (Plant No.53-21) was used for backcrossing (Table 3). A total of 285 BC 3 F 1 backcross derivative progenies were produced by backcrossing the plant showing 91.82% recurrent genome with the recipient parent, Jalmagna. Twenty eight BC 3 F 1 plants were positive for Xa 21, 35 for xa 5 and 14 for xa 13. Eighteen BC 3 F 1 plants showed the presence of Xa21 and xa13 resistance genes while 14 plants showed the presence of Xa21 and xa5 resistance genes and 14 plants showed the presence of xa13 and xa5 resistance genes. Only fourteen plants showed the presence of three resistance genes Xa21, xa13 and xa5. These BC 3 F 1 plants showed recurrent genome content of Jalmagna ranging from 91 to 97% with an average of 92.38% (Table 3). BC 3 F 1 derivative SPJ53-21-77 and and SPJ53-21-25 showed more than 95% genome content of recipient parent were self pollinated to obtain the derivatives of BC 3 F 2 generation. In BC 3 F 2 generation, plants homozygous for three and two bacterial blight resistance gene combinations were identified. It is observed that 26 plants containing Xa21, xa13 and xa5 genes; 31 plants with Xa21 and xa5; 31 plants with Xa21, xa13 and 30 with xa13 and xa5 amongst the BC 3 F 2 derivatives. The plants with three and two genes were grown as BC 3 F 3 lines. Bioassays Bioassays conducted against eight isolates of Xoo confirmed the resistance and susceptible reaction of the donor (CRMAS 2232-85) and the recurrent (Jalmagna) respectively with the donor showing smaller range of At each backcross generation, fewer than expected triple heterozygotes were obtained. This is due to the fact that some of the putative backcross progeny were obtained by inadvertent selfing of Jalmagna (the female parent in these backcrosses). c At each backcross generation, genomic DNA was isolated from derivative lines that are triple heterozygotes for BB resistant gene linked markers. Microsatellite markers that are polymorphic between the parental lines were then used, as described in Materials and Methods, to identify the plant with maximum recurrent parent genome contribution. d As per Mendelian ratios for independent gene action. average lesion lengths (2.1-2.8 cm) while on Jalmagna, the lesion lengths were longer (9.4-12.8 cm) ( Table 4). The results indicated that the pyramided lines were better as compared to recurrent parent, Jalmagna with regard to bacterial leaf blight tolerance. Screening of the BC 3 F 3 pyramided lines against Xoo isolates exhibited that all the pyramid lines were more effective in comparison to the recipient parent. The lesion lengths observed on the lines containing Xa21 + xa13 gene combination varied from 3.1 to 3.9 cm ; for Xa21 + xa5 combination, 3.5-4.8 cm ; for xa5 + xa13 combination 4.9 to 5.7 while 1.4 to 2.9 cm lesion length present in pyramided line containing xa5 + xa13 + Xa21 combination. The individual values for the donor parent and recurrent parent are in the range of 1.7-3.3 and 9.0-13.3 cm respectively. Though all the gene combinations tested did not show any susceptible reaction to any of the eight isolates employed, the gene pyramids with three genes displayed higher levels of disease resistance with shorter lesion lengths against all BB isolates. Results indicated that the degree of severity of the disease from the data, the order of gene combinations in conferring Yield and agro-morphological traits of the pyramided lines Fourteen three-gene pyramid and six two genes pyramid lines at BC 3 F 3 generation along with the donor and recipient parents were evaluated during wet season, 2013 at CRRI, Cuttack. The recipient parent, Jalmagna recorded mean grain yield of 17.35 g/plant, while the donor parent (Swarna BB pyramided line) recorded 20.5 g/plant. The test entries viz., SPJ23, SPJ25, SPJ50, SPJ51, SPJ52 and SPJ77 showed grain yields higher than recurrent parent, Jalmagna (Table 5). Many test entries did not show any significant variation as compared to Jalmagna in terms of flowering duration, panicles/m 2 , plant stature as well as other characters that are considered under distinctness, uniformity and stability (DUS) tests. The genetic distance coefficient on 14 agromorphologic traits of 20 pyramids and two parental lines revealed that two clusters were observed and it is interesting to note that all the pyramided lines are similar to Jalmagna -12.6 ± 1.7 11.4 ± 1.4 12.8 ± 1.5 9.4 ± 1.2 11.6 ± 1.6 9.8 ± 1.8 10.2 ± 1.7 11.6 ± 1.8 S Figure 4A). Background selection The background selection was carried out for estimating the recurrent parent's genome content in the pyramided lines. Background selection was performed by using 60 SSR markers among the lines possessing three resistance gene combinations in BC 1 F 1 , BC 2 F 1 and BC 3 F 1 generations. At BC 3 F 1 generation, a total of 120 alleles from 60 markers were observed. The similarity co-efficiency among all lines ranged from 0.791 to 0.952 suggesting a high level of genetic similarity between the pyramids and Jalmagna. The dendrogram generated using the SSR data grouped the 14 three-gene pyramid lines into two major clusters ( Figure 4B) with cluster I having CRMAS Analysis of genome introgression on the carrier and noncarrier chromosomes In CRMAS2232-85/Jalmagna combination, 5-6 microsatellite markers on each of three carrier chromosomes in the genomic region flanking to xa5, xa13 and Xa21 were polymorphic. Based on six markers analysis, all the 14 lines showed heterozygosity for donor segment introgression of xa5 between marker HYV59 and HYV5-37 in BC 3 F 1 generation while exhibited homozygocity for Jalmagna genome and no drag to xa5 gene was observed. In the flanking region of xa13, for five polymorphic markers, nine lines showed introgression of the donor segment of marker HYV14. In case of Xa21, six pyramid lines showed genetic drag of donor segment with the marker segment RM144 ( Figure 5). Discussion Integration of molecular markers to the backcross breeding was highly effective for transfer of three bacterial blight resistance genes. Phenotypic selections in three backcrossing and two selfing generations coupled with SSR based background selection was sufficient for transfer of Xa21, xa13 and xa5 genes into popular deepwater variety Jalmagna background. Jalmagna is a very long duration and strongly photo-sensitive variety. Again under deepwater situation, control of the disease using chemicals was a very difficult task. Here, integrating molecular markers and using very less generations and advancement of some generations under RGA helped to obtain the broad spectrum BB resistant lines for deepwater ecology. The transferred genes in the recombinants did exhibit high level of resistance against the most virulent BB isolates that is comparable to the reaction level of CRMAS 2232-85, the donor parent and the results are similar to earlier reports (Huang et al. 1997;Sanchez et al. 2000;Singh et al. 2001;Shanti et al. 2001;Bharatkumar et al. 2008;Hu et al. 2008;Perez et al. 2008;Sundaram et al., 2008;Rajpurohit et al., 2011;Dokku et al., 2013;Suh et al., 2013). The three gene combination pyramided lines expressed higher levels of resistance in comparison to parental lines, two and single gene combination. The results suggest that two gene combinations with Xa21 + xa13 was most effective with shorter lesions lengths followed by Xa21 + xa5 while lines with xa13 + xa5 were relatively less effective. Lines with Xa21 in combination with either xa5, xa13, or both have shown promise advocating the utility of Xa21 in achieving higher levels of resistance in rice as reported earlier (Singh et al. 2001;Sanchez et al. 2000;Sridhar et al. 1999;Huang et al. 1997) suggesting that synergistic action and/or quantitative complementation between the resistant genes might result in enhanced levels of resistance (Sanchez et al. 2000). All the three resistance genes that have been considered in the present work have been cloned and characterized. Xa21 is a dominant resistance gene that encodes a receptor kinase containing NBS-LRR domains (Song et al. 1995), while xa5 is a recessive resistance gene and encodes a variant form of transcription factor cIIa (Iyer and McCouch 2004). The xa13 resistance gene is also recessive in nature and has been shown to be a mutation in the promoter region of a gene that is a homolog of the nodulin MtN3 (Chu et al. 2006b). In rice lines containing the dominant (susceptibility) allele of the gene, the expression of the nodulin homolog is up regulated upon infection with Xoo. It appears that the increased expression of this gene is necessary for Xoo to grow on rice. This up regulation does not occur in rice lines containing the resistance (recessive) xa13 allele (Yang, 2006). The apparently different modes of action of the three resistance genes used in this work might contribute to make the resistance in the three-gene pyramid lines quite durable. There is a variation in the theoretically expected value of contribution from the recurrent parent genome to the BC 1 F 1 plants and in other backcross generations. As per reports of Sundaram et al. 2008, there might be exercising a "pull" for introgression of the Xa21, xa13 and xa5 genes during selection, which favors inheritance of additional unlinked loci from the donor genome in BC 1 F 1 plants and BC 2 F 1 generation. But, we found no pull effect during the transfer of Xa21, xa13 and xa5 genes to different backcross generations. Selection of plants similar to the recurrent parent from BC 3 F 1 stage was the strategy followed in the study and the graphical genotyping data supports that view as genotype SPJ53-21-77 had 97% of the recurrent parent genome having donor segments of target resistance genes xa5, xa13 and Xa21 and further no linkage drag in regions flanking Xa21, xa13 and xa5 is observed. The high recurrent genome recovery observed in many pyramid lines may be due to the use of more number of polymorphic microsatellite markers. Similar results were obtained in case of Sundaram et al. 2008;Dokku et al. 2013;Suh et al. 2013 suggesting more number of background markers. No genetic linkage drag was observed for the transfer of genes Xa21, xa13 and xa5 ( Figure 5) may be due to mega variety used as the donor source for BB resistance genes. The mega variety, Swarna is a highly adapted variety for the favorable ecology. Results indicated that a broad based highly adapted variety as source of donor may give better performance and less drag as compared to the wild and land races as donor. It is expected that all the favorable genes are accumulated in the mega variety and subsequently transfer of some of these genes is improving further the background of the pyramided lines. The approach used in the study ensured the realization of the major objective resulting in the release of a cultivar with enhanced resistance to BB and accelerated recovery of recurrent genome with better yield. Yield and agro-morphologic data of 20 pyramided two parental lines revealed that the pyramided lines possessed excellent features of recurrent parent and also yielding ability with tolerance to bacterial blight resistance. This indicates that some pyramid lines are very close to the recurrent parent and some are even better than the recurrent parent with respect to yield. The higher yield of the pyramided lines may be due to inheritance of some yield traits or QTLs of mega variety (Swarna BB pyramid) used here as the donor parent, besides the recurrent parent Jalmagna to the pyramided line. The complete recovery of the yield and grain quality characters of Jalmagna along with transfer of three BB resistance genes of CRMAS 2232-85 is a very significant achievement. This is particularly so because yield and agro-morphologic traits is multigenic traits encoded by loci that are distributed across the rice genome. The traits recovery of Jalmagna was due to integration of many polymorphic markers in the backcross breeding program. As per our analysis, we find that there is a variation from the theoretically expected 75% contribution from the recurrent parent genome to the BC 1 F 1 plants. All the selected BC 1 F 1 plants had a recurrent parent genome contribution more than the expected 75%. Again in BC 2 F 2 , it had highest gemone content of 91.8% along with the target genes. During BC 3 F 1 , the genome content of Jalmagna in selected derivative (SPJ53-21-77) was as high as 97%. The background selection with many markers accelerated the recovery of recurrent genome suggests that selection for introgression of the Xa21, xa13 and xa5 genes has no antagonistic effects for yield and other traits. The field evaluation of BC 3 F 3 progenies showed that the best entry had better yielding than the Jalmagna parent. Besides, BB resistance, the pyramided line. SPJ53-21-77 was better yielder than its recurrent parent and equivalent to agro-morphological traits and grain quality features of the recurrent parent. The high levels of resistance to BB and the absence of any yield penalty due to accumulation of resistance genes in the pyramids provides us a successful example of the integrated approach of selection at both molecular and phenotypic levels for transfer of the desired trait(s) and recovery of the recurrent parental genome. Development of broad-spectrum resistance against BB in the Indian subcontinent is a major challenge due to the rich diversity of the agroclimatic zones where rice is cultivated, as well as the presence of a number of genetically distinct virulent Xoo strains in different geographical areas of India. Deployment of a three gene combination like xa5 + xa13 + Xa21 can achieve durable and broad-spectrum resistance in many BB prone rice growing areas in India including the deepwater ecosystem. The study clearly establishes the utility of MAS in pyramiding recessive genes like xa5 and xa13, and dominant gene Xa21 to present a multiple gene barrier against one of the most destructive diseases of rice in a long duration, photosensitive and deepwater rice. Conclusion Marker-assisted backcrossing using functional markers reduce the risk of false selection in recombination between the molecular marker and the gene of interest. We were successful in identifying superior recombinations for three BB resistance genes (Xa21, xa13 and xa5) in the homozygous condition in a long duration, photosensitive and deepwater rice variety. The pyramided genotypes can be further be used for multilocation testing to be released as variety in the country or be used as potential BB resistance donors. The BB pyramided deepwater breeding lines, which are developed through MAS and phenotypic selection, will be of practical value in providing durable bacterial blight resistance in the deepwater growing region where control through chemicals under deepwater situation was less effective. These BB pyramided lines are expected to have a high impact on the yield stability and sustainability of deep water rice production. Plant materials and breeding method The donor parent CRMAS 2232-85, a derivative of Swarna and IRBB 60 cross contains three BB resistance genes xa5, xa13 and Xa21 in the background of mega variety Swarna. The donor parent was developed at Central Rice Research Institute (CRRI), Cuttack, India (Sundaram et al. 2014). The recurrent parent was Jalmagna, a highly popular variety of deepwater ecosystem of India but highly susceptible to bacterial blight disease. Jalmagna was hybridized with CRMAS 2232-85 and F 1 plants were backcrossed with recipient parent Jalmagna. Marker-assisted backcross method was followed up to BC 3 generation and around 200 plants/lines were genotyped at each generation for the presence of the target genes and only positive plants having the resistance alleles were advanced to the next generation. Foreground selection continued till BC 3 F 3 to identify pure homozygous lines for all three target genes while background selection was up to BC 3 F 1 generation. Selection based on foreground, background and morphological traits was practiced from BC 1 F 1 onwards for identification of lines that were similar to the recurrent parent. Rapid Generation Advancement (RGA) facility was used during dry season as Jalmagna was a strongly photo-sensitive and very long duration variety. The schematic diagram for development of BB pyramided lines is presented in Figure 6. Screening for bacterial blight resistance For field evaluation against BB, the inoculums of eight predominant Xoo isolates of Orissa prepared by suspending the bacterial mass in sterile water to a concentration of aproximately10 9 cells/ml (Kauffman et al. 1973). Four leaves from four different plants of each entry were clip inoculated at the maximum tillering stage and lesion lengths (LL) were recorded after 15 days. The disease symptoms were scored as resistant (R, LL ≤ 3.0 cm), moderately resistant (MR, 3.0 cm < LL ≤ 6.0 cm), moderately susceptible (MS, 6.0 cm < LL ≤ 9.0 cm) or susceptible (S, LL > 9.0 cm) (Amante-Bordeos et al. 1992). Characterization for agro-morphological traits Thirty days' old seedlings of the BC 3 F 3 pyramid lines and the parents (Jalmagna and CRMAS 2232-85) were transplanted in three rows with twenty five plants per row per entry at 15 × 20 cm spacing under a randomized complete block design with two replications at the experimental farm of Central Rice Research Institute (CRRI), Cuttack. Data were recorded on ten plants from each entry and replication for agronomic traits like plant height, tillers/plant, panicle length, number of filled grains/panicle, 1000-grain weight, flag leaf, 2 nd leaf length and breadth while days to 50% flowering was recorded on whole plot basis data analysis was performed using SAS statistical software (SAS Institute Inc. 2010). DNA isolation and PCR amplification Mini scale DNA isolation for PCR analysis was carried out as per Dellaporta et al. (1983). The PCR reaction mixture contained 50 ng templates DNA, 5 pico mole of each of the primers, 200 μM dNTPs, 1 X PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, and 0.01 mg/ml gelatin) and 0.6 unit of Taq DNA polymerase in a volume of 20 μl and amplification of target sequences were as per earlier reports ( Table 1). The PCR products of STS marker RG 136 were digested with restriction enzymes HinfI as per manufacturer's instructions. The PCR products and the DNA fragments produced by restriction digestions were separated by gel electrophoresis and gel images were analyzed on gel documentation system (SynGene). Marker analysis The primers employed for the three target genes were all from published reports (Table 1). Of the 236 SSRs markers used for parental polymorphism survey, 120 were found to be polymorphic between the parents (range 4-6 per chromosome) and 60 were used for background selection. Data were analyzed and similarity matrix was constructed from binary data with Jaccard's coefficients and dendrogram was generated with unweighted pair group method arithmatic average (UPGMA) algorithm, using FreeTree software (Hampl et al. 2001;Pavalíce et al. 1999) and the dendrograms were visualized by Treeview 32 software (Page 1996). Graphical Geno Types (GGT) Version 2.0 (Van Berloo 1999) software programme was used for the assessment of the genomic contribution of the parent in the selected recombinants based on SSR marker data.

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THE ROLE OF MINERAL NUTRITION ON YIELDS AND FRUIT QUALITY IN GRAPEVINE, PEAR AND APPLE 1 – Fertilization of temperate fruit trees, such as grapevine ( Vitis spp.), apple ( Malus domestica ), and pear ( Pyrus communis ) is an important tool to achive maximum yield and fruit quality. Fertilizers are provided when soil fertility does not allow trees to express their genetic potential, and time and rate of application should be scheduled to promote fruit quality. Grapevine berries, must and wine quality are affected principally by N, that regulate the synthesis of some important compounds, such as anthocyanins, which are responsible for coloring of the must and the wine. Fermenation of the must may stop in grapes with low concentration of N because N is requested in high amount by yeasts. An N excess may increase the pulp to peel ratio, diluting the concentration of anthocyanins and promoting the migration of anthocyanins from berries to the growing plant organs; a decrease of grape juice soluble solid concentration is also expected because of an increase in vegetative growth. Potassium is also important for wine quality contributing to adequate berry maturation, concentration of sugars, synthesis of phenols and the regulation of pH and acidity. In apple and pear, Ca and K are important for fruit quality and storage. Potassium is the most important component of fruit, however, any excess should be avoided and an adequate K:Ca balance should be achieved. Adequate concentration of Ca in the fruit prevents pre- and post-harvest fruit disorders and, at the same time, increases tolerance to pathogens. Although N promotes adequate growth soil N availability should be monitored to avoid excessive N uptake that may decrease fruit skin color and storability. INTRODUCTION Temperate fruit trees, such as the grapevine (Vitis spp.), apple (Malus domestica Borkh.), and pear tree (Pyrus communis L.) should be fertilized whenever the soil cannot provide a sufficient amount of nutrients to feed the plant for maximum yield. However it appears that this simple concept is not yet fully understood. In general, in the most traditional fruit production regions, fertilization programs are based on soil fertility, tree requirements and crop nutritional status. The results of soil and tissue analyses are compared with critical nutrient ranges according to the probability that plants respond to additions of nutrients. Some systems also consider other recommendation criteria, such as expected yield, plant growth, which may be estimated by the length of new shoots, and fruit analysis, as is the case of trees (CQFS-RS/SC, 2004). Not all fertilizer recommendation guidelines for temperate fruit crops are based on results of long term regional studies. Some recommendations for fruit trees, such as those established in the first versions published in the Fertilization and Liming Manual for the States of Rio Grande do Sul and Santa Catarina (2014), lack of results collected regionally (CQFS-RS/SC, 2004). Fertilization is considered one of the most effective tool to increase the profits in fruit tree cropping; the relatively low cost of fertilizers and the positive response of tree vegetative and reproductive organs to added nutrients triggered a general use of fertilizer with low attention to actual tree requirements. However, in view of the increasing global market, it is now strategic to improve fruit quality offering a product that meets customer demand in terms of organoleptic characteristics, functional properties and environment. Excessive nutrient availability compared to plant requirements convey negative results such as excessive plant vigor, decreased yield and fruit skin color, abortion of flowers (MARTÍN et al., 2004;JONES, 2013;MARTÍN et al., 2004), as well as increased incidence of fungal diseases on leaves and fruit (HUBER; THOMPSON, 2007). Often, an increase in fruit yield is not observed because soil nutiernt availability is already within the adequate range (MENGEL; KIRKBY, 2001); above upper threshold, toxicity symptoms are expected. Sometimes even when a soil nutrient availability is lower than lower threshold, trees do not respond to fertilization because the nutrient reserves build up in perennial organs in previous years (BRUNETTO et al., 2014a). The practice of fertilization may affect fruit quality from morphological, physical, chemical, and organoleptic points of view. In particular, fruit quality with reference to chemical composition may be estimated through macro-and micronutrients, pH, total soluble solids (TSS), total or titratable acidity, organic acids, anthocyanins, total polyphenols, vitamins, etc. (SMART, 1991;CHADHA;SHIKHAMANY, 1999). The quality of fruit must meet the standards for fresh market, storage or fruit processing. Consequentely fertilization management should be adjusted to the final destination. The aim of this review is to provide information regarding the effects of the application of nutrients on yields and fruit chemical composition of three temperate climate species, namely grape, pear and apple. Nitrogen The application of nitrogen (N) on grapevines should be undertaken with caution because of its conflicting effects on vegetative growth, yield, and chemical composition of the grapes, must and wine (BELL;HENSCHKE, 2005;BRUNETTO et al., 2007). High rates of nitrogen fertilizers as NO 3 --N and NH 4 + -N, may stimulate vegetative growth hence reducing solar radiation within the plant canopy, favoring the incidence of fungal diseases on the leaves and fruits, reducing the number of pollinated flowers, producing a lower number of berries per bunch and, in the end, delaying leaf senescence and plant dormancy (KELLER et al., 1999;DUCHêNE et al., 2001;BRUNETTO et al., 2007BRUNETTO et al., , 20092012b). In addition, the excessive vigor of grapevines and the lack of light within the canopy may reduct the activity of enzymes that regulate the synthesis of some important compounds, such as anthocyanins (red-colored phenolic compounds) which color the must and the wine. An increase of the pulp to peel ratio dilutes the concentration of anthocyanins and stimulates the migration of anthocyanins located in berries to growing plant organs, including the new shoots (KELLER; HRAZDINA, 1998;KELLER et al., 1999;TESIC et al., 2007;BRUNETTO et al., 2009). As a result, the yield and composition of the grape, must and wine could be affected. The effect of N on grape yield depends mostly on soil physico-chemical characteristics. In sandy soils with low organic matter content, a yield increase is normally expected in grape after the addition of N fertilisers (GOLDSPINK; GORDON, THE ROLE OF MINERAL NUTRITION ON YIELDS AND FRUIT... 1991;BELL;ROBSON, 1999), although exceptions are also possible, considering the low N requirement of grape, N accumulated reserves in the plant and organic N mineralization (BRUNETTO et al., 2011(BRUNETTO et al., , 2014a. For example in an experiment carried out in the region of Campanha in Rio Grande do Sul, South Brazil, applications of 0, 15, 30, 45, 60, and 85 kg N ha -1 as urea (BRUNETTO et al., 2007) and compost in an Hapludalf soil did not influence the yield and quality of the 'Cabernet Sauvignon' grapevine. The lack of response of grape to added N was expected expecially in clay or clay loam soils with medium to high organic matter content, that generally provides sufficient amounts of mineral N though mineralization (DELAS et al., 1991;DAL Bó, 1992). Nitrogen fertilization influences the different aspects of grape quality from cluster morphology (length, width and weight of bunches to number of berries per bunch, weight of the berries, etc.), juice chemical composition (pH, TSS, total titratable acid, organic acids, anthocyanin, and total polyphenols), and organoleptic properties (CHADHA; SHIKHAMANY, 1999). For example, tartaric and malic acid represent more than 90% of all the acids in the berry and indicate the stability and longevity of the wine. The anthocyanins are found in larger quantity in the peel and determine the color of the grape, the juice, and the wine. Total polyphenols, also found in greater amounts in the peel, normally increase as response to N fertilization and contribute to color intensity, tonality, and taste characteristics of grape and wine (CHADHA;SHIKHAMANY, 1999). In the central region of Washington State, USA, the pH, total N, and ammoniacal N in grape and must increased linearly with rates of N (0, 56, 112, and 224 kg N ha -1 ) (SPAYD et al., 1994), but showed effect on K concentration, TSS, and malic and tartaric acids in the must. Other reports (SPAYD et al., 1995), showed a negative effect of 0, 30, 60, and 90 kg N ha -1 on TSS. In California, rates of 56 and 112 kg N ha -1 reduced TSS in the grape and the must (CHISTENSEN et al., 1994). In Portugal, tartaric and malic acid, and total acidity responded positively to added N (0, 30, 60, and 90 kg N ha -1 ) (PACHECO et al., 1997). In largest grapevines and wine producing region in Brazil, 'Cabernet Sauvigon' showed a decrease in the anthocyanin concentration in the must , with increasing rates of soil applied N in the form of urea. In the same area of Southern Brazil, the addition of organic compost as N source, did not affect pH, TSS, total titratable acidity, and tartaric and malic acids in the grape (MELO et al., 2012). The highly variable effects of added N reported in literature may be due to the contrasting soils and organic matter contents. Thus, discussion of the results is normally restricted to the amount of N applied and its impact on the yield and composition of the grape and must. Application of N increased concentration of total N, ammoniacal N, and biotin in the must (OUGH et al., 1968), especially on 'Merlot' (BERTRAND et al., 1991) grown in a sandy soil grafted on different rootstocks. Fermenation of the must may stop in when using grapes with low concentration of N forms because, after C, N is used in greatest amount by yeasts and bacteria. Thus, the concentration of N in the must has an impact on microbial biomass, the rate and time of fermentation, and the final products of the microbial metabolism (CANTARELLI, 1957;BISSON, 1991) such as alcohols and aroma-producing compounds in wine (RAPP; VERSINI, 1991). Potassium Potassium (K) is one of the macronutrients most required by grapevines and a large amount is exported through havest. Potassium availability to grape may be assessed by leaf analysis at full bloom and at berry veraison. However, it is not always possible to determine K accurately in the leaves, because most K is present free that can be redistributed rapidly to growing organs (i. e. berries), or stored in reserve organs such as branches and roots (TAGLIAVINI; SCANDELLARI, 2013). Leaf K is not always related to yield (BOOTERM et al., 2010) because plants, in general, take up an amount of K greater than their metabolic needs, and accumulated it into cell organelles in luxury consumption . However, few studies reported an increase in the number and weight of bunches, where was combined with N or when the K fertilizations was applied to soil with a K availability below the critical level for sufficiency (BRUNETTO et al., 2012b). As much as 50% of total K taken up by the grapevines accumulates in berries. Its functions in the fruit are related to synthesis reactions and enzymatic activation, directly contributing to fruit maturation, sugar synthesis, and the maintenance of cell turgor. In addition, through its mobility in the phloem and xylem, K is important in the transport of solutes, the partition of assimilates, and the synthesis of polyphenols responsible for fruit color and aroma. Nevertheless, an excess of K may be undesirable in terms of adequate pH and total titratable acidity in the must (KODUR, 2011). The pH indicates the ionization capacity of the must and normally G. BRUNETTO et al. should remain below 3.5, while total titratable acidity represents the amount of free acids. Both are related to the quality (organoleptic and visual characteristics) and stability of the must and wine (oxidation capacity) (MPELASOKA et al., 2003;WALKER;BLACKMORE, 2012). Under conditions of high K concentration, for example, there may be a stoichiometric exchange of the protons of tartaric acid and K, leading K bitartrate, a salt that precipitates and decrease organoleptic quality of the wine. This leads to a reduced transport of malic acid to the cytoplasm, a decrease of its degradation rate (MPLEASOKA et al., 2003). As a result tartaric:malic acid ratio decreases, resulting in the increase of pH, and wine susceptibility to oxidation and microbiological damages, Phosphorus Phosphorus (P) is needed for the formation of cell membranes, carbohydrate metabolism, protein synthesis, photosynthesis, respiration sugar metabolism, energy storage and transfer (FREGONI, 1980). In addition, P may confer special characteristics to the wines produced in a given region. For example, Zalamena et al. (2013ab) observed that cover plants increased the P concentration in leaves and the P the concentration of anthocyanins in wine. In contrast, P concentration considered adequate for wine-making promotes the fermentation of the must, hence the organoleptic quality of the wine in terms of aroma and flavor (POMMER, 2003). In geral, the P demand and the amount exported through harvesting is small. In addition, the mutualistic symbiosis between plant roots and endomycorrhizal fungi may facilitate P acquisition and limit the need for P fertilization (SIQUEIRA; MOREIRA, 2001). In Brazil, soils have a low content of available P. For that reason, when soil analysis shows P deficiency, phosphate fertilizers are applied at pre-planting and throughout the production cycle. If soil P is not sufficient to meet plant requirements, grapevines may show symptoms of P deficiency such as low vigor, yellowing or reddening of the basal leaves and early leaf drop. When P added blindly, P accumulation may occur, in the soil and the proportions of P forms change in the soil. As available P forms accumulate in the soil, the P could be transferred through surface runoff or underground flow (SCHMITT et al., 2014). Furthermore, high P contents in the soil may reduce the availability of Zn, because of the formation of Zn phosphate; consequently, symptoms of Zn deficiency may be observed in grape leaves (SKINNER; MATTHEWS, 1989). C a l c i u m , m a g n e s i u m a n d s o m e micronutrients In the acid soils of Southern Brazil, limestone is often applied before grape planting with the aim to increase soil Ca and Mg contents and to reduce Al toxicity. Liming increases base saturation of the cation exchange capacity at the expense of Al saturation. An increase of Ca tissue concentration is expected after soil application of lime leading to the activation of phosphatase and peptidase and increase rigidity of the cell walls of fruit and other grape organs (LECOURIEUX et al., 2006). An increase in Mg contents is also expected to improve chlorophyll synthesis (POMMER, 2003). Some studies reported that in soils with high levels of exchangeable K, symptoms of Mg deficiency in grape leaves and even desiccation of the rachis may occur resulting in physiological disturbance and yield decrease (HALL et al., 2011). In the uppermost soil layers of the most important areas for grapevine industry worldwide, an accumulation of Cu and Zn arises from the application of fungicides and the addition of organic residues as a source of N, P and K, (BRUNETTO et al., 2014b). The accumulation of Cu and Zn in the soil modifies the distribution of the forms of those elements, increasing the most labile forms that possibly contaminat surface and ground waters, especially in soils with a sandy texture and with low organic matter content (CASALI et al., 2008). Excessive accumulation in the soil is toxic to many crop species . In addition, an excess of Cu and Zn may be toxic to young growing grapevines and cause physiological and biochemical stress to grapes . This may impair the production and the chemical composition of the grape and, consequently, of the must and the wine. However, the actual negative impact of excessive Cu and Zn on yield and grape composition should be evaluated in relation to soil properties. PEAR Nitrogen Optimal concentration of N in fruits allows a proper development of skin color, fruit size and flavor (Table 1). Fruit N concentration depends on grafting combination (cultivar and rootstock), environment and orchard management (Table 2), with reported value of 2.5 g N kg fruit dw -1 for 'Abbè Fetel' in Italy (QUARTIERI et al., 2002) to 4.5 g N kg fruit dw -1 for 'Conference' in Belgium (DECKERS et al., 2011), to 9.8 g N kg fruit dw -1 in THE ROLE OF MINERAL NUTRITION ON YIELDS AND FRUIT... 'Bartlett' grown in Argentina (SANCHEZ, 2002). Fruit N concentration is higher during the first stage of fruit development (cytochinesis) and decreases thereafter during fruit growth until harvest (Table 2). An excessive soil N availability in the summer may delay fruit maturation, have a negative impact on TSS, and decrease plant tolerance to pests and diseases as psylla (Cacopsylla pyri L.) symptoms on 'D'Anjou' (RAESE; STAIFF, 1989) and ' Bartlett' (DAUGHERTY et al., 2007), fire blight (Erwinia amylovora) (VAN DER ZWET; KEIL, 1979) and post harvest blue mold (Penicillum expansum) on 'Conference' (SUGAR et al., 1992). Optimum N availability at the end of winter dormancy allows a prompt leaf development that provides assimilates for new fruit and shoot growth. Early N deficency may reduce leaf area, with the ensuing negative effect on fruit yield and quality. If N deficiency persists during the season, fruits may remain small in size and return poor flavor (Table 1). As already reported for apple (MILLARD, 1995;TOSELLI, 2000), and pear (TAGLIAVINI et al., 1997), spring growth relies on N stored in permanent organ such as roots, trunk, branches and twigs during the previous year. As a result, if the N supply has been adequate in the previous season, N application can be postponed after floral petal drop. As in the case of apple, N applied late in the season, immediately before harvest, is not partitioned to the fruits but is stored then remobilized in the following spring during flower development (SUGAR et al., 1992). Under the climatic conditions of the Po valley in Italy, 39% of pear fruit N at harvest, comes from previous year storage, 50% is taken up between March and May (first 2 months after bloom), and only 11% of N is from soil-uptaken from end of June to harvest, during the last 2 months of fruit growth (QUARTIERI et al., 2002). Consequentely pre harvest (4-5 weeks before harvest) N applications do not increase the N:Ca ratio and pear susceptibility to pre-and post-harvest decay (SUGAR et al.,1992;TOSELLI et al., 1998). Potassium Potassium (K) is the most aboundant nutrient in the fruit where it affects positively the size, firmness, skin color, TSS, acidity, juiciness and aroma. This nutrient is also important during storage since an imbalanced K:Ca ratio may promote cork spot in 'D'Anjou' (CURTIS et al., 1990) and 'Alexander Lucas' (TOMALA;TRZAK, 1994). Because pear trees generally show higher absorption and transport of Ca to the fruit than apple (MARCELLE, 1995), the negative effect of K on quality of stored pear fruits is less frequent than in apple fruits. Potassium concentration in fruits is stable during the growing season ranging between 0.8 to 1.0%, and it is 30-40% higher in the peel than to the pulp. As observed for N:Ca, the K:Ca ratio is also higher in the flesh (often >30) than the peel (<10). Because K is antagonist to both Ca and Mg, if K increases in the soil over the normal range, both Ca and Mg uptake may decrease, as reported in 'Abbè Fetel' in Northern Italy. Phosphorus and magnesium Due to little plant requirement, deficiency is unlikely to occur. Phosphorous concentration decreases toward the interior of the fruit (FAUST et al., 1967); in the pear fruit, the optimum P concentration range lies between 700 and 1000 mg P (kg dw -1 ). Magnesium uptake can be strongly depressed by K + , NH 4 + , Ca 2+ (MARSCHNER, 1995). Deficiencies in plant tissues are common in acid soils, particularly where the soil is high in plant-available K. In pear fruits, Mg ranges between 350 and 1000 mg kg dw -1 depending on fruit stage ( Table 2). The Mg accumulates linearly at a slow rate throughout the growing season (TAGLIAVINI et al., 2000). Calcium Calcium differs from other nutrients because it is transferred to fleshy fruit in amounts much smaller than leaves (SAURE, 2005). Despite Ca sufficiency in most orchard soils, localized Cadeficiency-related disorders such as bitter pit in apple and pear fruit may become a serious problem. The dynamics and factors affecting Ca transfer to the fruit are still not fully understood (SAURE, 2005). Some authors reported that Ca uptake by the fruit occurs only during the first part of fruit growth (FAUST, 1989) or linearly until harvest (ZAVALLONI et al., 2001). Regardless the dynamics of fruit Ca-accumulation, Ca has a low vascular mobility. Consequentely, its uptake and partitioning to the fruits is most consistent in the first stage of fruit development. For this reason it is essential to promote Ca uptake early in the season, approximately within the first 40-50 days after blooming (SHEAR; FAUST, 1971;SCHLEGEL;SCHOENHERR, 2002). Calcium is involved in cell physiology, the integrity and stability of cell membranes, organization of the cell wall, and tolerance against fungal and bacterial infections (BATEMAN; LUMSDEN, 1965), because Ca is associatedto polygalacturonic acid as exchangeable Ca pectate (MARSCHNER, 1995). The Ca pectate is considered to be the Ca fraction best associated with fruit suitability for G. BRUNETTO et al. storage. The Ca pectate was found to range between 25% in untreated trees to 30% in soil fertilized In fruits of 'Abbè Fetel' the Ca pectate (TOSELLI et al., 2012). On the other hand, high Ca concentration inhibits the activity of polygalacturonase and delays ripening of fruits (MARSCHNER, 1995). Optimum fruit Ca concentration promotes fruit firmness, increases disease tolerance and reduces storage related disorders. Fruit Ca accumulation is higher at the beginning of fruit development (during fruit cytochinesis), reaches 1800-2000 mg Ca kg dw -1 , and decreases thereafter throughout the season until harvest, ranging from 200 to 1000 mg Ca kg dw -1 with average values of 300-400 mg Ca kg dw -1 (Table 2). Many physiological disorders and susceptibility to pre-and post-harvest fungal decay are related to the Ca status of pear fruits. In 'Doyenne du Comice', a negative correlation was found between Ca fruit content and fruit susceptibility to superficial scald (MARCELLE, 1995). It has been reported that storage disorders such as internal breakdown of 'Passacrassane', senescent breakdown of 'Abbè Fetel' and 'Bosc' (GORINI, 1988), corkspot of 'D'Anjou' (CURTIS et al., 1990) and 'Alexander Lucas' (TOMALA; TRZAK, 1994).pre-and postharvest diseases such as brown spot (Stemphylium vesicarium), particularly frequent in 'Abbé Fetel' and 'Conference' can be reduced through increased fruit Ca concentration up to a threshold of 1000 mg Ca kg -1 (TOSELLI et al., 2012). Side rot (Phialophora malorum) in 'Bosc' was reduce applying three mid-summer sprays of 3.6 and 6 g Ca L -1 as CaCl 2 (SUGAR et al., 1992). Considering the low mobility of Ca and the little permeability of the fruit cuticle during most phenological stages, foliar and soil applications of Ca fertilizers often show a low effectiveness in promoting Ca accumulation in fruit in both controlled and field studies on calcareous soils in Northern Italy (TOSELLI et al., 2012). Under such environmental conditions, largest effects were obtained with early (bloom) soil applications. Foliar Ca applications have been reported to be effective in sandy, low in organic matter, soils of the US Pacific Northwest (SUGAR et al., 1992), as well as in silt loam soils of Poland (GASTOL; DOMAGALA-SWIATKIEWICZ, 2009). Foliar applications of Ca promoted Ca accumulation in fruit of 'Conference' from 93 to 125 mg kg -1 fw (fresh weight basis) in 2004 and from 76 to 101 mg kg -1 fw in 2005.. Other variables may affect fruit Ca composition such as genotype and rootstock. It was observed that fruits of 'Abbè Fetel' grafted on quince 'Sydo' were more tolerant to brown spot than when grafted on seedling clone 'Fox 11', with a higher fraction of Ca pectate compared to 'Fox 11' ( Table 3). 'Sydo' rootstock was led higher leaf and fruit Ca concentrations (Table 3) (QUARTIERI et al., 2013). According to Tomala and Tszark (1994) cork spot (pear bitter pit) in 'Alexander Lucas' could be predicted using the following equation R 2 = 0.84): X= 5.46 + 1.33 (fruit Mn) -0.13 (fruit Ca) + 0.11 (fruit Mg) where: Mn, Ca and Mg are expressed in mg (kg dw -1 ). In the Hood river valley of Oregon, threshold for cork spot in 'D'Anjou' was found to be peel N:Ca ratio > 6.3 (CURTIS et al., 1990). On the other hand it was reported (SUGAR et al., 1992) that where fruiting is dense, N is diluted while Ca concentration increases, in agreement with the concept that a smaller leaf:fruit ratio must lead to a higher fruit Ca concentration (SUGAR et al., 1992). Hence, where fruits occur to be sparse, the fruit N:Ca ratio is likely to be relatively high and the fruit to be more susceptible to defects (SUGAR et al., 1992). In mature fruits, Ca concentration increases towards the core (SAURE, 2005); the peel is approximately 4 times higher in Ca than the pulp. At the same time, peel N is usually twice as high as flesh N; as a result, the N:Ca ratio is lower in peel (<10) than in pulp (>10) (GASTOL; DOMAGALA-SWIATKIEWICZ, 2009). The pear trees take up NH 4 + -N preferentially to NO 3 --N (MALAGUTI et al., 2001). Because Ca 2+ competes with NH 4 + -N and K + ; high application of ammonium may inhibit Ca root uptake. This is why ammonium fertilizers should be applied at least 40 days after blooming (FAUST, 1989), and split-applied at 3 occasions such as fruit set, fruit cell enlargement and end of summer (immediately before havest or post-harvest). The application rates should be calibrated on the basis of soil mineral-N (NO 3 and NH 4 + ) (TAGLIAVINI et al., 1996). The N can also be dissolved in fertigation water and applied at regular intervals from flower petal drop to harvest. Micronutrients Among micronutrients, boron (B) is one of the most critical in pear orchards. Indeed, it is believed that pear trees have a high B requirement (WOJCIK; WOJCIK, 2003). Typical symptoms of B deficiency are the reduction of fruit set and yielding, as well as small, deformed, cracked and corked fruits (Table 1). Optimum fruit B concentration at harvest may range between 16 and 20 mg B kg -1 dw -1 (RAESE, 1989). An adequate B concentration promotes Ca mobility, regulates flowering and fruit set, and contributes to a stable fruit production. In Poland, foliar application of B to 'Conference' increased fruit Ca concentration THE ROLE OF MINERAL NUTRITION ON YIELDS AND FRUIT... and storability by reducing the permeability of cortex cells and the incidence of internal browning (WOJCIK;WOJCIK, 2003). Since B is mobile in the trees of the Rosacee family (BROWN et al., 1999), the most appropriate application period is the end of summer when B is stored in permanent organs and can be remobilized to the developing fruits the following spring (SANCHEZ et al., 1998). Despite the abundance of iron (Fe) in soils, Fe acquisition by fruit crops is often impaired, compromising fruit yield and quality. The occurence of Fe chlorosis depends on several environmental and agronomic factors (TAGLIAVINI; ROMBOLà, 2001) such as soil, climatic conditions, grafting, etc. Iron concentration in pear fruits is low, ranging from 20 to 35 mg Fe kg dw -1 (Table 2). In calcareous soils, Fe can be a yield-limiting factor when trees are grafted on 'quince', the most common rootstock in Italy, that has no ability to solubilize Fe from Fe(OH) 3 . To avoid yield losses, it is a common practice to fertilize pear trees grafted on 'quince' with synthetic Fe chelates and to include sustainable orchard management practices (TAGLIAVINI; ROMBOLà, 2001;ÁLVAREZ-FERNÁNDEZ et al., 2004) that includes grass intercropping, soil organic matter buildup, improvement of soil drainage, etc. Manganese (Mn) concentration in pear fruits is very low ranging from 2 to 4 mg Mn kg dw -1 ( Table 2). The Mn deficiency can severely reduce fruit yield and shows symptoms similar to Fe chlorosis; it may occur in alcaline and calcareous soils, but also in soils with a high content of organic matter (MARSCHNER, 1995). The Mn deficiency in pear fruits is more common with 'quince' rootstocks. Pear is considered to be a (Zn) sensitive species FAUST, 1980). At low level of soil Zn availability, plant growth is impaired, and fruit set and yields are limited. Under such conditions, fruits are small, deformed and sour, and ripen early. Pear responds promptly to Zn foliar application. Where Zn deficiency occurs, pre-bloom Zn sprays are successful to increase Zn concentration in flowers while post-bloom Zn sprays are effective in promoting leaf and fruit Zn levels. However, fruit trees require little Zn in addition to Zn-based sprays to controlling diseases; in this case, Zn foliar sprays are not successful in improving plant growth and fruit yield and quality (WóJCIK; POPIñSKA, 2009). Copper concentration in pear fruits is low, ranging from 5 to 13 mg Cu kg dw -1 (Table 2). Pear is one of the crops most subjected to Cu sprays for controlling diseases in low impact agricultural systems (TOSELLI et al., 2009). As a result, soil Cu accumulation is frequent and may lead to Cu toxicity symptoms. Copper uptake by roots and plant susceptibility to Cu toxicity depend on soil texture, pH, hydrous oxide content, and clay mineralogy (BRUN et al., 2001;EPSTEIN;BASSEIN, 2001;MENGEL;KIRKBY, 2001;TOSELLI et al., 2008). High soil Cu concentration was found to reduce the photosynthetic activity of pear trees, indirectly by inhibiting the uptake of Mn and Zn -. Copper toxicity is common in sandy, low pH soils; in loam to silt loam soils high in organic matter, the toxicity threshold exceeds 1000 mg Cu kg -1 (TOSELLI et al., 2008). APPLE Nitrogen All other factors but N being equal, the higher the N availability in the soil and the higher the N uptake, the higher will be the vegetative growth of apple trees. Shoot growth responds more to enhanced soil N availability than root growth and crop productivity. Annual N uptake data based on estimates of tree growth crop productivity and mineral nutrient concentration of tree organs suggest that N removals from soil are in the range of 60 to 75 kg N ha -1 yr -1 , depending on yields (TAGLIAVINI; SCANDELLARI, 2013). The N allocation to fruits for yields from 40 to 60 t FW ha -1 are often in the range 20 to 30 kg N ha -1 (Table 4). From growth resumption in spring and until two weeks from full bloom, apple trees use mainly N derived from remobilization, while root N uptake becomes the main N source thereafter. As recently indicated in Zanotelli et al. (2014) N uptake occurs at highest rates from 37 to 81 days after full bloom then starts to decrease (Table 5). Foliar-applied N is absorbed rapidly with higher efficiency and therefore represents an interesting mean to supplement soil N supply (TOSELLI et al., 2004), especially in postharvest, when soil N supply often results in increase risks of N leaching due to autumn and winter rainfall. Apple nutrient concentration depends on cultivars and years, being often in the range of 300-400 ppm. Excessive soil N availability causes luxury consumption and depresses apple fruit color in redcolored varieties mainly because of increased shading caused by excessive shoot growth; it can moreover depress shoot hardening (lignification), making them more susceptible to winter frost damages. Because N is highly mobile in the soil, the higher is the N supply the higher the risks of N losses by leaching or by volatilization. Potassium In mature and highly productive apple orchards, K is often the nutrient absorbed at highest rates. Apple fruit, in fact, is a strong sink for K and it normally contains significant amounts of K, with concentration ranging from 0.55 to 0.8 kg K Mg -1 fruit d.w. (ZAVALLONI et al., 2001). Therefore annual K uptake strongly depends on fruit yields and can range from 80 to 100 kg K ha -1 with yields ranging from 40 to 60 Mg fruit (f.w) ha -1 . The K deficient apple trees normally have leaf K concentration in summer less than 1% K (dw basis), show reduced leaf photosynthesis and produce fruits with less sugars. Optimum sugar to acid ratio and fruit size are often reported in soils well endowed with K. Trees under excessive K supply show sometimes Ca-related disorders and have excessive fruit K/Ca ratios. Similarly to N, the highest rate of K uptake by apple trees occurs after cell division and lasts for at least 5 weeks (Table 5); K uptake rates remain relatively high until fruit harvest. Due to its high phloem mobility, K allocation to fruit remains relatively stable from fruit set to fruit maturity (ZAVALLONI et al., 2001). Calcium Main issues related with Ca uptake and partitioning as well as the physiological role of Ca in fruits have been discussed in details in the preceding section. In apple, Ca is involved in the development of physiological fruit disorders such as bitter pit especially in post-harvest. On the other hand, increased fruit Ca content by pre-harvest Ca spray applications reduced the infection by Gloeosporium, an internal breakdown and softening of apples. Post-harvest CaCl 2 applications are also known to reduce the decay caused by Penicillium and other fungal diseases. Total fruit Ca concentration is often in the range of 200-400 ppm (dw), but differs between the peel and the flesh, where it can reach much higher concentrations (>700 ppm). Apple cultivars differ as to their susceptibility to Ca-deficiency related bitter pit, with 'Gala' and 'Golden Delicious' being less susceptible than 'Breaburn', 'Red Decilious' and 'Stayman'. Soils in the main apple districts worldwide are often well endowed with Ca and Ca-related disorders are the consequence of internal problems of proper fruit Ca allocation rather than in Ca uptake. Total Ca uptake by apple trees can be comparable N; for instance, Scandellari et al. (2010) estimated that 74 kg Ca ha -1 are taken up annually by 'Gala' trees, then allocated as follows: 11% in the tree framework, 4% in fruits, 60% in leaves and 25% in pruned wood. Several susceptible apple varieties (e.g. 'Cox's Orange Pippin') accumulate most Ca within 4-6 weeks from full bloom, but Ca flux into fruit can continue (even at lower rates) until 120-140 days in 'Golden D. ', 'Breaburn' and 'Fuji' (ZAVALLONI et al., 2001), provided that Ca partitioning to vegetative growth is not excessive. When fruits reach large sizes (e.g. in off years) the amount of fruit Ca becomes very diluted and fruits are more prone to develop bitter pit. Pre-harvest as well as post-harvest Ca sprays (usually in the form of CaCl 2 or Ca (NO 3 ) 2 ), in the range of 0.3-0.6% w:v) are the most widely means for preventing bitter pit. Cuticle Ca uptake rate per unit of fruit surface is higher when fruits are young, and decreases thereafter (SCHöNHERR, 2002); considering that larger fruits in a later stage of development have larger cuticular surface, both early and late sprays are effective and often included in the management pratices where susceptible varieties are grown. Thus, grapevine berries, must and wine quality are affected by the addition of nutrients, principally by N, that regulate the synthesis of some important compounds, such as anthocyanins, which are responsible for coloring of the must and the wine. Fermenation of the must may stop in grapes with low concentration of N because N is requested in high amount by yeasts. Nitrogen excess may increase the pulp to peel ratio, diluting the concentration of anthocyanins and promoting the migration of anthocyanins from berries to the growing plant organs. Potassium is also important for wine quality contributing to adequate berry maturation, concentration of sugars, synthesis of phenols and the regulation of pH and acidity. In apple and pear fruit, Ca and K are important for fruit quality and storage. Potassium is the most important component of fruit, however, any excess should be avoided and an adequate K:Ca balance should be achieved. Adequate concentration of Ca in the fruit prevents pre-and post-harvest fruit disorders and, at the same time, increases tolerance to pathogens. Nitrogen availability should be monitored to avoid THE ROLE OF MINERAL NUTRITION ON YIELDS AND FRUIT... excessive N uptake that may decrease fruit skin color and storability. (2003) Mg (mg kg -1 ) 700-1000 350-700 Quartieri et al. (2002) Sanchez (2002) Fe ( Alexandria, v.124, p.347-352, 1999.

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Effects of Dietary Fermented Chlorella vulgaris (CBT®) on Growth Performance, Relative Organ Weights, Cecal Microflora, Tibia Bone Characteristics, and Meat Qualities in Pekin Ducks Fermented Chlorella vulgaris was examined for its effects on growth performance, cecal microflora, tibia bone strength, and meat qualities in commercial Pekin ducks. A total of three hundred, day-old male Pekin ducks were divided into three groups with five replicates (n = 20 ducklings per replicate) and offered diets supplemented with commercial fermented C. vulgaris (CBT®) at the level of 0, 1,000 or 2,000 mg/kg, respectively for 6 wks. The final body weight was linearly (p = 0.001) increased as the addition of fermented C. vulgaris into diets increased. Similarly, dietary C. vulgaris linearly increased body weight gain (p = 0.001) and feed intake (p = 0.001) especially at the later days of the feeding trial. However, there was no C. vulgaris effect on feed efficiency. Relative weights of liver were significantly lowered by dietary fermented C. vulgaris (linear effect at p = 0.044). Dietary fermented C. vulgaris did not affect total microbes, lactic acid bacteria, and coliforms in cecal contents. Finally, meat quality parameters such as meat color (i.e., yellowness), shear force, pH, or water holding capacity were altered by adding fermented C. vulgaris into the diet. In our knowledge, this is the first report to show that dietary fermented C. vulgaris enhanced meat qualities of duck meats. In conclusion, our study indicates that dietary fermented C. vulgaris exerted benefits on productivity and can be employed as a novel, nutrition-based strategy to produce value-added duck meats. INTRODUCTION Chlorella vulgaris (C. Vulgaris) is a genus of single-cell green algae belonging to the Phylum Chlorophyta and has been widely acknowledged as a functional food worldwide. It is rich in protein with balanced essential amino acid profiles, lipid, carotenoids, vitamins and minerals (Kay, 1991). Due to its rapid multiplication, it is considered as a useful protein candidate among future food resources (Kang et al., 2004). Earlier, intact C. vulgaris was known to have low protein digestibility, which was mainly attributed to the presence of rigid cell walls (Shelef and Soeder, 1980). However, protein digestibility of C. vulgaris fermented with beneficial bacteria or yeasts was dramatically improved in rats (Buckenhüskes et al., 1990;Keijiro, 2011). In addition to the use of the protein resource for humans, C. vulgaris was also tested as a replacement of antibiotics in foodproducing animals (Yan et al., 2012). Dietary C. vulgaris was reported to possess immunemodulatory activity in broiler chickens (Kotrbacek et al., 1994), to increase growth performance in pigs (Kim, 2011;Yan et al., 2012), and to enhance egg quality in laying hens (Zheng et al., 2012). Despite there being several experiments investigating fermented C. vulgaris in broiler chickens, laying hens and pigs, the utilization of fermented C. vulgaris as a duck feed additive has never been reported. In the present study, we investigated the effects of fermented C. vulgaris on growth performance, meat qualities, cecal microflora, relative weights of organs, and tibia bone characteristics in Pekin ducks. vulgaris product (CBT ® , Celltech, Co. Ltd, Eumsung, Korea) was used which was produced by the patent fermentation process as described earlier (Zheng et al., 2012). Briefly, C. vulgaris was inoculated with baker's yeast and lactic acid bacterium. After 72 h of dry fermentation with cereal broth, fermented C. vulgaris was then dried at low temperature and ground into powder. The analyzed chemical contents of the CBT ® were as following; crude protein (CP), 24.85%; crude fiber, 5.36%; ether extract, 2.42%; Ca, 0.20%; and P, 0.65%. Experimental design, animals and management Three hundred day-old male Pekin ducks were divided into three dietary groups with five replicates (n = 20 ducklings per replicate) in a completely randomized design model. The control basal diet was formulated to meet or exceed the nutrient recommendations of the NRC (1994) and Korean feeding standard for poultry (2012). The starter basal diet in a crumble form was fed from hatch to 3 wk, and contained 22% CP and 2,950 kcal nitrogen corrected true metabolizable energy (TMEn)/kg of diet. The grower basal diet in a pellet form was provided from 3 to 6 wk, and had 18% CP and 3,100 TMEn/kg of diet. The formula and chemical compositions of the experimental diets are shown in Table 1. The supplement CBT ® was added into the basal diet to the inclusion rate of 0, 1,000, or 2,000 mg/kg. The diet and water were freely available. The ducklings were initially reared at 33°C, and the room temperature was gradually decreased by 4°C weekly to reach 22°C and kept constant thereafter. They were housed on floor pens with rice husks as a bedding material with a 23 h light and 1 h dark cycle throughout the experimental period. All animal care procedures were approved by the Institutional Animal Care and Use Committee at Konkuk University. Sampling and measurements The body weight (BW) and feed intake on a pen basis were recorded weekly and used to calculate gain:feed ratio. At the end of experimental period, eight ducks per pen were randomly selected, weighed individually and euthanized by cervical dislocation. At necropsy, right breast and leg meats, liver, spleen and abdominal fat were immediately removed, blotted onto dry towel, weighed and expressed as relative weights per 100 g of BW. For microbial test, cecal content per bird was aseptically sampled into preweighed sterile tubes and homogenized followed by serial 10-fold dilution with sterile phosphate buffered saline as described elsewhere (Miller and Wolin, 1974). Bacterial total microbes were enumerated on nutrient agar (Difco, BD science, Detroit, MI, USA), presumptive lactic acid bacteria on MRS agar (Difco, BD science, Detroit, MI, USA) and presumptive coliform bacteria on MacConkey agar (Difco, BD science, Detroit, MI, USA). All inoculated plates were incubated aerobically at 37°C for 24 h. The results obtained were presented as base-10 logarithm colony-forming units (cfu) per gram of cecal content (Tuohy et al., 2002). To determine the cooking loss, 60 g of fresh breast meat was boiled in polyethylene bag, immersed in 80°C water bath for 30 min, and cooled to room temperature for 30 min. The cooking loss was calculated as the difference between the uncooked and cooked meat (Bouton et al., 1971). The water holding capacity was estimated according to the filter paper pressed method (Grau and Hamm, 1953). Approximately, 300 mg of breast meat was weighed on a Whatman filter paper (No. 2, UK) and the sample was pressed between two plexiglass plates for 3 min. The areas of pressed sample and water were measured using planimeter (Type KP-21, mitutoyo, Kawasaki-shi, Japan). The pH of breast meat was measured in triplicate with a pH meter (Model 340, Mettler-Toledo, Urdorf, Switzerland). Briefly, 1 g of breast meat was cut into small pieces, homogenized with 9 mL of distilled water for 1 min in an Ultra-Turrax (Model No. T25, Janke and Kunkel, Staufen, Germany). The instrumental colors of fresh meat, including lightness (L*), redness (a*) and yellowness (b*), were measured by a reflectance colorimeter (CR 210, Minolta, Tokyo, Japan) using illuminant source C. Color was measured in triplicate on the bone-side surface of each sample. The colorimeter was calibrated using a standard white ceramic tile. The right tibia (meat removed) was used to analyze the breaking strength using an Instron Materials tester (model 3342, Instron Corp., Canton, MA, USA) with Automated Materials Test System software version 4.2. The weights, diameters, and lengths of right tibia were measured. Tibia diameters were calculated as the mean value of both narrow and wide parts. The deformation rate was 5 mm/min. Tracing of force was recorded at a constant rate. The graphs showed the plateau curve of applied maximal force (KN) to measure the bone strength as expressed as energy stored in the bone. After measuring the bone breaking strength, the bones were dried at 105°C, weighed, ashed in a muffle furnace at 600°C for 24 h, cooled in a desiccator, and weighed again. Statistical analysis The experimental data were analyzed using Generalized Linear Model procedures of SAS (SAS Institute, 2002). Orthogonal polynomials contrasts were used to determine the linear and quadratic effects of the dietary CBT ® supplementation according to the following general model: Y = μ+α+ε, where Y was the observed response variables; μ was the overall mean; α was the effect of diet and ε was the random error. The pen was considered as an experimental unit. An alpha level of 0.05 was considered the statistical significance. Growth performance The effect of dietary fermented C. vulgaris on growth performance is presented in Table 2. The final BW was linearly (p = 0.001) increased as the inclusion rate of fermented C. vulgaris into diets increased. Similarly, dietary C. vulgaris linearly increased the BW gain (p = 0.001) and the feed intake (p = 0.001), especially from 1 to 42 days post-hatch. However, there was no significant effect on feed efficiency between the treatment and control groups. Thus, it is concluded that the increased BW in C. vulgaris-treated ducks is the consequence of the increase in feed intake, but not by efficient nutrient utilization. It has been suggested (Zheng et al., 2012) that fermentation of C. vulgaris may improve its flavor as the underlying mechanism of increased feed intake in laying hens. However, it is well understood that taste sensors in poultry are less developed compared with those of the mammals. Thus, further study is needed to clarity the increased feed intake by fermented C. vulgaris in Pekin ducks as seen in this study. Relative weights of various organs The effect of dietary fermented C. vulgaris on relative weights of the various organs and tissues are presented in Table 3. Relative weights of liver were significantly lowered by dietary fermented C. vulgaris (quadratic effect at p = 0.091). At this stage, a clear explanation of the decreased relative weights of liver in response to feeding fermented C. vulgaris is unknown and requires further study. Our finding is in contrast to the previous study (Zheng et al., 2012) which showed no effect of C. vulgaris at the inclusion rate of 1,000 or 2,000 mg/kg in diet on the relative weights of liver in 80-wk-old laying hens compared with those of the control group. The difference in the relative liver weight between our study and Zheng et al. (2012) could be the breed difference as we used the rapidly growing meat-type duck while the latter used slow-growing laying hens. On the other hand, spleen, abdominal fat, and the breast and leg muscles of ducks when adjusted to 100 g of BW were not affected by supplementation of the fermented C. vulgaris. Cecal microflora The findings by Pratt et al. (1944) who reported that chlorellin, the active component in Chlorella, has an antibiotic effect, and by Amaro et al. (2011) who reported that methanol extracts of C. vulgaris lowered E. coli and Salmonella, prompted us to measure the population of cecal microflora in this study. As shown in Table 4, dietary fermented C. vulgaris did not affect total microbes and lactic acid bacteria in cecal contents (Table 4). However, the population of cecal coliform bacteria in ducks fed diet with 2,000 mg/kg fermented C. vulgaris tended to be lower compared with their control-diet fed counterparts (linear effect at p = 0.064), indicating that C. vulgaris may have a positive effect on improving cecal microflora. Janczyk et al. (2009) reported that when 23 wk-old brown-egg laying hens were fed with powder and pellet forms of C. vulgaris at 5 g/kg of diet, the number of Lactobacilli spp. increased in the crop and the ceca. In this study, we did not attempt to measure the active components such as chlorellin, which is known to have the antibiotic effect (Pratt et al., 1944) or biomolecules which might be produced during the C. vulgaris fermentation process with baker's yeast and lactic acid bacterium. It should be pointed out that the concentrations that we used in this study would be sufficient to exhibit the biological effect, if any, on cecal microbiota. In any events, a clear explanation regarding the lack of effect by dietary fermented C. vulgaris on cecal microbiota is not readily available. Physicochemical properties of tibia Dietary fermented C. vulgaris did not affect tibia length (Table 5), but it significantly increased breaking strength of the tibia (linear effects at p = 0.001 and quadratic effects at p = 0.01). Although not significant (linear p = 0.080), dietary fermented C. vulgaris numerically increased ash content in tibia. Calcium metabolic analysis can be measured by either compressive strength, ash content, mineral content, or bone density measurement (Crenshaw et al., 1981;Garlich et al., 1982;Akpe et al., 1987;Watkins and Southern, 1992). Thus, increased tibia strength as seen in this study might be related to the enhanced calcium storing capacity of the tibia in ducks fed diets containing the fermented C. vulgaris. In humans, short-or long-term intake of Chlorella increased bone density and bone metabolism (Kim et al., 2002), which may support our latter assumption on C. vulgaris and calcium metabolism. In any event, further studies on apparent metabolizabilities of calcium and/or phosphorus could explain the observed effect of dietary fermented C. vulgaris on the calcium utilization in Pekin duck. Meat quality It is reported that C. vulgaris contains carotenoids (Kay, 1991), which are well-known as coloring agents in skin pigmentation. Thus, various meat quality parameters were measured including meat color intensity, shear force, pH, cooking loss and water holding capacity. The effect of dietary fermented C. vulgaris on meat quality is presented in Table 6. Dietary C. vulgaris linearly increased (p = 0.012) breast meat yellowness, but no effect on lightness or redness was monitored. In leg meats, dietary C. vulgaris significantly increased meat lightness (linear effect at p = 0.002) and yellowness (quadratic effect at p = 0.008). The shear force (p = 0.048) and pH (p = 0.004) of breast meats, but not leg meats were linearly increased by C. vulargis. The cooking loss of the breast or leg meats was not affected by dietary C. vulgaris. Finally, water hold capacity was increased quadratically (p = 0.044) in breast meat, but not in leg meat, when ducks were fed diets supplemented with C. vulgaris. As expected, the yellowness of duck meats were substantially increased as C. vulgaris increased in diets, indicating efficient transfer of active carotenoids present in C. vulgaris. In general, poultry skin pigmentation (i.e., yellowness) can be increased by feeding natural or synthetic pigments such as carotenoids in diets (Castaneda et al., 2005), which was the case in this study. According to the study by Park et al. (2005), the quality of edible meat can be determined by pH, which is associated with water retention capacity and color. Indeed, we also observed that the pH and water hold capacity of the breast meat increased as C. vulgaris increased in diet. In our knowledge, this is the first report that fermented C. vulgaris enhanced duck meat qualities. CONCLUSIONS In conclusion, these results indicated that dietary fermented C. vulgaris increased growth performance, tibia bone strength, and enhanced meat qualities in Pekin ducks. It is demonstrated that fermented C. vulgaris has strong potentials to produce value-added duck meats and can be introduced as a novel feed additive in duck industry.

v3-fos

2018-08-08T22:16:37.553Z

2015-06-01T00:00:00.000Z

51965142

s2

Biological control of banana black Sigatoka disease with Trichoderma Black Sigatoka disease caused by Mycosphaerella fijiensis is the most severe banana disease worldwide. The pathogen is in an invasive phase in Brazil and is already present in most States of the country. The potential of 29 isolates of Trichoderma spp. was studied for the control of black Sigatoka disease under field conditions. Four isolates were able to significantly reduce disease severity and were further tested in a second field experiment. Isolate 2.047 showed the best results in both field experiments and was selected for fungicide sensitivity tests and mass production. This isolate was identified as Trichoderma atroviride by sequencing fragments of the ITS region of the rDNA and tef-1α of the RNA polymerase. Trichoderma atroviride was as effective as the fungicide Azoxystrobin, which is recommended for controlling black Sigatoka. This biocontrol agent has potential to control the disease and may be scaled-up for field applications on rice-based solid fermentation. INTRODUCTION Black Sigatoka, caused by Mycosphaerella fijiensis (anamorph Pseudocercospora fijiensis), is considered the most destructive banana disease in Brazil and in several other Latin American countries (SENHOR et al., 2009). The spread of the pathogen to areas where its occurrence was not yet detected is expected to happen in the near future. Black Sigatoka may typically cause losses of around 50%, but it may reach 100% under certain conditions (PLOETZ, 1991). The pathogen is considerably more difficult to control than yellow Sigatoka, caused by M. musicola and has a wider host range (PLOETZ, 1991). The disease is occurring in almost all Brazilian States, except for Rio de Janeiro, Espirito Santo, Goiás, Distrito Federal and the Northeastern States (MATOS & CORDEIRO, 2011). In Amazonas State, many plantations were abandoned due to heavy losses caused by the pathogen after its arrival at the municipalities of Tabatinga and Benjamim Constant, where susceptible cultivars such as Prata, Maçã, Nanica, Prata graúda and plátano D'Angola were grown (GASPAROTTO et al., 2006). Fungicide spraying has been the control method most employed for controlling black Sigatoka in banana plantations around the world. In Brazil, owing to the high cost, fungicides are only used in plantations with high levels of technology and yields (GASPAROTTO et al., 2006). In the Amazonian region, fungicide utilization is not feasible because of the high costs and environmental impacts. The use of resistant cultivars is the ideal strategy, however, factors such as the time frame to develop new cultivars in face of the banana life cycle and its narrow genetic base and the variability of the pathogen that could rapidly adapt to new resistance genes due to its sexual reproduction are complicating factors. Biological control is an alternative to the use of chemicals (ALABOUVETTE et al., 2006) that may be feasible for some systems. Fungi of the genus Trichoderma are extensively employed as biological control agents of many plant pathogens (RAKHOLIYA, 2010). Among the advantages of using biological agents is the low risk of adverse impacts on the environmental and human health associated with the lower cost of this technology in Brazil. In one of the few studies available on the effect of biological agents against black Sigatoka, Trichoderma isolates were able to inhibit 45% of M. fijiensis mycelial growth in vitro (ARZATE et al., 2006). However, little is known on the effect of biocontrol agents against this disease under field conditions. In this study, Trichoderma isolates were tested for their effectiveness in field experiments. The most promising isolate was selected for molecular identification, sensitivity to fungicides and mass production. Isolates and inoculum production All isolates of Trichoderma used in this study were obtained from soil and are preserved in silica-gel (DHINGRA & SINCLAIR, 1995) and deposited in the microorganism collection of the Institute for Amazonian Research (INPA), in the laboratory for Phytopathology, under accession numbers listed in table 1. Isolate 2.047 was also deposited under number URM 6702 in the culture collection URM from Pernambuco Federal University (Recife, PE). To produce spores for field applications, the isolates were grown on Potato Dextrose Agar Medium (PDA) for 5 days under 25°C. Spore suspensions were adjusted to 10 7 conidia mL -1 and used for field applications. Screening Trichoderma isolates under field conditions Two-month-old banana plantlets cultivar 'Prata Anã' free of disease symptoms were produced at Embrapa Amazônia Ocidental and used for the field tests. To favor the natural occurrence of disease, plantlets were planted between rows of an old banana plantation heavily infected with M. fijiensis established with a spacing of 3m between rows and 1.5m from each other in the rows. The experiment was installed in a completely randomized design with five replicates of one plant and 30 treatments, composed of 29 isolates of Trichoderma plus one control with distilled water. Leaves 0, 1, 2 and 3 were sprayed to run-off with Trichoderma spore suspensions or distilled water. Sprayings were done always after 16:00 h on both faces of the leaves and were repeated every 10 days for 3 months. Disease severity was evaluated at 60 and 90 days after the first application with the disease index described by GAUHL (1990) that ranges from 0 to 6, where (0) represents no symptoms; (1) less than 1% of the leaf tissue lesioned or a maximum of 10 lesions; (2) 1 to 5% of lesioned leaf tissue; (3) 6 to 15%; (4) 16 to 33%; (5) 34 to 50% and (6) 51 to 100% of lesioned leaf tissue. A second experiment was installed with the four isolates that showed the best results in the first assay. Banana plantlets and spore suspensions were prepared as described for the first assay. However, spraying intervals were reduced to every seven days for a period of 60 days. The experiment was installed in a completely randomized design with ten replicates of one plant and six treatments composed of an untreated control, one fungicide treatment and four isolates of Trichoderma spp. The fungicide Azoxystrobin was applied in the concentration recommended for banana, 250mg L -1 . Severity was evaluated 60 days after the first application according to the disease index described above. For statistical analyses, severity data of the first experiment, on the 5 th leaf at 60 and 90 days after the first spray were transformed with arc sin √X (100) -1 . Severity in the 6 th leaf evaluated in the second experiment, 60 days after the first spray was transformed with arc sin √X+0.5 (100) -1 . Comparison of the means was done with Tukey's test at 5% probability. Species identification Isolate 2.047 was selected for having the highest potential to control black Sigatoka and identified on the basis of morphological characters and by sequencing the ITS region of the ribosomal DNA and a fragment of the translation and elongation factor of the RNA polymerase (tef-1α). DNA extraction, amplifications, sequencing, alignments and dendrogram construction were done as described previously (SAMUELS et al., 2011). Sequences obtained for isolate 2.047 were deposited in EMBL under accession numbers HG325825 (ITS) and HG325833 (tef-1α). The following accession numbers of Trichoderma atroviride isolates were used in the comparative analyses performed in this study: GJS95-113 Sensitivity to fungicides The fungicides Clorotalonil at 1200mg L -1 , Flutriafol at 75mg L -1 , Copper Oxychloride at 1400mg L -1 and Azoxystrobin at 250mg L -1 were tested on conidial germination. These concentrations **Plants were treated with Trichoderma or distilled water (control) and severity was scored at 60 and 90 days after the inoculation on a 1-to-6 scale described by GAUHL (1990). Data were transformed by arc sin vX (100) -1 . ***Means followed by the same letters are not significantly different by Tukey's test at 5% probability. are recommended by their respective manufacturers for controlling fungal diseases. Conidial suspensions of isolate 2.047 containing 10 7 conidia mL -1 were prepared in each fungicide and sterile distilled water was used as control. Samples of 100µL were taken at 0, 30, 60, 90, 120 and 180min, spread on PDA plates and incubated at 25 o C in three replicates per incubation time. This experiment was installed in a randomized design, with a 6x6 factorial scheme, where the first factor represents the 6 incubation periods and the second the fungicides and the control, with three replicates per treatment. Sixteen hours after spreading the suspensions on the plates, conidia germination was determined under the microscope. Conidia germination data was transformed with arc sin √X+0.5 (100) -1 and the means of the treatments compared with the Tukey test at 5%. Mass production Isolate 2.047 was grown in plastic bags containing 200g of autoclaved rice with different humidity. The experiment was installed in a randomized design with a factorial scheme 2x2x3, where the first factor was polypropylene bags that were either kept closed or open after three days of mycelial growth; the second factor was addition or not of 0.03g mL -1 of CaCO 3 ; and three humidity levels: 30, 50 and 70%, with three replicates per treatment. Five 5-mm diameter mycelial discs were added to each bag and every two days the chunks of rice were manually broken to allow better aeration. Conidia were counted with a Neubauer chamber after seven days of incubation at 25 o C and a photoperiod of 12h of light. Comparison of the means was done with Tukey's test at 5%. RESULTS AND DISCUSSION Among the 29 Trichoderna isolates tested under field conditions to control black Sigatoka, only four were able to reduce disease severity to values lower than 20% at 90 days after the beginning of the experiment (Table 1). This reduction in severity provided by the antagonistic isolates was equivalent to 34-86% in relation to the untreated control (Table 1). In a second experiment, these four isolates were tested with a higher frequency of application. In this field experiment, disease severity did not differ significantly between isolate 2.047 and the chemical treatment with fungicide azoxystrobin (Table 2). Severity reductions provided by isolate 2.047 and the fungicide azoxystrobin were 66 and 82%, respectively. The severity reductions obtained by isolate 2.047 were the highest among all Trichoderma isolates in both experiments. The few studies conducted to evaluate isolates of Trichoderma to control M. fijiensis in banana were done in vitro (ARZATE et al., 2006) or under greenhouse conditions (BIZERRA et al., 2006). The results reported herein are the first to our knowledge obtained in an attempt to control black Sigatoka under field conditions. It is recognized that further studies still need to be carried out to gather more information on the activity of this isolate in other phases of banana development, including fruit set. Several species of the genus Trichoderma are known to be antagonists of pathogenic fungi, but their antagonistic potential depends on the species and on the isolate. Trichoderma species exert biocontrol activity by mechanisms such as micoparasitism, competition for space and nutrients, and induction of resistance (GUEDEZ et al., 2009). The studies reported herein were focused on the activity of Trichoderma isolates under field conditions without determining the mechanism of action at this moment. Further studies should be directed to reveal the mechanisms by which isolates such as 2.047 utilize to suppress M. fijiensis. Nevertheless, recent studies show that Trichoderma is essentially mycotrophic (ATANASOVA et al., 2013) and thus, mycoparasitism is expected to be involved in the activity of this antagonist against M. fijiensis. Coincidentally, among the three species profiled in their mycoparasitic activity, T. atroviride was shown to be the most aggressive (ATANASOVA et al., 2013). In the field experiments, high concentrations of M. fijiensis inoculum were maintained throughout the entire experimental period. Trichoderma was sprayed only on the experimental 13.8 c * Treatments were applied at every 7 days and severity was scored on a 1-to-6 scale described by GAUHL (1990). Data were transformed by arc sin vX+0.5 (100) -1 . **Means followed by the same letters are not significantly different by Tukey's test at 5% probability. plots but all plants growing around were continuously releasing spores of the pathogen. Although isolate 2.047 was as effective as the chemical fungicide, the high inoculum pressure in the field may have limited its capacity to further reduce disease severity. Only isolate 2.047 was selected for identification by morphological and molecular methods because of its superior performance as a biocontrol agent. Morphological characteristics were similar to Trichoderma harzianum but sequences of the ITS region of the ribosomal DNA of isolate 2.047 were 100% identical to other sequences of T. atroviride deposited in public databases (e.g. KC895539; KC506274; JF694930). Sequences of a fragment of the translation and elongation factor 1-α were 99% identical to other sequences of T. atroviride from public databases (e.g. DQ307548; DQ307550; DQ307547). The combined analyses of these two fragments confirm the identity of isolate 2.047 as T. atroviride. It is also possible to observe that there is a considerable diversity in populations of T. atroviride from different geographic origins (Figure 1). The highest production of conidia by isolate 2.047 was obtained in autoclaved rice amended with CaCO 3 and with 50% humidity kept in bags that were opened at 3 days after inoculation. Maximum amount of conidia produced was 2.3x10 9 spores g -1 of rice ( Figure 2). This spore concentration is similar to what was obtained by HANADA et al. (2009) with Trichoderma martiale on rice with different amendments. Solid fermentation on rice as a substrate is commonly employed to produce fungal spores in Brazil due to its effectiveness, ease of finding it in local markets and reduced cost (POMELLA et al., 2007). Addition of calcium increased the sporulation in these studies and was also observed by other authors (WUYEP et al., 2003). This increase is probably due to pH stabilization and mineral nutrition (KRISHNA, 2005). Although no economical evaluation was done in this study, the production of conidia on rice does not require a significant investment. This process could be scaled-up by using artisanal methods and this characteristic may facilitate the commercialization of the biocontrol agent. The germination of conidia of isolate 2.047 was not inhibited by the fungicides Chlorotalonil, Flutriafol, Copper Oxychloride, and Azoxystrobin after an exposition of three hours (Data not shown). This exposure time was normally enough to perform the preparation and application of the spores under field conditions. This suggests that combined applications of the biocontrol agent with fungicides may be performed when required from the standpoint of the integrated disease management of the disease. CONCLUSION Trichoderma atroviride 2.047 is the first isolate of this genus to show potential to control banana black Sigatoka under field conditions. This isolate was selected among 29 others for being able to reduce the severity of back Sigatoka to levels similar to the ones provided by the fungicide Azoxystrobin. Spores of this isolate may be produced in rice-based solid fermentation for field applications.

v3-fos

2016-10-09T20:37:22.850Z

2015-01-15T00:00:00.000Z

2677262

s2

Organochlorine pesticides residue in breast milk: a systematic review. Background: Chlorinated pesticides have been used in pest control for several decades in the world. These compounds are still applied in many regions, and their continuous usage has resulted in their bioaccumulation and residue in the food chain. These residues could transfer to food products and accumulate in fat tissues. Undoubtedly, the breast milk could be a significant biomarker for estimation of these residues in the human body. This study was conducted to review and compile the results of the studies undertaken in the world which surveyed the organochlorine pesticides residue in breast milk. Methods: A total of 710 national and international articles and texts related to the focused subject were extracted from the virtual databases using the following key words: Chlorinated pesticides, residue and breast milk. Thirty articles published between 1980 and 2013 were selected and reviewed. Results: The majority of the reviewed articles indicated the presence of two or more organochlorine pesticides in the collected samples of breast milk. Based on the reviewed studies, dichlorodiphenyltrichloroethane (DDT) had the highest level of concentration in the collected samples of breast milk. Moreover, there was a statistically significant positive correlation between mother’s age, her multiparty and concentration of chlorinated pesticides in breast milk. Conclusion: The organochlorine pesticides are still applied in some developing countries including some regions of Iran. Thus, it seems essential to inform the community about the adverse effects of this class of pesticides; and most importantly the governments should also ban the use of such compounds. Introduction Increase in population size as well as development in agricultural activities have led to the increased usage of pesticides for plant protection. Moreover, pesticides are widely used to control the vectors of various diseases such as malaria and contribute to prevention of vector borne diseases. However, these compounds are considered as chemical pollutants and might be viewed as a serious threat for human health and environment. Agric ultural pests are becoming widespread in the world, and huge pesticides are being used for pest control in many regions. Hence, concerns about environment pollution by these chemical compounds used in agriculture and in vector control have been recently justified. Some workers investigated the pesticide residues in food and environment; for instance, the residues of different pesticides have been reported in streams, groundwater, soil, fish, wild and domestic animals, vegetables and even breast milk (1,2). Among different classes of pesticides, organochlorines have been widely used worldwide because of their persistence, low cost and their toxicity against various pests. For example, the main antimalaria measures have been the use of chlorinated hydrocarbons as a residual spraying against the adults and larvae of malaria vectors in some areas through several previous decades (3)(4)(5). Obviously, it could be suggested that the major concern has been directed to this class of pesticides. Environmental impact of these chemical compounds are of high concern because of their resistance to heat, humidity, radiation and biodegradation; and thus, they are extremely persistent in water, soil and can be a real danger to wildlife. Also, they could enter the food chain due to their hydrophobic chemical structure. Organochlorine pesticides, based on their bioaccumulative nature, are also persistent in animal and plant tissues (6). Continues application of this class of pesticides against harmful insects has resulted in toxic effects on the environment and accumulation of high level of residues in agriculture and dairy products (7). The first chlorinated hydrocarbon DDT was successful in controlling louse borne typhus and malaria during world war Π. However, just like most of the chlorinated hydrocarbons, this pesticide was banned in 1972 in many countries due to its toxic effect (such as bioaccumulation and bioconcentration) on the environment. It has been revealed that the concentration of DDT could be increased by 1000 fold through the trophic levels (1,8). However, the organochlorine pesticides are still used in some developing countries to control the vectors of diseases and agricultural insect pests (2,9,10). The organochlorine pesticides are usually divided into three main groups including DDT and its derivates, gama isomer of hexachlorocyclohexane, HCH and chlorinated cyclodiene such as aldrin and dieldrin. Exposure of the man and animals to organochlorine pesticides and polychlorinated biphenyls can cause some side effects including reproductive toxicity, teratogenic effects and preterm labor. In addition, it has also been suggested that long-term exposure of humans to organochlorine pesticides may damage their central nervous system, liver, kidney and bladder (6). Humans could be exposed to these chemical compounds via several routes including breathing polluted air, dermal penetration and ingesting contaminated food. The two former routes comprise approximately less than 2% of the total absorption of pesticides, whereas residues of pesticides remained in the crops might appear in food products; and therefore the ingesting contaminated food is considered as the main source of human exposure to pesticides (11). The levels of residues of these compounds in living organisms are based on their habitat and their positions in the food chain (12). The most important human health problem is associated with chlorinated hydrocarbons, and their tendency to accumulate in fatty tissues; and the occurrence of residues of these poisons in fatty tissues can be considered as the best indication for the presence of these compounds in the human body (13). Considering the adverse effects of the organochlorine pesticides on humans' health due to their highly significant position in the food chain as the secondary consumers, we deemed necessary to carry out a systematic review of the literature on the residues of these chemical compounds in breast milk and on the factors influencing their concentration levels. The aim of this study was to conduct a systematic review on the residues of this group of pesticides in breast milk and to survey the influencing factors in different countries. Methods This systematic review was conducted based on the national articles which were extracted from three virtual databases of Iranmedex, Irandoc and SID and international articles and texts extracted from the following virtual databases: Google Scholar, Scopus, Science Direct, Index Medicus /WHO/EMdR, Elsiver, Directory of Open Access Journal and PubMed. A total of 710 articles and texts were collected based on the following key words: Chlorinated pesticide, residue, breast milk; and finally 32 articles and texts published between 1980 and 2012 were reviewed. From these articles, the list of 17 selected studies, classified according to the characteristics of the population, intervention, comparison and outcomes of each paper (PICO frame), was prepared (Table 1). In this study, the data bases and the extracted documents were evaluated and checked by the authors in order to make the study more valid and reliable. Fig. 1 illustrates the search process. Results The selected studies were conducted to investigate the residue levels of organochlorine pesticides in breast milk by residue analytical methods based on the age of the mother, body mass index and body weight. The majority of these studies used gas chromatograph equipment (GC) to measure detectable quantities of organochlorine compound. Table1 demonstrates the results of the studies measuring the content of residue of organochlorine pesticides in breast Table 1 shows the PICO description of 17 selected papers in the order of year of publication. The reported data from each country was separately evaluated. The Concentration of Chlorinated Poisons in Breast Milk in Asian Countries In our review, the concentration of chlorinated poisons in breast milk in eight Asian countries was surveyed (14)(15)(16)(17)(18)(19)(20)(21). Based on the results from measuring the concentration of these compounds in breast milk in those countries, the maximum quantity 3210 ng/g and the minimum quantity 19 ng/g of DDT were detected in India and Taiwan, respectively (15, 19). The maximum 3780 ng/g and minimum 0.8 ng/g quantities of HCH were detected in Iran and Taiwan, respectively (17,19). The maximum 930 ng/g and minimum 3 ng/g quantities of HCB were detected in Iran and Vietnam, respectively (17,18). The maximum 1560 ng/g and minimum 35 ng/g quantities of PCBs were detected in Iran and Bangladesh, respectively (17,21). Mishra and Sharma (2011) suggested that organochlorine pesticides have been used extensively against malaria vectors as well as agricultural pests in India despite the worldwide prohibition of usage of these compounds. Based on their findings, the average detected quantities of DDTs and HCHs in breast milk were 3210 and 2870 ng/g, respectively (19). According to Burke et al. ( 2003), there was a significant difference between the levels of residue of organochlorine pesticide in breast milk in urban and rural regions in Indonesia; and 60% and 40 % of breast milk samples collected from the urban and rural regions contained HCB, respectively. Moreover, dieldrin was detected in 80% of breast milk samples which were collected from urban regions. Their results were similar to those found in Japan and England. Moreover, in a study conducted by Chao et al. (2006), the level of residue of organo-chlorine pesticides in breast milk in Taiwan were considerably lower than those of some Asian countries such as China, Thailand, Indonesia and Vietnam. Therefore, the authors suggested that those values were comparable to the reported values from some European countries such as Sweden and England (15). In contrast, Subramanian et al. (2007) proposed that in Chennai (India) the levels of both DDTs and HCH in breast milk had an increasing trend through the two last decades. In a study by Fujii et al. (2012), the levels of chlorinated cyclodiene pesticides in breast milk samples collected from Japan, China and Korea were compared. The levels of chlordane and PCBs in Japan and Korea were 0.8 -4.5 ng/g and 0.2 -4.7 ng/g, respectively. However, the levels of these compounds in breast milk in China were lower than 0.1 ng/g. Heptachlor, 2012), investigated the occurrence and level of organochlorine compounds in breast milk in Bangladesh and found that the level of PCBs in breast milk of the women whose major employment was agriculture was low, but the levels of DDT, DDE and their metabolites were higher than those of the other countries. The Concentration of Chlorinated Poisons in Breast Milk in European Countries In this review, the concentration of chlorinated poisons in breast milk in six European countries was surveyed (22)(23)(24)(25)(26)(27). Among these countries, the maximum and minimum amount of detected DDT were 660 ng/g and 41 ng/g in Russia and Norway, respectively (22,25). The maximum and minimum amount of detected HCH were 880 ng/g and 0.063 ng/g in Russia and Poland, respectively (22,23). The maximum and minimum amount of detected HCB were 12 ng/g and 0.086 ng/g in Po-land and Sweden, respectively (22,23). The maximum and minimum amount of detected PCBs were 113 ng/g and 8.07 ng/g in Sweden and Turkey, respectively (26,27). According to Szyrwin´ska & Lulek 2007, the amounts of detected HCB and HCHs in breast milk were consistent with those of the other European countries; however, the amounts of DDT and DDE in breast milk were slightly higher than those of other European countries (23). Podler et al. (2003) indicated that the detected quantities of рo DDE and βHCH in breast milk were 810 ng/g and 660 ng/g, respectively in Russia. Podler et al. (2008) conducted a study to determine the levels of chlorinated pesticides and PCBs in breast milk in northern and southern Norway. They suggested the order of the levels of studied compounds based on their detected quantities as follows: DDTs>PCBs> HCHs>HCBs>Mirex. It was also suggested that HCBs were only detected in breast milk samples which were collected from northern Norway. In a study conducted in the vicinity Mediterranean city Mersin, Turkey, Çok et al. The Concentration of Chlorinated Poisons in Breast Milk in other Countries The measured quantities of DDT and HCB in breast milk were 1163 ng/g and 286 ng/g, respectively in an African country, Tunis (25). The measured quantity of DDT in breast milk was 9362 ng/g in an American country, Brazil (28). The results of a study conducted in Australia indicated that the quantities of organochlorine pesticides including DDT, HCH and HCB in breast milk were 7, 21 and 14 ng/g, respectively (29). Ben Hassine et al. (2012), in a study determining chlorinated pesticides and polychlorinated biphenyls in breast milk, showed that the ratio of ρο DDE / ρο DDT was low, suggesting the high quantities of ρο DDT in breast milk in Bizerte, Tunisia. High usage of DDT could increase the quantities of ρο DDT in breast milk in this region. In general, the order of quantities of chlorinated compounds in breast milk was as follows: DDTs>PCBs>HCB>HCHs> Dieldrin (30). In a study carried out by Azeredo et al. (2008) in Theamazon (Brazil), it was indicated that the quantity of DDT in approximately 87% of the collected breast milk samples was higher than the WHO standard. The authors suggested that Theamazon was in the vicinity of Maderia river, so DDT was extensively used as an antimalaria measure because of the high incidence of this disease. However, it has been frequently revealed that the organochlorine pesticides such as DDT had toxic effects on the environment and wildlife and cause contamination of the fishes. On the other hand, the consumption of fish was the main dietary regime in the region. Mueller et al. (2008) determined the level of organochlorine pesticides in collected samples of breast milk in Australia. The levels of DDT and HCH in breast milk were 279 and 21 ng/g, respectively. These compounds were also detected in all samples of breast milk; and based on the obtained results, it was suggested that the levels of organochlorine pesticides in breast milk continued to increase from 1980 to1990 (29). The Factors Influencing the Amount of Chlorinated Pesticides in Breast Milk Our review on the studies undertaken in the world showed that DDT and its metabolites had the highest levels in breast milk. It can be suggested that high human exposure to these chemical compounds and their extensive usage have likely increased their amount in breast milk (15,17,19,22,25). Nevertheless, it should be mentioned that DDT was the first chlorinated pesticide whose use was forbidden in different countries (17). According to Burke et al. (2003), it was suggested that the level of residue of organochlorine pesticides in breast milk was lower in regions where anti-malaria program had been performed (14). In a study conducted by Chao et al. (2006), the results indicated that the level of DDT in breast milk has been reduced from 3595 ng/g to 333 ng/g during the two previous decades as a consequence of limited usage of organochlorine pesticides and application of substituted compounds against agricultural pests (15). Organochlorine Pesticides Residue in Breast Milk Mueller et al. (2008) concluded that the levels of dieldrin and heptachlor were higher in breast milk in areas where these pesticides had been used for controlling termite than the other areas (29). The measured Med J Islam Repub Iran 2015 (7 July). Vol. 29:228. http://mjiri.iums.ac.ir quantity of DDT in breast milk was 9362 ng/g in an American country, Brazil (28). They observed that the level of this pesticide in breast milk was very high in this country due to the use of organochlorine pesticides against vectors of malaria. The majority of the performed studies revealed a positive relation between the age of the mother and the level of pesticides in breast milk, and this may be due to the more accumulation of fat tissues in older women and potentially higher accumulation of organochlorine pesticides in those tissues (14,15,22). In study of Mishra and Sharma (2011), a statistically significant positive correlation was found between the level of organochlorine pesticides in breast milk and the age of the mother; and it was indicated that the estimated daily intake (EDI) of DDTs and HCHs in the body of children who consumed breast milk were higher than the acceptable daily intake (ADI) because the unacceptable quantities of these compounds could transfer to the body of children through breast milk (19). In a study by Fujii et al. (2012), the level of endosulfan and chlordane in breast milk in China and southern Korea were correlated with the age of the mother. Also, there was a significant positive relation between the body mass index (BMI) of the mothers and the levels of toxaphen and oxychlordane in breast milk in Japan (20). Polder et al. (2009) found that the age of mother was also one of the factors influencing the levels of pesticides in breast milk (25). Moreover, in a study carried out by Azeredo et al. (2008), a direct relation was found between the increased age of the mother and increased level of DDT in breast milk in Brazil (28). Multiparty has been surveyed in all the performed studies, and the increased levels of pesticides in breast milk of multipar mothers were specified (8)(9)19). Podler et al. (2008) found that the levels of organochlorine pesticides in breast milk of multipar mothers were higher than those of nulipar ones. Moreover, the results indicated that the determined quantities of the studied pesticides have been decreased by 50 to 60% compared to a study conducted in these regions in 1991 (24). Ben Hassine et al. (2012) suggested that the age of mother and her multiparty influenced the level of organochlorine in breast milk; therefore, the levels of these compounds were high in multipar mothers (30). By the worldwide review of the articles we found that the levels of chlorinated pesticides in breast milk in rural areas were higher than the others. These areas were in the vicinity of farms, so the extensive use of pesticides is likely to have influenced the levels of pesticides in breast milk (14,19). Subramanian et al. (2007) suggested that several factors could influence the levels of organochlorine pesticides in breast milk in Chennai as follows: The region and the river in the vicinity, high agricultural activities, high incidence of malaria and extensive application of organochlorine pesticides including DDT against agricultural pests and malaria vectors have caused the increment of the levels of DDTs and HCH in breast milk (16). In a study by Glynn et al. (2011), it was suggested that several factors including health, genetics and life style influenced the levels of chlorinated pesticides in breast milk. The results revealed that regional differences in levels of chlorinated pesticides in breast milk were due to geographical factors and differences between the age and body mass index (BMI) of mothers (26). The results obtained in some of the studies indicated a direct relation between diet and the levels of pesticides in breast milk. It has been revealed that the residues of chlorinated pesticides in food products could transfer to different tissues after ingesting the contaminated food (14,19). Podler et al. (2003) suggested that nutritious habits and exposure through pesticides residue in food are likely to cause the enhancement of the levels of these compounds in breast milk (22). Dahmardeh et al. (2009) indicated that the level of organochlorine pesticides in breast milk of mothers who consumed fish once a weak was significantly lower than those of mothers who consistently consumed it in their diet. These authors proposed that the organochlorine pesticides such as DDT might be leached from the treated farms and finally transferred to rivers and seas (17). Finally, the results of this review revealed that the amounts of chlorinated pesticides in breast milk in Asian, African and Sought American countries were higher than those of European countries. However, in general, other studies than what carried out in Chenai (India) suggested that the residues of these compounds in breast milk have been decreasing during the recent years. It was also suggested that the levels of these pesticides in breast milk in developed countries such as Australia and Russia were lower than those of developing and African countries and this may be due to the fact that these developed countries banned the use of these pesticides much earlier than other countries. Mishra and Sharma (2011) suggested that the values of organochlorine pesticides were very higher than those of developed countries (19). Bergkvist et al. ( 2012) suggested that illiteracy of the women and use of a large amount of pesticides have resulted in enhancement of the levels of residue of DDTs and their metabolites in Bangladesh (21). Conclusion Although at the present time the developing agricultural activities have aimed at increasing agricultural and dairy products, the extensive use of pesticides in plant protection has resulted in environment pollution. Among the different classes of pesticides, the main concern has been directed to the application of organochlorine pesticides due to their bioaccumulative nature and persistence in animal and plant tissues. Obviously, the bioaccumulation of organochlorine pesticides in humans is of high concern, and the conducted studies sug-gested that breast milk could be a significant biomarker for estimation of these residues in the human body. The organochlorine pesticides are still applied in some developing countries including some regions of Iran, against agricultural pests or vectors of diseases such as malaria. Thus, it seems essential to inform the community about the adverse effects of these chemical compounds. Also, governments should ban the use of such pesticides.

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A Novel Soybean Intrinsic Protein Gene, GmTIP2;3, Involved in Responding to Osmotic Stress Water is essential for plant growth and development. Water deficiency leads to loss of yield and decreased crop quality. To understand water transport mechanisms in plants, we cloned and characterized a novel tonoplast intrinsic protein (TIP) gene from soybean with the highest similarity to TIP2-type from other plants, and thus designated GmTIP2;3. The protein sequence contains two conserved NPA motifs and six transmembrane domains. The expression analysis indicated that this gene was constitutively expressed in all detected tissues, with higher levels in the root, stem and pod, and the accumulation of GmTIP2;3 transcript showed a significant response to osmotic stresses, including 20% PEG6000 (polyethylene glycol) and 100 μM ABA (abscisic acid) treatments. The promoter-GUS (glucuronidase) activity analysis suggested that GmTIP2;3 was also expressed in the root, stem, and leaf, and preferentially expressed in the stele of root and stem, and the core promoter region was 1000 bp in length, located upstream of the ATG start codon. The GUS tissue and induced expression observations were consistent with the findings in soybean. In addition, subcellular localization showed that GmTIP2;3 was a plasma membrane-localized protein. Yeast heterologous expression revealed that GmTIP2;3 could improve tolerance to osmotic stress in yeast cells. Integrating these results, GmTIP2;3 might play an important role in response to osmotic stress in plants. INTRODUCTION Lack of water resources is an important factor restricting the development of agriculture. Plant growth and development depend on water uptake and transport regulation across cellular membranes and tissues. In the past, it was thought that water moved across cell membranes by free diffusion through the lipid bilayer. However, its transport is now thought to be highly and selectively regulated by aquaporins. Aquaporins (AQPs) belong to the ancient major intrinsic protein (MIP) family found in animals, microbes, and plants. Since the discovery of AQP1 Abbreviations: GFP, Green Fluorescent Protein; GUS, Glucuronidase; TIP, Tonoplast Intrinsic Protein; QRT-PCR, Quantitative reverse transcriptase Chain Reaction; CDS, Coding sequence; PEG, Polyethylene glycol; ABA, Abscisic acid. (Denker et al., 1988), many aquaporin genes have been found in plants including 35 AQPs in Arabidopsis (Quigley et al., 2002;Boursiac et al., 2005), 31 in Zea mays (Chaumont et al., 2001), and 33 in Oryza sativa (Sakurai et al., 2005). Guo et al. (2006) further analyzed the expression and function of the rice plasma membrane intrinsic protein (PIP) gene family. Other scholars found 23 AQPs in Physcomitrella patens (Danielson and Johanson, 2008), 37 in Solanum lycopersicum (Sade et al., 2009), 66 in soybean (Zhang et al., 2013), 47 in tomato (Reuscher et al., 2013), 71 in Gossypium hirsutum (Park et al., 2010), and 53 in Chinese cabbage (Tao et al., 2014). Plant AQPs can be categorized into major four subfamilies based on localization and expression patterns: plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin26like intrinsic proteins (NIPs), small and basic intrinsic proteins (SIPs) (Chaumont et al., 2001;Kaldenhoff and Fischer, 2006), and uncategorized X intrinsic proteins (XIPs) (Danielson and Johanson, 2008). AQPS play important roles in various physiological processes in plants, such as growth, development, and response to biotic and abiotic stresses. Srivastava et al. (2015) also reviewed the versatile functions of aquaporins as molecular conduits in the plant response to abiotic stresses. For example, Guenther and Roberts (2000) isolated two major intrinsic membrane proteins from Lotus japonicus, named LIMP1 and LIMP2. Functional analysis using the Xenopus oocytes system indicated that LIMP1 appeared to be a member of the TIP subfamily and LIMP2 was a nodulin 26 ortholog protein. Rodrigues et al. (2013) investigated a gene encoding a root-specific tonoplast intrinsic aquaporin (TIP) from Eucalyptus grandis named EgTIP2, whose expression was induced by PEG and mannitol treatments but was downregulated by abscisic acid, suggesting that EgTIP2 might be involved in the eucalyptus response to drought. Wang et al. (2014) cloned and characterized a tonoplast AQP gene (TsTIP1;2) from the halophyte Thellungiella salsuginea and reported that it mediated the transduction of both H 2 O and H 2 O 2 across the membranes and might contribute to the survival of T. salsuginea under multiple stresses. Ligaba et al. (2011) studied the expression patterns of 7 MIP genes from barley under different abiotic stresses using quantitative realtime PCR (RT-PCR), indicating that abiotic stress modulates the expression of major intrinsic proteins in barley. Zelazny et al. (2007) by using FRET imaging analysis showed that plasma membrane aquaporins could interact to regulate their subcellular localization in living maize cells. Tomato SiTIP2;2 expressing in transgenic Arabidopsis could enhance the plant's tolerance to salt stress and interact with its homologous proteins SiTIP1;1 and SiTIP2;1 (Xin et al., 2014). Gao et al. (2010) overexpressed TaNIP, a putative aquaporin gene from wheat, and found that it could enhance salt tolerance in transgenic Arabidopsis. Wang et al. (2011) cloned the novel Glycine soja tonoplast intrinsic protein gene GsTIP2;1, and the overexpression of GsTIP2;1 in Arabidopsis repressed/reduced tolerance to salt and dehydration stress, suggesting that GsTIP2;1 might mediate stress sensitivity by enhancing water loss in plants. In this study, a novel tonoplast intrinsic aquaporin from soybean, GmTIP2;3, was cloned and characterized. Protein structure analysis showed that GmTIP2;3 possesses typical aquaporin characteristics, such as six transmembrane domains and NPA motifs. The expression analysis indicated that it was constitutively expressed in all tissues tested, especially in the root, stem, and pod, and exhibited responses to ABA and PEG treatments at certain time points. Subcellular localization showed it to be localized in the cell plasma membrane. The promoter activity assay demonstrated that the core sequence for this gene was 1000 bp upstream from the ATG start codon. Yeast heterologous expression revealed that GmTIP2;3 could improve osmotic tolerance in yeast cells. Integrating these results, GmTIP2;3 plays an important role in response to osmotic stress in plants. Plant Materials Glycine max var. Willimas 82 was selected for the experiments, which included growth of seedlings, flowering, podding, extracting total RNA for GmTIP2;3 cloning, and tissue expression and induced expression analysis. Lotus japonicus was used to transfer the promoter sequence for activity testing and Arabidopsis ecotype Col-0 was used for transformation. Protoplasts were grown in a 7:2:1 (v/v/v) mixture of vermiculite:soirite:perlite under a 16-h light/8-h dark regime, and the day and night temperatures were 23 • C / 20 • C, respectively. The plants were watered every week. Gene Cloning and Sequence Analysis The gene primers were designed based on the full-length coding sequences, and RT-PCR (reverse transcriptase-polymerase chain reaction) was performed to isolate the genes from soybean tissues. The neighbor-joining (NJ) tree was constructed from soybean GmTIP2;3 and from other plant TIPs based on alignment using the Clustalx and MEGA 5.0 software, and used to explore the evolutionary relationships of soybean and other plant TIPs. qRT-PCR Analysis Soybean samples from the seedling, flowering, and podding stages (root, stem, leaf at young seedling stage; root, stem, leaf, and flower at flowering stage; and root, stem, leaf, and pod at podding stage) were harvested and frozen in liquid nitrogen for RNA extraction. Soybean roots were collected from plants treated with PEG6000 (polyethylene glycol) for 0, 2, 4, 12, and 48 h and with 100 µ M ABA (abscisic acid) for 10, 20, 30, 45, 60, 90, and 120 min. The total RNA for all samples used in this study was isolated using TRIzol R reagent (Invitrogen) following the manufacturer's instructions and used for qRT-PCR analysis. The qRT-PCR analysis was conducted according to the method described by Zhang et al. (2013) and was repeated three times. The primers used are given in Table 1. Subcellular Localization PCR-generated Hind III-BamH1 fragments containing the open reading frame of GmTIP2;3 were subcloned upstream of the GFP gene in plasmid pJIT166GFP. All constructs were validated by sequencing. The primers used are listed in Table 1. Arabidopsis protoplasts were isolated according to Yoo et al. (2007). The CDS of GmTIP2;3 without stop codons was used to create an in-frame fusion with GFP gene inserted into the pJIT166-GFP vector. The resulting fusion construct and an empty vector as a control (p35S::GFP) were introduced into Arabidopsis protoplasts by the PEG4000-mediated method (Abel and Theologis, 1994). After incubation of transformed Arabidopsis protoplasts for 18-24 h at room temperature, GFP signal was detected by confocal fluorescence microscopy (Zeiss, LSM510 Meta, Carl Zeiss AG). Promoter Analysis A 2081 bp-long region (named P1) located upstream of the ATG start codon was cloned from soybean genome DNA using primers described in Table 1, and sequence analysis was performed using the PlantCARE online software (http:// bioinformatics.psb.ugent.be/webtools/plantcare/html/). To find the core promoter region of GmTIP2;3, seven truncated fragments (P2-P8) were cloned from P1 and transformed into A. rhizogenes strain K599 for GUS activity detection. Soybean hairy root transformation was performed according to the method given by Subramanian et al. (2005). The primer pairs are listed in Table 1. Histochemical and Fluorometric GUS Assay For histochemical staining of GUS, fresh tissue samples including whole transgenic lotus plants, soybean hairy roots, and dissected leaves from positive plants that had undergone osmotic stress (20% PEG6000 and 100 µMABA), salinity (200 mM NaCl solution), and wounding for 2 h were immediately dipped into X-Gluc solution, as previously described (Jefferson et al., 1987). After overnight incubation at 37 • C in the growth chamber, stained samples were bleached with 70% (v/v) ethanol, washed several times with ddH 2 O, and observed under a Zeiss Stemi 2000-C microscope, Germany. A quantitative fluorometric GUS assay was conducted as described by Jefferson et al. (1987). The protein concentrations from a series of truncated constructs pGUSP1-P4 in transgenic soybean hairy roots were assessed by Bradford method, using bovine serum albumin (BSA) as a standard. GUS activity was normalized to the protein concentration of each sample and calculated as nmol of 4-MU per milligram of soluble protein per minute. Generation of Transgenic Lotus japonicus Plants The resulting pGUS-GmTIP2;3 promoter(p3):GUS plasmid was introduced into Agrobacterium rhizogenes strain K599 and used FIGURE 1 | Phylogenetic tree of GmTIP2;3 and reported TIP proteins from Arabidopsis thaliana TIPs (AtTIPs), Oryza saliva TIPs (OsTIPs), Zea mays TIPs (ZmTIPs), and Medicago sativa TIPs (MtTIPs). The Glycine max TIP cloned in this paper showed the highest similarity to TIP2-Type proteins from other plants. Therefore, TIP was designated as GmTIP2;3. to transform small Lotus japonicus seedling to produce hairy roots, as in the soybean hairy root system. The hairy roots were transferred to MS medium with 0.5 mg/L 6-BA for 20 days to generate adventive buds, and 1-2 cm high regeneration seedlings without roots were transferred to 1/2 MS medium to produce roots. Finally, the whole seedlings were transferred to pots. Yeast Transformation The novel pYES2-GFP was reconstructed via the recombination of pYES2 and pJIT166-GFP using the same double-digestion by Hind III and EcoR I. The CDS with Hind III and BamH I digestion for the forward primer and reverse primer, respectively (Table 1), was inserted into the pYES2-GFP vector digested with the same enzymes. The resulting pYES2-GmTIP2;3:GFP plasmid was introduced into S. cerevisiae INVSc1 strain cells using the lithium acetate method, with the empty vector pYES2-GFP as a control. S. cerevisiae INVSc1 strain cells transformed with the empty vector PYES2-GFP alone and with pYES2-GmTIP2;3:GFP were induced with galactose and spotted on the SC-Ura medium in 0, 10, 100, 1000, and 10,000-fold-dilutions, and the drought tolerance of yeast cells expressing GmTIP2;3 was tested by 30% PEG6000 treatment for 40 h. The GFP in yeast was observed using a fluorescence microscope (Olympus BX61). All experiments were repeated three times. Frontiers in Plant Science | www.frontiersin.org (NP_001105036.1) from Zea mays were used for phylogenetic tree analysis. Statistical Analysis The data were analyzed by ANOVA testing using the EXCEL software. Significant differences among means were determined by the LSD at P < 0.05, and a-f represent the different significant levels. RESULTS The isolation and characterization of GmTIP2;3 The gene locus Glyma07 g02060 from the QTL region between the markers Satt590 and Satt567 on chromosome 7 in soybean (Specht et al., 2001) was selected as a candidate gene and further isolated by RT-PCR method. BLAST X showed that this locus encoded a protein with 89% identity to TIP2-1-like from Cicer arietinum. The phylogenetic trees were created using GmTIP and Arabidopsis thaliana TIPs (AtTIPs), Oryza saliva TIPs (OsTIPs), Zea mays TIPs (ZmTIPs), and Medicago sativa TIPs (MtTIPs). The phylogenetic tree showed that GmTIP had the highest similarity to TIP2-type proteins from other plants (Figure 1). Therefore, GmTIP was designated as GmTIP2;3. SMART software analysis showed that its protein sequence possessed two conserved NPA motifs and six transmembrane domains, indicating that it was a typical aquaporin protein (Figure 2). Expression Analysis of GmTIP2;3 The temporal and spatial expression patterns of GmTIP2;3 in various tissues/organs of soybean cv. Willimas 82 plants were examined using quantitative RT-PCR. GmTIP2;3 appears to be expressed in most parts of the plant, with the highest expression in the root, stem, and pod, Moreover, the expression patterns of GmTIP2;3 in different developmental stages of the same tissue, namely in different organs in the three-leaf, blooming, and podding stages, showed that the transcript abundance of GmTIP2;3 in the stem exhibited a slight increase at the blooming stage, then significantly decreased in both the root and stem at the podding stage except in the pod tissue ( Figure 3A). To test whether GmTIP2;3 responds to drought stress, soybean seedling roots were treated with PEG and ABA. Then, the expression of GmTIP2;3 was analyzed by quantitative real-time RT-PCR. The results indicated that the expression of GmTIP2;3 decreased within 2 h after PEG-6000 (20%) treatment, and then the mRNA level continuously increased from 4 to 12 h and reached a maximum at 12 h (Figure 3B-1) However, ABA treatment (100 µM) initially significantly suppressed GmTIP2;3 expression after 10 min treatment, reached its minimum at 30 min (p < 0.05), then increased from 30 to 45 min and continuously decreased from 45 to 120 min, followed by a stable expression level (Figure 3B-2). The Promoter Activity Analysis of GmTIP2;3 To analyze the elements contained in the promoter region and the promoter activities of GmTIP2;3, a more than 2 kb (2081 bp)-long promoter region located upstream of the ATG start codon was amplified and inserted into the pGUSP vector by the T/A cloning method. The resulting construct was transformed into Lotus japonicus, and the transgenic plant was successfully obtained (Figures 4A,B). The non-transgenic plant was used as a negative control ( Figure 4C). GUS staining revealed that GmTIP2;3 was mainly expressed in the root (stele), stem (stele), and leaf (Figures 4D-F). Moreover, transgenic soybean hair roots using this construct also showed higher expression at the stele of the root (Figure 4G), which was consistent with its function as a water transporter. The GmTIP2;3 expression patterns in transgenic plants or hairy roots were identical to the patterns in different organs in the soybean plant. In addition, promoter sequence analysis using the PlantCARE online software indicated that it contained many light responsive elements, such as Box4, G-Box and I-Box, GATA-motif, MBS, and GARE-motif ( Table 2). To further dissect the core region of the GmTIP2;3 promoter and explore the impact of external factors on its expression, a series of 8 truncated vectors were constructed, 2081, 1524, 1035, 935, 835, 735, 663, and 581 bp in length, named P1-P8, and transformed into Agrobacterium rhizogenes strain K599 to generate soybean hairy roots. The GUS staining and quantity assays demonstrated that only P1 and P3 exhibited GUS activities, and the activity of P3 was stronger than for P1 ( Figure 5A). P5-P8 had no GUS signal, indicating that the core promoter region of GmTIP2;3 was 935 bp long from the ATG site. Interestingly, no GUS signal or GUS activity was detected for P2, implying that the inhibitor sequence occurred between P1 and P3, which also explained why the activity of P3 was stronger than for P1. Meanwhile, the expression of GmTIP2;3 was down-regulated under dark, drought (PEG and ABA), and salinity treatments for 2 h but showed no response to wounding treatment in transgenic lotus plants ( Figure 5B). These results were consistent with the results of the expression patterns after treatments with ABA and PEG in soybean roots. Plasma Membrane Localization of GmTIP2;3 To examine the localization of the GmTIP2;3 protein, the coding sequences were fused in frame with the coding region of the N-terminal side of green fluorescent protein (GFP). The fusion genes were expressed under the control of the CaMV 35S promoter. GFP fluorescence was evident in the cell plasma membrane transformed with the GmTIP2;3::GFP fusion plasmid ( Figure 6A), whereas GFP fluorescence (control) was detected throughout the cells transformed with GFP control plasmid ( Figure 6B). Heterologous Expression GmTIP2;3 Improved Osmotic Stress Resistance in Yeast Yeast cells carrying pYES2-GmTIP2;3:GFP or PYES2-GFP (control) were treated with PEG6000 for 40 h, and the survival state was detected. The results revealed that GmTIP2;3 was specifically expressed at the yeast cell membrane, and the heterologous expression of GmTIP2;3 in yeast cells could improve the survival efficiency under osmotic stress (Figure 7), indicating that GmTIP2;3 played an important role in osmotic tolerance in eukaryotes. DISCUSSION In this study, we isolated and characterized GmTIP2;3, an MIP family protein showing the highest similarity to Arabidopsis, rice, and corn TIP5. SMART software showed that GmTIP2;3 contains six transmembrane domains, single "AEFH" and "NWIYWVGP" motifs, and two conserved NPA motifs. Fujiyoshi et al. (2002) reviewed the structure and function of water channels in mammalian aquaporins, reporting that the sequence alignment of aquaporins shows several highly conserved motifs including two "NPA" sequences and single "AEFL" and "HW[V/I][F/Y]WXGP" sequences. Here, we found that plant TIPs contain AEFI or AEFV/H, and TIPs from other plants do possess HW[V/I][F/Y]WXGP, but the soybean TIP5 had the motif NWIYWVGP, thereby implying the differences in function and localization between GmTIP2;3 and other plant TIPs. Spatial and temporal expression analysis showed that GmTIP2;3 was constitutively expressed in all tested organs, with higher expression in the root and stem, indicating that it can absorb water from the soil through the root and then transport water through the stem to other organs, such as the leaf, flower, and pod. Tungngoen et al. (2009) cloned and characterized two aquaporins, HbPIP2;1 and HbTIP1;1, and induced expression analysis found that HbTIP1;1 was downregulated in liber tissues but up-regulated in laticifers in response to bark Ethrel treatment. Regon et al. (2014) also analyzed the expression patterns of 100 TIP aquaporin genes from dicots and monocots and indicated that the expression of TIP genes varies during different developmental stages and under stressed conditions. da Silva et al. (2013) identified and analyzed the expression patterns of sugarcane aquaporin genes under water deficit, thereby finding the aquaporin transcription in sugarcane to be potentially genotype specific. These findings demonstrated that TIP expression was organ specific or genotype specific and performed different regulator roles in different tissues. Recently, Lee et al. (2015) showed that the expressions of barley HvTIP1;2 and HvTIP3;1 were regulated by gibberellic acid (GA) and ABA and that these two hormones were involved in the fusion of protein storage vacuoles in aleurone cells, indicating that TIP plays another role in vacuole formation and transportation. When subjected to drought stress (ABA and PEG), the expression of GmTIP2;3 showed a dynamic trend at different time points, with an increase after PEG and ABA treatments for 48 h and 45 min, respectively, indicating that the expression of GmTIP2;3 exhibited a response to osmotic stress. In fact, GmTIP2;3 should be a plasma membrane intrinsic protein (PIP). It was predicted to be localized at the plasma membrane by the online software http://www.predictprotein. org/, and this subcellular localization was proven using Arabidopsis protoplasts, yeast cells, and onion epidermal cells (data not shown) harboring GFP. However, BLAST result at NCBI showed GmTIP2;3 to be a tonoplast intrinsic protein (TIP). Analysis of the promoter activity of GmTIP2;3 indicated that the activity of P3 (∼1000 bp in length) was stronger than the activity of P1 (∼2000 bp), implying that the inhibitor region occurred between these two regions, and P4 (∼550 bp) exhibited no GUS activity. To further determine the core or minimum region for the GmTIP2;3 promoter, five truncated constructs at 100 bp intervals between P3-P4 were prepared, but no GUS signal was detected. Therefore, we concluded that the core promoter region for GmTIP2;3 was located +1000 bp FIGURE 6 | Subcellular localization of GmTIP2;3 protein in Arabidopsis protoplasts. The fusion construct of the GmTIP2;3-green fluorescent protein (p35S::GmTIP2;3-GFP) in the pJIT166-GFP vector without a termination codon to create an in-frame fusion between the CDS and GFP, and the GFP control plasmid (p35S::GFP), was transformed into Arabidopsis protoplasts by PEG4000-mediated method. The transformed Arabidopsis protoplasts were incubated for 18-24 h at room temperature and observed under a confocal fluorescence microscope. GmTIP2;3 was mainly located at the cell membrane (A). However, the GFP control was distributed throughout the whole cell (B). Scale bars = 10 µm. FIGURE 7 | Osmotic response of yeast cells in heterogeneously expressing GmTIP2;3 S. cerevisiae INVSc1 strain cells transformed with the empty vector pYES2-GFP alone (Con) and pYES2-GmTIP2;3:GFP. GFP signal indicated that GmTIP2;3 was mainly located at the cell membrane, and the empty vector was distributed throughout the cells. S. cerevisiae INVSc1 strain cells transformed with the empty vector pYES2-GFP alone and pYES2-GmTIP2;3:GFP were spotted on SC-Ura medium in 0, 10, 100, 1000, and 10,000-fold dilutions, repeated twice for two different positive clones of pYES2-GmTIP2;3:GFP. The results showed the osmotic tolerance of yeast cells expressing GmTIP2;3. All experiments were repeated three times. upstream of the ATG start codon containing the 5 ′ UTR region of the GmTIP2;3 gene. The promoter-GUS system was used to detect the GUS activity changes of transgenic Lotus leaf under different treatments, including ABA, Nacl, dark, wounding, and PEG for 2 h. The results showed that the expression of GmTIP2;3 decreased under all treatments except wounding. The plant CARE software revealed that the promoter region contains many light-responsive elements, so the down-regulated expression under dark conditions was reasonable. Moreover, GUS activity under drought treatment for 2 h was consistent with the expression patterns after ABA and PEG treatments for 2 h. Lee et al. (2015) detected the promoter activity of HvTIP3;1 in response to ABA and revealed that the ABA responsiveness of the HvTIP3;1 promoter is likely to occur via a unique regulatory system distinct from the one involving the ABAresponse promoter complexes. Therefore, the mechanism of the ABA responsiveness of GmTIP2;3 should be further examined. Here, we can hypothesize that the plants first reduce the water hole number or close water channels to reduce the loss under stress by decreasing the transcription level of GmTIP2;3, and then when the plants have adapted to the stress environment, the expression of GmTIP2;3 recovers to its original level and continues to increase its transcript abundance to respond to stressed conditions. The plant response to drought is dependent on the SPAC (Soil-Plant-Air-Continuum). Root absorption and soil play important roles in plant adaption to drought stress (Shao et al., 2009). Higher expression of aquaporin proteins in plants can allow them to effectively absorb water from the soil using the roots and then transport water by the stem to other organs, such as the leaf, flower, and seed, especially under osmotic stress (Devi et al., 2015;Ding et al., 2015;Miniussi et al., 2015;Olaetxea et al., 2015). Here, the higher expression of GmTIP2;3 in the steles of the root and stem might promote and speed up water transportation from the roots to other organs under osmotic stress, improving plant tolerance to osmotic stress. Azad et al. (2009) analyzed water channels by yeast heterologous expression of tulip petal plasma membrane aquaporins from Pichia pastoris and monitored their water channel activity (WCA) by in vivo spheroplast-bursting and hypo-osmotic shock assays, suggesting that P. pastoris can be employed as a heterologous expression system to assay the WCA and to monitor the AQP-mediating channel gating mechanism of aquaporins. The yeast heterologous expression assay in this study showed that GmTIP2;3 could effectively improve the tolerance of yeast to drought stress. Previously, we performed this assay using salinity and drought treatments simultaneously, but the results indicated that yeast cells expressing GmTIP2;3 did not show improved survival rates under salinity stress, implying that GmTIP2;3 had the ability to transport water but not ions.

v3-fos

2017-06-26T06:24:53.388Z

2015-11-04T00:00:00.000Z

11781682

s2

Identification of QTL for resistance to Mediterranean corn borer in a maize tropical line to improve temperate germplasm Background A QTL mapping study for maize resistance to the Mediterranean corn borer (MCB) was performed with a RIL population derived from the cross B73 × CML103. To develop commercial inbreds of maize resistant to the MCB for use in Europe, it would be useful to transfer resistance from tropical germplasm like the subtropical inbred CML103 to temperate lines. The inbred B73 was chosen as representative of the Stiff Stock heterotic group, a major heterotic group used in hybrid grown in both North American and Europe. The objectives were to study the architecture of genetic factors for resistance to MCB and to check the feasibility of using marker-assisted selection (MAS) for transferring those genetic factors. Results Eight quantitative trait loci (QTL) were declared significant for resistance traits and eight QTL were located for agronomic traits. Alleles from CML103 at QTL significant for tunnel length could reduce tunnel length made for MCB in inbred B73 in more than 8 cm; favorable alleles for yield were also found in CML103 and no genetic correlation coefficient between tunnel length and yield was detected. Conclusions MAS for transferring resistance genes to corn borer attack from CML103 to B73 could be successful based on cross validation results and a negative effect on yield would not be expected. Electronic supplementary material The online version of this article (doi:10.1186/s12870-015-0652-9) contains supplementary material, which is available to authorized users. Background Sesamia nonagrioides Lef., commonly called Mediterranean corn borer (MCB), is the most important pest of maize (Zea mays L.) in the Mediterranean area [1]. The use of Bt hybrids seemed the most efficient method for controlling this pest, but transgenic crops are not authorized in many European countries and are not allowed for organic production [2]. Therefore, breeding for resistance to corn borers based on maize genetic variability for resistance would be valuable to the European and organic seed markets. Also, recent studies have reported a reduction of efficacy as some important pests have evolved resistance to Bt [3,4]. In this context, the stacking of several resistant genes has been proposed as one of the means to delay insect adaptation, and maize natural sources of resistance to stem borers could bring promising genes [5]. In a previous research a collection of 121 inbred lines was evaluated for resistance to MCB in a two-year experiment; the inbred B73 was classified as moderately resistant [6]. B73 is an inbred developed from the Iowa Stiff Stalk Synthetic population with great historic importance to breeders because the hybrid B73 × Mo17 has been widely used and is currently relevant as many commercial inbreds have B73 in their pedigrees [7]. The Iowa Stiff Stalk Synthetic population was constituted by 16 inbred lines resistant to stalk breakage [8] and the borer resistance exhibited by inbreds developed from this population could be consequence of mechanical resistance [9]. However, resistance to MCB attack of the inbred B73 is far from attaining the threshold required by farmers. In a previous study, we looked for quantitative trait loci (QTL) for stem tunneling by MCB in an array of recombinant inbred lines (RIL) developed from the cross B73 × Mo17 [10], B73 and Mo17 are both inbreds with some resistance to MCB attack [6], and limited gains will be obtained by combining resistance factors from both parents. This result is most likely due to common resistance factors among temperate germplasm. In recent studies the subtropical inbred CML103 [11] has shown better performance under MCB attack than B73 (unpublished data). CML103 has also demonstrated high general combining ability; therefore CML103 appears to be a promising candidate to donate novel genes for MCB resistance to temperate germplasm. In this study, QTL analysis for MCB resistance and agronomic traits was performed in a population of RIL derived from the cross B73 × CML103. In addition, the feasibility of using marker-assisted selection (MAS) for transferring those genetic factors was explored by testing the bias of each QTL by cross validation test. For the first time, QTL for resistance to MCB has been detected in a segregating population derived from a cross between inbreds with high and moderate resistance. Previous studies focused on crosses between moderately resistant and susceptible inbreds (EP39 × EP42), between two inbreds moderately resistant (B73 × Mo17) and between two susceptible inbreds (A637 × EP42) [10,12,13]. Results obtained until now have widened our knowledge about the genetic architecture of maize resistance to MCB, but lacked applicability. However, breeders around the world could benefit from the release of a version of B73 with increased resistance to MCB by transferring resistance factors from CML103 because some mechanisms of maize resistance could be common for corn borers [12]. Such resistance mechanisms could include chemical defense systems like benzoxazinoids, mayzin, protease inhibitors, etc. or physical defense traits related with cell wall components like lignin or silica [14]. Results Significant difference between B73 and CML103 were found for two resistance traits, tunnel length and stalk damaged (Table 1). Heritabilities for resistance traits ranged from low to moderate while for agronomic traits ranged from moderate to high (Table 1). Moderate genetic correlation between tunnel length and plant height (r g = 0.63) and high genetic correlation coefficients between tunnel length and stalk damaged (r g = 0.87) were found ( Table 2). The genetic map covered a length of 1388.5 cM. The average interval between markers was of 10.1 cM. No segregation distortion from the expected ratio was observed in the analyses for any marker. In a preliminary fit of the model selection, putative QTL for stalk lodging, kernel resistance, shank resistance, plant height, and days to silking were identified and mapped to different genetic positions (indicated by a gray arrow as suggestive QTL in Fig. 1). The LOD peaks that exceeded the LOD threshold chosen by permutation test (for each trait) indicated the presence of putative QTL ( Fig. 1) but several of them were excluded after a final fit of the model controlled by the Bayesian information criterion (BIC) was performed. For tunnel length, days to silking, and yield under infestation with MCB, all the LOD peaks that exceed the previously fixed LOD threshold were selected as real QTL in the final fit. Eight QTL for resistance traits were identified in this RIL population (Table 3, Fig. 2). Three QTL for tunnel length were located on chromosome 1, 5 and 6 and accounted for more than 50 and 25 % of the total genetic and phenotypic variance, respectively, with a percentage of estimation bias between 22 and 67 %. The additive effects ranged from 3 to 4 cm for each QTL with a bias estimation between 6 and 45 %. For stalk damaged two QTL were located on chromosome 1 and 6, each explained more than 30 and 10 % of the genetic and phenotypic variance, respectively, but the estimation biases for those parameters were too high (>95 %). The additive effect estimated in the test set ( α TS , more detailed explanation is in methods section) for both QTL for stalk damage were less than 1 %. A QTL for stalk lodging was located on chromosome 5 which accounted for 17 and 5.8 % of the genetic and phenotypic variance, respectively, with an estimation bias higher than 90 %. The absolute value of the additive effect estimated in the test set (TS) for this QTL was 2.2 %. Another QTL on chromosome 5 was located for kernel resistance which accounted for 10 and 3.5 % of the genetic and phenotypic variance, respectively. However the estimation of these parameters was completely biased as suggested by the results of the cross validation (CV) test (Table 3). In addition, the additive effect estimated in the TS was very small (0.02 point in the subjective scale from 1to 9). For shank resistance, one QTL of small additive effect ( α TS = 0.19) was located on chromosome 2. This QTL explained 66 and 7 % of the genetic and phenotypic variance, respectively, with an estimation bias of 58 %. Eight QTL were identified for agronomic traits (Table 3, Fig. 2). Two QTL for plant height were located on chromosome 5 and 7. These QTL accounted for 23 % and 19 % of the total genetic and phenotypic variance, respectively, and the bias estimation of these parameters was of 38 and 82 % for the QTL on chromosomes 5 and 7, respectively. The absolute value of additive effect estimated in the TS was 8.31 cm for the QTL on chromosome 5 and 5.7 cm for the QTL on chromosome 7. The detection frequency of the QTL on chromosome 5 was higher (0.90) than that observed in the QTL of chromosome 7 (0.38). Three QTL were detected for days to silking on chromosome 6, 8, and 9. The proportion of the genetic and phenotypic variance explained by the three QTL was 24.2 and 19.5 %, respectively, with an overestimation from 69 to 94 %. The additive effect estimated in the TS for each QTL was less than 1 day. For yield, three QTL were located on chromosomes 1, 6, and 7. The proportion of genetic and phenotypic variance explained by the three QTL was 33 and 23 % and the estimation bias of the genetic variance explained by each QTL ranged from 30 to 96 %. The additive effect estimated in TS ranged from 0.1 to 0.6 Mg ha −1 . The detection frequency through the CV runs was of 34, 95, and 17 % for the QTL in chromosomes 1, 6, and 7, respectively. Discussion The heritabilities observed for tunnel length by MCB was intermediate and compare favorably to those obtained with other RIL populations under similar conditions of infestation with MCB [10,12]. Heritability for kernel resistance observed herein (h 2 = 0.35) is inferior to that obtained (h 2 = 0.5) by Ordás et al. [10]. In addition, the heritability for shank resistance was not different from zero which is in agreement with Samayoa et al. [12]. The heritabilities for agronomic traits were similar to those obtained by other authors in numerous, diverse RIL populations [18][19][20]. As the goal is to detect reliable QTL, most discussion will be focused on results from the final fit of the model selection that is conditioned by the Bayesian information criterion (BIC) [17]. Although this model selection criteria tend to find slightly fewer QTL compared with other criteria it minimizes the risk of selecting spurious QTL [21]. No QTL for stalk tunneling by MCB were previously reported in bins 1.07-1.08, 5.03, and 6.05-6.06 [10,12,13,22]. However, QTL for tunnel length by the European corn borer (ECB, Ostrinia nubilalis) have been previously mapped to chromosomes 1 and 5 [18,[23][24][25]. Krakowsky et al. [26] and Orsini et al. [27] also localized QTL for tunnel length and stalk breakage by ECB in the bin 6.05. As all favorable alleles for tunnel length in this study came from the subtropical line CML103, this inbred could clearly enhance the resistance of the line B73 by providing new alleles of resistance in chromosomes 1 and 5 and, even, in the chromosome 6 where it is known that the B73 line carries alleles associated with resistance to tunnel length by ECB [26]. In addition, the additive effects for the three QTL detected for tunnel length were, in general, higher (α = 3-4.1 cm) than those reported in the studies mentioned above (α = 0.5-1.2 cm) and, most importantly, the CV analysis revealed that the reliability of QTL for tunnel length was moderate to high. Significantly different from zero at 0.05 probability level QTL for tunnel length and plant height were colocalized in the same region of the chromosomes 5. In addition, a significant and moderate genetic correlation between tunnel length and plant height was found agreeing with results of previous QTL studies with artificial infestation both with MCB [12,13] as with ECB [24,28,29]. In addition, in a recent association mapping for resistance to MCB attack it was observed an intermediate and positive genetic correlation between tunnel length and plant height but no significantly associated SNP was co-localized for both traits [22], therefore it remains necessary to carry out deepest studies to elucidate if these findings are due to linkage or pleiotropy. QTL for tunnel length and days to silking were also co-localized on chromosome 6. Opposite signs of the additive QTL effects for these traits indicate that flowering time could be slightly delayed when transferring resistance alleles from CML103 to B73 but it was not supported by the genetic correlation between these traits. Yield would not be significantly modified because no genetic correlation was found between tunnel length and yield under infestation unlike to other studies in which selection to reduce tunnel length made by corn borers has resulting in an important reduction of yield probably due to linkage between certain alleles for resistance and some alleles affecting maize yield. [30][31][32]. The final fit for days to silking revealed the presence of three QTL in chromosomes 6, 8 and 9; one of them was also detected by Buckler et al. [33] in the same RIL population and genotyping data but different data analysis method. The QTL for days to silking on chromosomes 8 and 9 detected herein were not detected by those authors; while a QTL in chromosome 3 detected by Buckler et al. [33] was found in the preliminary fit but it was not retained in the final fit. These discrepancies between our results and those provided previously by Buckler et al. were probably due to QTL × environment interaction effects and stressed the importance of making phenotypic evaluations in environments similar to those for which breeding materials are intended. We identified three novels QTL for grain yield under infestation with S. nonagrioides in chromosomes 1, 6, and 7. In previous studies, QTL for yield under Fig. 1 Whole-genome scans to detect QTL for resistance and agronomic traits. Solid black line represents the LOD curve obtained with QTL scan using a marker interval of approximately 10 cM and the red dashed line indicates the LOD threshold chosen by permutation test to declare the presence of a significant. Gray arrows indicate the presence of putative QTL which were detected in the preliminary fit but not in the final fit of the model selection. infestation with MCB were located on chromosomes 4, 5, and 8 [12,13], and QTL for grain yield under infestation with ECB have been reported on chromosomes 2, 4, 6, 8, 9, and 10 [18-20]. Alleles from the line B73 at QTL in chromosomes 6 and 7 increased grain yield, but the allele from CML103 for the QTL in chromosome 1 could be used to improve yield of the inbred B73. Although this QTL would need to be tested in hybrid for efficacy since yield QTL in inbreds versus hybrids are poorly correlated. In general, the additive effects estimated in TS of each of the three QTL (0.1 -0.6 Mg ha −1 ) were higher than those obtained by authors mentioned above (0.2 -0.3 Mg ha −1 ). The QTL located in chromosome 6 is especially interesting because it explained a high proportion of the genetic variance (17 %) with the lowest estimation bias for this parameter and with a high detection frequency (95 %) through CV runs. Although no QTL for yield under infestation with corn borers have been previously found in this region (bin 6.01-6.02) several studies have reported important QTL for grain yield and its components in physiological conditions in the same region in other mapping populations [34][35][36][37][38]. Even the results of a fine mapping study suggest that a pleiotropic locus could be affecting grain yield and related traits in this region of chromosome 6 [39]. Conclusion The inbred CML103 could enhance the resistance of the inbred B73 without reducing its yield under infestation by providing new alleles of resistance in chromosomes 1, 5 and 6 where it is known that the inbred B73 carries alleles associated with resistance to tunnel length by ECB. The inbred CML103 could also potentially provide favorable alleles for yield under infestation with MCB on chromosome 1. Three novels QTL for yield under infestation with MCB were found in this RIL population, the highly reliable QTL of chromosome 6 with an additive effect of 0.6 Mg ha −1 being particularly important. Cross validation analyses confirmed the moderate to high reliability of QTL detected for tunnel length and supported the use of markers associated to these QTL for performing marker-assisted selection in order to transfer resistance alleles from CML103 to B73. Methods The 178 RILs obtained from the cross B73 × CML103 are part of the nesting association mapping population with genotype at 1478 SNPs provided [40,41]. Based on preliminary analysis with all the markers and the conclusion of previous research comparing the use of high vs. low density marker map [42,43] we constructed a genetic map using MAPMAKER software with a subset of 147 markers (see Availability of supporting data section) to obtain an average marker interval of 10 cM. One hundred percent of the genome was within 20 cM of the nearest marker in the genetic map. The 178 RILs were evaluated in 2011 and 2012 along with the parental inbreds B73 and CML103 using a 14 × 14 single lattice design with two replication per year. The trials were hand planted and each experimental plot consisted of one row spaced 0.8 m apart with 13 two-kernel hills spaced 0.18 m apart. Plots were overplanted and thinned, obtaining a final density of~70,000 plant ha −1 . The evaluations were performed under artificial infestation with MCB eggs obtained at the Misión Biologica de Galicia by rearing the insect as described by Eizaguirre and Albajes [44] and Khan and Saxena [45]. Before flowering, five plants of each plot were infested with~40 MCB eggs placed between the stem and the sheath of a basal Individual phenotypic data (per year) was analyzed in SAS software using the mixed model procedure (PROC MIXED) [46] considering replications and blocks within replications as random effects and RILs as fixed effects. Then, combined analysis across year was conducted considering RILs as the only fixed effects. A best linear unbiased estimator (BLUE) was obtained to estimate each line mean phenotypic value both for individual as for combined data. Heritabilities (ĥ 2 ) across environments were estimated for each trait on a family-mean basis as described by Holland et al. [15]. The genetic (r g ) and phenotypic (r p ) correlations between traits were computed following Holland [47]. All previous analyses were made in SAS software version 9.4 (see Additional file 2 for more details of code). QTL analysis was performed using the software package PlabMQTL [17]. Composite interval mapping approach was conducted for QTL detection and to estimate QTL effects using the command cov SEL (Additional file 2). According to a previously executed permutation test with 1000 random reshuffles [48], LOD thresholds of 2.9 (with and empirical critical value of 25 %) were chosen to declare significant the presence of a putative QTL. The QTL mapping was conducted by a two-step procedure: in a first step an entire genome scanning is performed to draw the LOD curves and identify the peaks where the putative QTL are located, in this preliminary fit are estimated the additive effects of all preselected cofactors. In the second step the more important genetic effects of QTL of previous step are screened using the BIC as criteria of selection during the stepwise regression procedure [17]. Following Utz et al. [49], a five-fold cross validation (CV) approach was employed for obtaining unbiased estimation of the QTL parameters such as genetic (p ) and phenotypic (R 2 ) variance explained by each putative QTL and its respective additive effect (α ). For each trait, CV was performed for the whole data set (DS) of entry BLUEs across environments. A total of 142 entries were used as estimation set (ES) for calibration and 36 entries were used as the test set (TS) for validation. One thousand CV runs were performed in order to determine the QTL frequency and shrinkage of estimations for QTL effects and proportion of the genetic and phenotypic variance explained by the QTL detected in the original data set [50]. The magnitude of the bias of the estimation of p i explained by each individual QTL i was calculated as the difference between the average estimate of p obtained in ES and the corresponding estimate in TS ( p i ES − p i TS:ES ) divided by p i ES . In the same way the biases for the estimates of additive effectsα i were obtained. A bias of 50 % was established as cutoff to consider low or high the estimation bias of each parameter. The Grep utility [51] was employed to extract, in each CV run, the proportion of genotypic (p i ES andp i TS ) and phenotypic (R 2 i ES andR 2 i TS ) variances of the ES and TS explained by each individual QTL i detected and also the additive effects (α i ES andα i TS ). Availability of supporting data Genotypic data of a RIL population derived from B73 × CML103 used in QTL analysis in this research are available in the Digital CSIC repository in http:// hdl.handle.net/10261/123685. This array was taken from the NAM population [40,41] genotyping data set (phased and fully imputed genotypes at 1 cM resolution) available in http://mirrors.iplantcollaborative.org/ browse/iplant/home/shared/panzea/genotypes/GBS/v23/ NAM_phasedImputed_1cM_AllZeaGBSv2.3_allChrs.zip. Competing interests The authors declare that they have no competing interests. Authors' contributions LFS carried out field experiments, performed statistical analysis of the data and made the draft of the initial manuscript. AB and MDM conceived the study, MDM generated the vegetal materials, AB and RAM assisted LFS in field experiment design and data collection, AB also participated in statistical analysis and in drafting the initial manuscript. All authors have read and approved the final version of the manuscript.

v3-fos

2016-05-04T20:20:58.661Z

2015-10-20T00:00:00.000Z

13580842

s2

Responses of soil microeukaryotic communities to short-term fumigation-incubation revealed by MiSeq amplicon sequencing In soil microbiology, there is a “paradox” of soil organic carbon (SOC) mineralization, which is that even though chloroform fumigation destroys majority of the soil microbial biomass, SOC mineralization continues at the same rate as in the non-fumigated soil during the incubation period. Soil microeukaryotes as important SOC decomposers, however, their community-level responses to chloroform fumigation are not well understood. Using the 18S rRNA gene amplicon sequencing, we analyzed the composition, diversity, and C-metabolic functions of a grassland soil and an arable soil microeukaryotic community in response to fumigation followed by a 30-day incubation. The grassland and arable soil microeukaryotic communities were dominated by the fungal Ascomycota (80.5–93.1% of the fungal sequences), followed by the protistan Cercozoa and Apicomplexa. In the arable soil fungal community, the predominance of the class Sordariomycetes was replaced by the class Eurotiomycetes after fumigation at days 7 and 30 of the incubation. Fumigation changed the microeukaryotic α-diversity in the grassland soil at days 0 and 7, and β-diversity in the arable soil at days 7 and 30. Network analysis indicated that after fumigation fungi were important groups closely related to other taxa. Most phylotypes (especially Sordariomycetes, Dothideomycetes, Coccidia, and uncultured Chytridiomycota) were inhibited, and only a few were positively stimulated by fumigation. Despite the inhibited Sordariomycetes, the fumigated communities mainly consisted of Eurotiomycetes and Sordariomycetes (21.9 and 36.5% relative frequency, respectively), which are able to produce hydrolytic enzymes associated with SOC mineralization. Our study suggests that fumigation not only decreases biomass size, but modulates the composition and diversity of the soil microeukaryotic communities, which are capable of driving SOC mineralization by release of hydrolytic enzymes during short-term fumigation-incubation. INTRODUCTION Soil microorganisms are the principal participants in most soil processes. The determination of microbial biomass can facilitate our understanding of microbial ecological functions and the magnitude of certain processes, such as soil carbon (C) and nitrogen (N) mineralization . Chloroform fumigation (fumigation) is a classic method used for determination of the soil microbial biomass. Jenkinson and Powlson (1976) described a fumigation-incubation method to estimate the soil microbial biomass. They proposed that, following fumigation, the extra CO 2 evolved from the fumigated soil compared to the similarly incubated but non-fumigated control soil during the first 10 days of incubation (termed Fumigation-incubation, FI) provides an estimate of the original soil microbial biomass (Jenkinson and Powlson, 1976). Subsequently, more analytically convenient, the fumigationextraction method to measure microbial biomass was developed from FI (e.g., Brookes et al., 1982Brookes et al., , 1985Vance et al., 1987;Wu et al., 1990). Previous investigations have observed an intriguing phenomenon that although fumigation destroyed 80-90% of the initial soil microbial biomass, following the fumigant removal, soil organic C (SOC) mineralization continued at the same rate as in the non-fumigated soil under appropriate incubation conditions for several weeks or even months (Jenkinson and Powlson, 1976;Wu et al., 1996;Kemmitt et al., 2008). Kemmitt et al. (2008) attempted to explain this phenomenon and developed the "Regulatory Gate" hypothesis. Firstly, the recalcitrant SOC was considered to be transformed into bio-available components via an abiotic process(es) (termed the "Regulatory Gate"), and this small trickle of bio-available C could then be mineralized by the soil microorganisms, independently of biomass size. Possible mechanisms of SOC transformation was considered to include chemical oxidation, chemical hydrolysis, desorption of absorbed organic matter or diffusion from within aggregates . There could be a combination of these parameters, or, indeed, none of them (Brookes et al., 2009). There is some support for the "Regulatory Gate" hypothesis. For instance, in mineral soils, physical access to occluded or adsorbed substrates by the microbial population is the rate-limiting process governing SOC mineralization (Schimel and Schaeffer, 2012). However, when considering the "Regulatory Gate" hypothesis, we must also consider different microbial communities associated with the functioning of SOC mineralization (Paterson, 2009). The bacterial community in an arable soil subjected to fumigation, followed by inoculation with a little fresh soil, was investigated by Dominguez-Mendoza et al. (2014), who considered that some bacterial groups (e.g., Micromonosporaceae, Bacillaceae, and Paenibacillaceae) had the capacity to metabolize the fumigantkilled soil microorganisms and partially recolonize a fumigated arable soil during a 10-day incubation. Microeukaryotes (e.g., fungi, protists, and metazoans) make important contributions to soil biogeochemical cycling and the maintenance of soil fertility because of their involvement in some key processes, such as C turnover and energy flow (Chen et al., 2012(Chen et al., , 2014Damon et al., 2012;Jing et al., 2014). By analyzing phospholipid fatty acids (PLFAs), Zelles et al. (1997) and Dickens and Anderson (1999) reported that the soil microeukaryotic biomass declined by 70-80% after fumigation followed by 10 and 28-day incubations. However, so far the changes in the composition, biodiversity and C-metabolic functions of the soil microeukaryotic communities are not well understood during the fumigation-incubation period. In the present study, we aimed to comprehensively survey the soil microeukaryotic communities, and further examine their changes in composition, diversity and functions in response to short-term fumigation-incubation. The following two hypotheses were tested: (i) fumigation would alter taxonomic composition and diversity patterns of the soil microeukaryotic communities, dependent on soil and incubation time, and (ii) such changed microeukaryotic communities would be still active or potentially active to drive the recalcitrant SOC mineralization. To test these hypotheses, a grassland soil was sampled from the Inner Mongolian prairie and an arable soil from Zhejiang in China. Both were fumigated with ethanol-free chloroform for 24 h, incubated aerobically for 30 days, and sampled at days 0, 7, and 30 of the incubation to determine the soil microeukaryotic community composition and diversity using a high-throughput sequencing approach. Microbial biomass, respiration rate, the metabolic quotient, potential, and specific activities of two C-acquiring enzymes (βglucosidase and invertase) were also measured and related to the fumigated microeukaryotic communities. Soil Description The grassland soil was acquired from Inner Mongolia Grassland Ecosystem Research Station of Chinese Academy of Sciences located in Xilingol Region (43 • 33 ′ N, 116 • 37 ′ E), Inner Mongolia, China. The Leymus chinensis (Trin.) Tzvelev grassland has been fenced since 1980, and experiences a temperate semiarid climate, with an annual mean temperature of 0.5 • C and annual average precipitation of 350 mm. The arable soil was taken from Dongyang Maize Research Institute of Zhejiang Academy of Agricultural Sciences in Dongyang County (29 • 27 ′ N, 120 • 23 ′ E), Zhejiang Province, China. Maize (Zea mays L.) has been continuously cropped twice a year for 10 years. Annual mean temperature and precipitation are 17 • C and 1350 mm, respectively. The two soils were collected on September 2014, after visible plant residues and stones were removed, airdried and sieved <2 mm. Basal soil physiochemical index were analyzed (Table 1). Soil Fumigation, Incubation, and Sampling Soils were pre-incubated at 60% of the maximum water-holding capacity (WHC) and 25 • C for 15 days, to allow microbial activity to stabilize after rewetting. Moist soil (200 g) was placed in a desiccator containing 20 ml of distilled water at the bottom (to maintain humidity), a beaker with 50 ml of ethanol-free chloroform and 50 ml of 1.0 M NaOH (to absorb CO 2 ). The desiccator was evacuated until the chloroform had boiled for (Nelson and Sommers, 1982). b Kjeldahl digestion (Bremner, 1965). c 1:2.5 soil and water suspension. d Laser particle characterization. e EDTA-ammonium acetate method (Lu, 2000). Cation exchange capacity is an important indicator for soil water and fertilizer-holding capacity, and soil buffering potential. 3 min, and then incubated in darkness for 24 h at 25 • C. The residual chloroform in the soil was then removed by repeated evacuations. The non-fumigated controls were treated similarly except that distilled water replaced ethanol-free chloroform in the desiccators and the soils were not evacuated. Fumigated and non-fumigated soil (200 g) was transferred to stoppered 1 l glass jars, and incubated at 60% WHC and 25 • C for 30 days. During the incubation period, soil moisture was controlled by weighing the jars and adding sterilized distilled water, and the air in the jars was refreshed every 2-3 days to maintain aerobic condition. Soil samples were collected at days 0 (after 24 h fumigation), 7 and 30 of the incubation. Samples were divided into two portions, one portion was stored at 4 • C to determine microbial biomass C, respiration rate, invertase and βglucosidase activities, and the other at −80 • C for DNA isolation and molecular analysis. The experiment consisted of four treatments (the grassland and arable soils with and without fumigation). All treatments were replicated three times. The grassland soil was designated "G, " the arable soil "A, " fumigation "F, " and incubation days "0, 7, and 30." Determination of Microbial Properties Microbial biomass C was extracted using the chloroform fumigation method (Vance et al., 1987). The C concentration was determined using a Multi C/N 3100 TOC analyzer (Analytik Jena AG, Jena, Germany), and a value of k EC = 0.45 (Wu et al., 1990) was used to calibrate biomass C content. Microbial respiration rate was analyzed using the alkali absorption method, and the trapped CO 2 concentration was measured by titration using an EasyPlus autotitrator (Mettler Toledo, Zurich, Switzerland). The metabolic quotient (qCO 2 ) was estimated by analyzing the hourly mean CO 2 emission per unit biomass C (Blagodatskaya and Anderson, 1998). Invertase activity was determined by a 3,5dinitrosalicylic acid method as described by Bandick and Dick (1999). Assay of β-glucosidase activity was adapted from Tabatabai (1994). In brief, 5.0 g of moist soil was suspended in 20 ml of modified universal buffer (pH 6.0), and 5 ml of 25 mM pnitrophenyl-β-D-glucopyranoside (Aladdin, Shanghai, China) was added as the reactive substrate. The suspension was reciprocally shaken at 200 rev min −1 and 37 • C for 1 h, and then 5 ml of 0.5 M CaCl 2 and 20 ml of 0.1 M Tris buffer (pH 12.0) were added to stop substrate degradation. The solution was centrifuged at 13,000 × g for 1 min and the concentration of paranitrophenol (PNP) in the supernatant was measured at 400 nm on a spectrophotometer (Puyuan, Shanghai, China). The same procedure was applied to the control, except that the substrate was added after the incubation and addition of the CaCl 2 and Tris buffer. DNA Isolation, Amplification, and Sequencing The total soil DNA was isolated and purified using a FastDNA spin kit (MP Biomedicals, Santa Ana, CA, USA), followed by an UltraClean DNA purification kit (MoBio, Carlsbad, CA, USA). The isolated DNA was dissolved in 50 µl of TE buffer, and the DNA quality and quantity were verified using electrophoresis on 1% agarose gels. To produce the eukaryotic amplicon library for highthroughput sequencing, the eukaryotic 18S rRNA gene fragments were amplified using the universal primers Euk1F (5 ′ -CTGGTTGATCCTGCCAG-3 ′ ) and Euk516R (5 ′ -ACCAGACTTGCCCTCC-3 ′ ) (Shen et al., 2014;Shi et al., 2015). The forward and reverse primers were tagged with adapter, pad and linker sequences. Each barcode sequence (5 mer) was added to the reverse primer for pooling of multiple samples in one run of MiSeq sequencing. For each sample, PCR amplification was performed in triplicate 50-µl reaction mixtures containing 0.5 µl (125 pmol) of each forward/reverse primer, 1 µl (approximately 50 ng) of DNA template, 23 µl of ddH 2 O, and 25 µl of Premix Taq (Takara, Dalian, China), which consisted of 1.25 U DNA polymerase, 2 × dNTP mixture (0.4 mM), 2 × buffer (3 mM Mg 2+ ), and the marker (Tartrazine/Xylene Cyanol FF). Thirty-five thermal cycles (95 • C for 45 s, 56 • C for 45 s, and 72 • C for 1 min) were carried out with a final extension at 72 • C for 7 min. PCR amplicons pooled from the triplicate reactions were purified using a QIAquick PCR purification kit (Qiagen, Shenzhen, China), and quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). Equimolar amounts of amplicons from all samples (each 200 ng) were combined into a mixed sample. According to the MiSeq reagent kit preparation guide (Illumina, San Diego, CA, USA), the purified mixture was diluted and denatured to obtain the 8 pM sample DNA library and mixed with an equal volume of 8 pM PhiX (Illumina). Finally, 360 µl of the mixture library was loaded with read 1, read 2, and the index sequencing primers on a 300-cycle (2 × 150 paired ends) kit and run on a MiSeq apparatus (Illumina). Bioinformatics and Data Analysis The 18S raw sequence data were processed using the Quantitative Insights Into Microbial Ecology (QIIME) 1.8.0-dev pipeline (Caporaso et al., 2010a) (http://qiime.org/). Poor-quality sequences (i.e., sequences of <200 bp with an average quality score of <25 and ambiguous characters) were discarded (Huse et al., 2007). Filtration of the sequences was done to remove erroneous operational taxonomic units (OTUs) due to sequence errors, and chimeras were detected using the UCHIME program (Edgar et al., 2011). Sequences were then binned into OTUs de novo at a 97% similarity level using the UCLUST algorithm (Edgar, 2010). The most highly connected sequence (i.e., the sequence with the highest similarity to all other sequences in the cluster) was chosen to represent each OTU (Hamady et al., 2008). All selected representative sequences were aligned by use of the PyNAST tool (Caporaso et al., 2010b). Taxonomy was assigned to eukaryotic phylotypes of the Silva 104 database (http://www.arb-silva.de/download/archive/qiime/). The variations in the main phylotypes induced by fumigation were expressed as log 10 -transformed odds ratio (Ganesh et al., 2014). We obtained between 5162 and 24,947 valid sequences per sample (mean 13,471) for soil samples with the exception of a sample from G-0 treatment (Table S1). To rarify all datasets to the same level of sampling effort, 5000 sequences per sample were randomly selected for the microeukaryotic αand β-diversity analyses. Phylogenetic diversity and phylotype richness (i.e., number of rarefied OTUs) indices were calculated by the QIIME toolkit, with rarefaction analysis of 250 bootstrap random sampling iterations and 4% incremental sampling efforts. For β-diversity analysis, dissimilarities of the microeukaryotic communities were calculated using principal coordinate analysis (PCoA) of normalized, weighted, pairwise UniFrac (Lozupone and Knight, 2005) distances between all samples, of which principal component eigenvalues were generated by the QIIME toolkit. Analysis of similarity (ANOSIM) based on 999 permutations was performed using the Bray-Curtis (Bray and Curtis, 1957) algorithm to quantitatively compare the community differences between different groups. Redundancy analysis (RDA) related microbial properties to the explanation of the fumigated communities. In addition, Mantel test revealed the correlations between microbial properties and the community composition of total microeukaryotes, fungi and protists in the fumigated soils. These analyses were completed in the package "vegan" of the R project (version 3.1.3) (http://www.r-project.org/). A heat map was constructed using the function "heatmap.2" from the R package "gplots." For better visualization, the original data were transformed following the formula log 2 (1000x + 1) (Lundberg et al., 2012), and hierarchical clustering was based on Bray-Curtis similarities with group-average linkage. A Venn diagram was employed to characterize the shared and unique microeukaryotic communities among different treatments. One-way ANOVA was performed using SPSS 16.0 software, and significant differences were determined using Bonferroni's multiple range test. Microeukaryotic co-occurrence networks were constructed using the online CoNet pipeline (http://apps.cytoscape.org/apps/ conet) to explore the internal community relationships. OTUs with less than 10 sequences were filtered to remove poorly represented OTUs and reduce network complexity (Barberán et al., 2012). All possible Spearman's rank correlations between OTUs were calculated. The valid co-occurrence patterns were considered with the Spearman's correlation coefficient r > 0.6 and significance P < 0.01 (Barberán et al., 2012). The nodes in the network represent the OTUs at 97% identity, and the connections correspond to a strong and significant correlation between nodes. The topological properties (i.e., average path length, cumulative degree distribution, network diameter, clustering coefficient, modularity, eccentricity, closeness, and betweenness centrality) were calculated in the platform Gephi (Bastian et al., 2009). Visualization of the network was also performed in the Gephi. Microbial Biomass and Activities After fumigation, the amount of microbial biomass C significantly decreased by approximately 70% in the grassland and arable soils during the incubation period, and it was significantly lower at day 30 compared to day 0 ( Table 2). Both fumigated soils at day 0 showed a significantly higher rate of microbial respiration than other treatments during the incubation period. The change in β-glucosidase activity in both soils was not significant during the incubation period. Both fumigated soils at days 0 and 7 had significantly higher invertase activities than other treatments. Fumigation enhanced the metabolic quotient in both soils at day 30. Specific β-glucosidase activity in the grassland and arable soils was increased by fumigation by average 4.5 and 4.4-fold, respectively, and specific invertase activity by average 5.7 and 8.2-fold, respectively ( Table 2). Taxonomic Assemblages of Microeukaryotes Across all soil samples, a total of 474,982 high-quality sequences (99.9% were retrieved from eukaryota), clustered into 6664 OTUs after trimming and filtration ( Table S1). The microeukaryotic communities were dominated by fungi, which accounted for 55.7-88.4% of the total sequences among different treatments. Ascomycota, Cercozoa, and Apicomplexa (belonging to fungi, Rhizaria, and Alveolata, respectively) were the major phyla. These phyla in the fumigated grassland soil showed no consistent changes during the incubation period, while in the fumigated arable soil, the relative frequency of Ascomycota (especially the class Eurotiomycetes, Figure 1B) increased while that of Apicomplexa decreased with increasing incubation time ( Figure 1A). The fungal community was dominated by Ascomycota (mainly the subphylum Pezizomycotina, Table S2) (80.5-93.1% of the fungal sequences), in which the classes Eurotiomycetes and Sordariomycetes showed high abundance ( Figure 1B). Overall, fumigation modulated taxonomic composition of the microeukaryotic communities at the phylum/class levels. Especially in the arable soil, the predominance of the class Sordariomycetes was replaced by the class Eurotiomycetes after fumigation at days 7 and 30 ( Figure 1B). During the incubation period, there were no significant differences in the number of total phyla in the grassland or arable treatment (Figure 1A), and the number of fungal classes in the arable treatment ( Figure 1B). OTU Distribution and Network Analysis We used a Venn diagram to observe shared and unique communities among different treatments at the end of incubation ( Figure S1). The Venn diagram was constructed based on a subset of 5000 sequences per sample and the average OTUs based on three replicates. The fumigated grassland and arable soils harbored 26 and 19 unique OTUs, respectively (accounting for 18.2 and 16.0% of the respective total OTUs), and they shared 96 and 76 OTUs with their corresponding non-fumigated controls, in which 39 and 38 were unique OTUs. Both fumigated soils exclusively shared 5 OTUs, only accounting for 3.5 and 4.2% of the respective communities. The 59 common OTUs were shared by all treatments (Figure S1). The co-occurrence patterns in the fumigated microeukaryotic communities through 30-day incubation were explored by construction of OTU networks (Figures S2, S3). The fumigated communities exhibited 190 significant correlations (connections) of 74 OTUs (nodes) in the grassland soil (Figure S2), and 192 significant correlations of 88 OTUs in the arable soil ( Figure S3). The fungal OTUs were shown to be important nodes closely related to other OTUs, and accounted for 56.8 and 51.1% of nodes in the fumigated grassland and arable soils, respectively. The average path lengths were 1.73 and 2.02 in the networks of the fumigated grassland and arable soils, respectively, and network diameters were both 5. These topological properties indicated that the microeukaryotic communities in both fumigated soils were highly connected and presented small-world networks (short network distance among most of nodes and their interconnections through several paths). Microeukaryotic α-and β-diversity Phylogenetic diversity and phylotype richness indices based on rarefaction to 5000 sequences were used to estimate the microeukaryotic α-diversity (Figures 2A-D). After fumigation, the grassland soil microeukaryotic α-diversity at days 0 and 7 significantly decreased, compared with the corresponding non-fumigated controls (Figures 2A,C). Compared with the FIGURE 1 | Taxonomic distributions of the microeukaryotic phyla (A) and the fungal classes (B) in different treatments during the incubation period. Each stripe represents the average frequency of three replicates. Percentages in panels (A,B) are the fungal frequency and the proportion of Ascomycota in the fungal community, respectively. Numbers on the right are the counts for total phyla (A) and fungal classes (B), with the respective standard deviation in parentheses, and the same letter indicates no significant difference within the grassland or arable treatment. G, grassland soil; A, arable soil; F, fumigation; number, incubation days. grassland soil, the arable soil exhibited different response of the microeukaryotic α-diversity to fumigation. During the incubation period, the microeukaryotic α-diversity between the fumigated and non-fumigated arable soil showed no statistical difference (Figures 2B,D). The profiles of the microeukaryotic community structure were plotted using PCoA of the normalized and weighted pairwise UniFrac distances between all samples ( Figure 2E). The fumigated microeukaryotic communities at the start of the incubation were not separated from the corresponding non-fumigated communities, which clustered well together during the incubation period. In the fumigated grassland soil, the microeukaryotic communities at day 30 were moderately separated from those at days 0 and 7. In the fumigated arable soil, the visible differentiations of community structure between different incubation time points occurred along the second coordinate axis (PCo2). The separation of the microeukaryotic communities in the first component (PCo1) implied that the two soils had different microeukaryotic community structures. The results of ANOSIM (Table 3) further confirmed the significant (P < 0.01) effects of fumigation and soil source on the microeukaryotic community structure. Fumigation had no statistical effect at day 0 but significant (P < 0.05) effect at days 7 and 30 on community structure in the arable soil (Table 3, Figure 2E). The Main Phylotypes in Response to Fumigation The microeukaryotic classes in which the relative frequencies exceeded 0.1% were selected to construct a heat map of distributions of the main microeukaryotes after fumigation ( Figure 3A). Figure 3B showed the log 10 -transformed odds ratio, which is the ratio of the odds of a given phylotype occurring in the fumigated soils to the odds of it occurring in the corresponding non-fumigated controls, based on OTU counts pooled across incubation days. The selected phylotypes made up 82.9-98.3% (91.0 ± 3.8%) of the total sequences in the fumigated samples ( Figure 3A). Hierarchical clustering demonstrated that FIGURE 2 | Microeukaryotic α-and β-diversity in different treatments during the incubation period. Phylogenetic diversity (A,B) and phylotype richness (C,D) were calculated based on rarefaction to 5000 sequences, the bars indicate ±1 standard deviations of three replicates, and different letters indicate significant differences at P < 0.05. Microeukaryotic community structure was indicated by principal coordinate analysis (PCoA) of the normalized and weighted pairwise UniFrac distances between all samples (E). G, grassland soil; A, arable soil; F, fumigation; number, incubation days. the fumigated treatments at day 30 clustered better than those at days 0 and 7. After fumigation, the classes Eurotiomycetes and Sordariomycetes were the core phylotypes (Figures 1B, 3A), with average 21.9 and 36.5% relative frequency, respectively (Table S3). Relationships between Microbial Properties and the Fumigated Microeukaryotic Communities After fumigation, irrespective of incubation time, the significantly varied microeukaryotic phylotypes (i.e., Eurotiomycetes, Sordariomycetes, Dothideomycetes, Coccidia, and uncultured Chytridiomycota, Figure 3B) in the two soils were used to relate microbial properties. RDA indicated that the variation in these microeukaryotic phylotypes was significantly explained by invertase activity, specific β-glucosidase activity, Statistic R and significance (P) of differences between different groups (fumigation and non-fumigation, grassland and arable soils, and different incubation time) were calculated by analysis of similarity (ANOSIM) using OTU-based Bray-Curtis distances. and biomass C in the fumigated grassland soil (Figure 4A), and by respiration rate and biomass C in the fumigated arable soil ( Figure 4B). The Mantel test revealed that invertase activity, specific β-glucosidase activity, and biomass C were significantly correlated with the community composition of total microeukaryotes, fungi, and protists in the fumigated grassland soil ( Table 4). Detailed Information on the Grassland and Arable Soil Microeukaryotic Communities The present study is the first work to extensively investigate the microeukaryotic communities in the fumigated soils by means of deep MiSeq sequencing of the eukaryotic 18S rRNA gene amplicons. A total of 474,982 quality filtered reads were clustered into 6664 OTUs across 36 sample datasets. These data provided detailed information on taxonomic composition and diversity patterns of the grassland and arable soil microeukaryotic communities, and further revealed their temporal evolution in response to fumigation. Compared with other studies in terms of soil eukaryotic microbiota, we obtained the more numerous sequences which were rarefied to a deeper level (5000 sequences per sample) for diversity analysis. For example, Chen et al. (2012) only measured 793 gene sequences for microeukaryotic community analysis in a continuous peanut-cropping area. In the studies of Shen et al. (2014) and Shi et al. (2015), the soil microeukaryotic datasets were only rarefied to approximately 1000 sequences per sample for diversity analysis. In addition, our information extends current knowledge of the grassland and arable soil microeukaryotic communities, which are derived from analyses of traditional genetic fingerprinting, clone library, and culture-dependent assays (e.g., Marschner et al., 2003;Moon-van der Staay et al., 2006;Lara et al., 2007;Tzeneva et al., 2009). In grassland ecosystems, the soil microeukaryotic community composition, and diversity are strongly influenced by aboveground vegetation structure (e.g., plant height, species diversity and richness, functional type, and composition) (Sugiyama et al., 2008;Prober et al., 2015). The arable soil microeukaryotic communities are affected by different agricultural management practices. In a long-term fertilization experiment, Lentendu et al. (2014) observed that the eukaryotic datasets were dominated by Streptophyta sequences, followed by fungal and microfauna sequences. The changes in soil pH, moisture and nutrient availability caused by fertilization affected the microeukaryotic community composition in the arable soil (Lentendu et al., 2014). In our study, the grassland and arable soil microeukaryotic communities were dominated by fungi, accounting for 55.7-88.4% of the total eukaryotic sequences. The majority of fungal sequences belonged to the phylum Ascomycota (Figure 1, Table S2), which is usual for soil habitats lacking ectomycorrhizal host plants (Schadt et al., 2003). Similar findings are observed in other grassland and arable soils using clone library constructing and molecular genetic fingerprinting based on the biomarker of fungal internal transcribed spacer (ITS) region gene (de Castro et al., 2008;Klaubauf et al., 2010;Karst et al., 2013). Previous results indicated that Ascomycota dominated the fungal community in a maize-wheat rotation soil during the process of straw decomposition (Chen et al., 2014). The majority of Ascomycota belong to the fast-growing fungal populations (or r-strategists) which preferentially metabolize easily degradable fractions of organic matter (Lundell et al., 2010), and are abundant in soils with relatively high N contents (Nemergut et al., 2008). However, other studies suggest that many Ascomycota groups have distinctive morphological features that confer extensive stress tolerance and permit survival in hostile environments (e.g., Sterflinger et al., 2012;Nai et al., 2013). These reports support our findings that Ascomycota similarly dominated the microeukaryotic communities in the fumigated soil ( Figure 1A). Actually, the phyla Ascomycota and Basidiomycota represent the main classified fungal decomposers in soils (Vandenkoornhuyse et al., 2002). The grassland soil analyzed by Anderson et al. (2003) was well colonized by Basidiomycota [60% of the clones in the combined small subunit (SSU) library and 47% in the ITS library], while their abundance was relatively low in our study ( Figure 1A, Table S2). By conducting a long-term elevated CO 2 (eCO 2 ) experiment on a secondary successional grassland (aCO 2 as control), Tu et al. (2015) observed that the fungal community was dominated by Ascomycota (77 and 81% of the fungal sequences for eCO 2 and aCO 2 , respectively), followed by Basidiomycota. In addition, two protist groups (i.e., Cercozoa and Apicomplexa) were moderately abundant in the grassland and arable soil microeukaryotic communities ( Figure 1A). In several German grassland soils, Domonell et al. (2013) found Cercozoa (abundance 32.4 ± 13.2%) as one of the dominant protists. Other studies also observed the existence of Cercozoa and Apicomplexa with moderate abundance in typical Chinese soils (Jing et al., 2014;Shen et al., 2014;Shi et al., 2015). Composition, Diversity, and C-metabolic Functions of the Microeukaryotic Communities in Response to Short-term Fumigation-incubation Overall, the number of total microeukaryotic phyla and fungal classes were not greatly decreased by fumigation during the incubation period (Figures 1A,B). Fumigation showed no significant effect on the observed OTU richness at the end of incubation ( Figure S1; Figures 2C,D). Therefore, fumigation does not sharply reduce the soil microeukaryotic taxa, but rather biomass size during short-term fumigationincubation (Zelles et al., 1997;Dickens and Anderson, 1999). Taxonomic composition of the microeukaryotic communities at the phylum/class levels was modulated by fumigation (Figures 1A,B). Fumigation significantly decreased the grassland soil microeukaryotic diversity and richness at days 0 and 7, and changed the arable soil community structure at days 7 and 30 (Figures 2A,C,E, Table 3). After fumigation, the successions of the arable soil microeukaryotic communities occurred with soil incubation (Figure 2E). Previous studies indicated that after fumigation the surviving microorganisms mineralized the necromass released from cell lyses within several days (Jenkinson and Powlson, 1976;Wu et al., 1996;Kemmitt et al., 2008). As nutrient conditions change (i.e., following fumigant removal there is a release of necromass), the redistribution of the microeukaryotic communities (i.e., copiotrophs and oligotrophs) probably occurs, leading to the changed community structure during the incubation period. Similarly, in a straw amendment incubation experiment, we also observed the redistributions of the arable soil bacterial and microeukaryotic communities as straw availability declined over time (Chen et al., 2014(Chen et al., , 2015. The main microeukaryotic phylotypes in response to fumigation were reflected by the base-10 logarithm of the odds ratio (Ganesh et al., 2014). Positive values indicate taxa that are more likely to occur in the fumigated treatments. Most phylotypes were inhibited by fumigation, only 3 and 4 phylotypes in the grassland and arable soils respectively were positively stimulated by fumigation. In the arable soil, fumigation showed a very significant stimulation of Eurotiomycetes ( Figure 3B). Many Eurotiomycetes species are adaptable and resilient in extreme ecosystems (e.g., heat, drought, oligotrophy, and hypersalinity) (Kis-Papo et al., 2001;Sterflinger et al., 2012;Nai et al., 2013). These abilities can facilitate their recolonization in acidic arable soils after fumigation. Eurotiomycetes, Glomeromycetes, Kickxellomycotina, and Mucoromycotina taxa in the two soils showed different responses to fumigation (Figures 2, 3B, Table 3). This can be ascribed to the distinctly different habitat conditions in the two soils. The grassland and arable soils contained 26.6 and 15.8 g kg −1 organic C respectively, with soil pHs of 7.1 and 4.4 ( Table 1). In soil microbiology, there is a "paradox" of SOC mineralization, which is that even though majority of the soil FIGURE 4 | Redundancy analysis relating microbial properties to the main microeukaryotic sequence patterns after fumigation. Panels (A,B) indicate the fumigated grassland and arable soils, respectively. The length of each arrow indicates the contribution of the corresponding parameter to the structural variation. * and ** mark significance at P < 0.05 and 0.01, respectively, based on 999 Monte Carlo permutations. microorganisms are killed by fumigation, SOC mineralization continues at the same rate as in the non-fumigated soil for several weeks or even months (Jenkinson and Powlson, 1976;Wu et al., 1996;Kemmitt et al., 2008). This phenomenon can be partly explained by the microeukaryotes that survive fumigation (mainly the phylotypes Eurotiomycetes and Sordariomycetes, Figure 4A), due to their metabolic functions in C turnover and energy flow (Chen et al., 2012(Chen et al., , 2014Damon et al., 2012;Jing et al., 2014). For instance, Eurotiomycetes belong to cellulolytic fungi and can produce extracellular cellulases based on fungal cellobiohydrolase (cbhl) gene characterization (Fan et al., 2012). The Sordariomycetes species are capable of decomposing the organic residues in soils, attributed to the excretion of carboxylases and amidolyases (Strope et al., 2011). Soil β-glucosidase and invertase activities are two useful indicators involved in the decomposition of organic C (Nannipieri et al., 2012). A flush of invertase activity occurred following fumigation (Table 2), as some intracellular enzymes were released into the soils during cell lyses. Some enzymes released during cell lyses can resist proteolysis and maintain their activities during and after fumigation (Renella et al., 2002). Specific enzyme activity, an activity index of microbial biomass, can be expressed as soil enzyme activity per unit biomass C (Waldrop et al., 2000). Specific β-glucosidase activity was greater in the fumigated soils compared to the non-fumigated soils ( Table 2), but those changes were not significant. In the fumigated grassland soil, the variation in the main microeukaryotes was significantly explained by invertase activity, biomass C and specific β-glucosidase activity, and they were well correlated with the community composition of total microeukaryotes, fungi, and protists ( Figure 4A, Table 4). The co-occurrence networks indicated that after fumigation the internal communities in the grassland and The bold values indicate the significant (P < 0.05) correlations and their corresponding Spearman's rank coefficients (r). arable soils were highly connected, mainly the connections of fungi to other taxa (Figures S2, S3), mirroring the diverse linkages between the microeukaryotic groups in terms of their ecological functions. Therefore, the fumigated microeukaryotic communities probably make a large contribution to SOC mineralization by release of hydrolytic enzymes and their activities. Our study is also supported by two basic principles in soil microbiology. Firstly, soil is considered to have a large excess pool of total microbial biomass, whereas only a small portion of the microbial biomass is active (excessive pool principle) (Morris and Blackwood, 2007). Secondly, many similar functions can be carried out by different microbial taxonomic groups (redundancy principle) (Stres and Tiedje, 2006). In our study, despite the greatly decreased biomass C after fumigation, the residual fraction of microeukaryotes that survive fumigation are still active to drive SOC mineralization. Different microeukaryotic phylotypes (e.g., Eurotiomycetes and Sordariomycetes) have similar functions in SOC mineralization by producing a variety of hydrolytic enzymes. Collectively, combined with previous studies based on PLFA analysis (Zelles et al., 1997;Dickens and Anderson, 1999), our study showed that short-term fumigation-incubation not only reduced the biomass size of the microeukaryotic communities, but changed their α-diversity in the grassland soil, β-diversity in the arable soil, and taxonomic composition in both soils. The co-occurrence networks indicated that after fumigation the internal microeukaryotic communities were highly connected, mainly the connections of fungi to other taxa. The fumigated microeukaryotic communities retain the ability to drive SOC mineralization by release of hydrolytic enzymes and their activities, despite the greatly decreased microeukaryotic biomass. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (41371246) and the National Basic Research Program (973 Program) of China (2014CB441003). The authors thank our lab colleagues for soil sampling and analysis, and two reviewers for insightful comments and constructive suggestions which improved this manuscript. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2015.01149 Figure S1 | Venn diagram illuminating the amounts of shared and unique OTUs among different treatments at day 30 of the incubation. OTUs were counted from a subset of 5000 sequences per sample, and the average OTUs of three replicates were enumerated. Figure S2 | Network analysis on the microeukaryotic communities in the fumigated grassland soil through 30-day incubation. Colored nodes were the OTUs at 97% identity, and the connections indicate significant correlations (r > 0.6, P < 0.01). The size of each node is proportional to the number of connections (degree). Figure S3 | Network analysis on the microeukaryotic communities in the fumigated arable soil through 30-day incubation. Colored nodes were the OTUs at 97% identity, and the connections indicate significant correlations (r > 0.6, P < 0.01). The size of each node is proportional to the number of connections (degree). Table S1 | Number of sequences and OTUs measured by QIIME processing.

v3-fos

2018-12-07T05:12:38.753Z

2015-09-28T00:00:00.000Z

55137112

s2

Effect of Coastal Sediment to Nutrient Availability and Maize Productivity on Entisols Entisols had a great potential for maize planting area expansion in increasing its production. The low soil fertility could be improved by application of coastal sediment. This current research examined the effect of different amount of coastal sediment on nutrient availability and maize production on Entisols, West Kalimantan. The research was conducted from July to November, 2013 in green house, Faculty of Agriculture, Tanjungpura University, West Kalimantan. The treatment conducted was the application of coastal sediment at dosage of: 0 Mg ha-1 (L0), 14 Mg ha-1 (L1), 28 Mg ha-1 (L2), 42 Mg ha-1 (L3), 56 Mg ha-1 (L4), 72 ton ha-1 (L5), 86 Mg ha-1 (L6) and 100 Mg ha-1 (L7). The treatments were arranged by completely randomized design with 3 replications. Research results showed that 42 Mg ha-1 coastal sediment was the best treatment and able to increase soil nutrients availability and maize productivity. The provision of coastal sediment increased the availability of K, Ca, Mg and Na, also the availability of nutrients in accordance with the increase of the dosage of coastal sediment. INTRODUCTION Maize (Zea mays L.) was the third most important cereal crop in the world after rice and wheat (Kage et al., 2013). It was one of the globe's most widely used cereal crops, which was not only as an important food crop for human, but also as a basic ingredient of feed and raw material for manufacturing of many industrial products (Orhun, 2013;Reddy et al., 2013), and biofuel as well. (Koçar and Civaş, 2013). According to Haryono (2012), In Indonesia, targeted maize production was 29,000,000 ton in 2014 and in order to achieve a sufficient amount of maize production, the area of production had to be expanded approximately to 4,999,000 ha and in line with that, the productivity had to be increased to 5.82 t ha -1 . Maize production in Indonesia could be increased by both intensifying cultivation practices and expansion of cultivable area (Lalu et al., 2012). One type of soil that could be used for the expansion of maize planting area in West Kalimantan province was alluvial soil which covered 2 million hectares or 10.29 percent (CBCI, 2011). In the Soil Taxonomy classification, alluvial soil groups were included in order of entisols (CRDALR, 2006). Entisols were formed from various materials deposited at flat to nearly flat slope by fluvial and/or colluvial processes, through water flow and gravity force. The processes led to variation in physical, chemical, and mineralogical properties, as well as nutrient accumulation (Brubaker et al., 1993). Entisols with the parent material of alluvial scattered at along the river side and sea-shores (Temenggung et al., 2009). These soils had properties that were less conducive to growth and crop production because it had low nutrient availability, acidic and high Al solubility (Syamsuddin et al., 2013). According to Hikmatullah and Jabri (2007), the soil CEC and exchangeable K were low. One effort to improve fertility and productivity of Entisols was by adding ameliorants which was easily be obtained, and relatively available and relatively cheap in this area (Sulistyowati and Suswati, 2010). Application of coastal sediment could improve soil productivity and fertility because it could increase pH and added nutrients to the soil simultaneously (Suswati, 2012). The purpose of this study was to determine the effect of coastal sediment as ameliorant on nutrient availability and maize productivity in Entisols of West Kalimantan. MATERIALS AND METHODS The research was conducted at green house, Faculty of Agriculture, Tanjungpura University, West Kalimantan, from July to November 2013. Entisols surface soil samples (0 -40 cm) were collected from Sungai Rengas Village in Sungai Kakap Subdistrict, Kubu Ray District; West Kalimantan Province. Coastal sediments were obtained from Kijing beach, West Kalimantan. Fertilizers required for maize were urea, SP-36, KCl, and applied based on calculation of soil analysis results and recommendations of N, P, and K to maize plants. The Pioner 21 hybrid variety of maize seed was used in this experiment. The experiment in greenhouse was conducted by using completely randomized design, with triplicates (Gomez and Gomez, 1984 . Pots were filled as much as 10 kg of Entisol soil, which were firstly mixed with urea, SP-36, KCl as basal fertilizer and appropriate level of coastal sediment treatments. After one week, soil samples for selected soil chemical properties were taken at depths of 0-10 cm at every pot, prior to maize planting time. In each pot, 2 seeds were sown at a depth of 2 cm, followed by reduction to one plants after 10 days. Each pot was watered twice a day with 200 ml of water for a period of 60 days (harvest). Every 5 days, the weight of the pots was checked and adjusted with water in relation to the initiate of experiment. Maize grains were harvested at best physiological maturity from each pot and dry grains of maize in each plot that were extrapolated to a hectare basis using plant populations to determine maize productivity. Selected physical and chemical properties of soil and ameliorant were determined using standard procedures. The coastal sediments texture was carried out using the International Pipette method (Sarkar and Haldar, 2005). The ameliorant pH determined in soil:water was (1:2.5). Distilled water suspension and KCl were used a glass of electrode. The content of organic carbon was determined using Walkley and Black method. Ameliorant cation exchange capacity (CEC) was determined by leaching 1 M ammonium acetate buffer adjusted to pH 7.0 followed by steam distillation (Pansu and Gautheyrou, 2006). Available phosphorus in ameliorant was extracted with NaHCO 3 (0.5 M) at pH 8.5 and colorimetrically determined after treated with ammonium molybdate and stannous chloride at a wavelength of 660 nm. The exchangeable base cations (K, Ca, Mg and Na) were extracted with 1.0 mol L −1 ammonium acetate (Pansu and Gautheyrou, 2006). After extraction, the cations were measured using atomic absorption spectrophotometry. The data obtained were subjected to a two way analysis of variance (ANOVA) followed by a Duncan Multiple Range Test (DMRT) at 5% level and some data were correlated. The data were analyzed using the Statistical Analysis System package (SAS Institute, 2003). The Characteristic of Entisols and Coastal Sediment Based on the standards of determining soil chemical properties (ISRI, 2005), the soil had acidic soil (pH 4.89). According to Tuyen et al. (2006), entisols soil had low pH value because entisols property was rich of SiO 2 and Al 2 O 3 . When silts deposited, the process of leaching of Ca, Mg and accumulating SiO 2 , Al, Fe happened; therefore, soil pH was low. Organic-C (4.43%) was high, whereas total N content (0.39%) was medium (Table 1). Organic matter was an important source of nutrients for plants, strong activities of microorganism and soil absorption ability (Nath, 2014). The medium content of total nitrogen could be accounted by the high organic matter content of soil which supplied about 75-85% of soil organic nitrogen (Grubinger, 2007). Table 1 showed that the content of available P was high (28.11 ppm), while the potential K was low (0.19 cmol(+)kg -1 ). CEC value was medium (17.21 cmol(+)kg -1 ) but bases saturation (BS) was very low (6.68%). The very low BS showed low basic cation availability, i.e. K, Ca, Mg and Na, this could be as a result of high average rainfall ranges from 2,500 to 4,500 mm per year which leaching those nutrients (Lusiana et al., 2008;Laird et al., 2010). It might inhibit equilibrium of nutrients, especially K, Ca and Mg in soil. (Suswati, 2009). The Effect of Coastal Sediment to the Availability of Nutrients in Entisols The availability of base cations in the entisols due to the provision of coastal sediment was measured using indicators of soil pH, K, Ca, Mg and Na available ( Table 2). The results of variance analysis showed that after incubation coastal sediment treatments significantly affected soil pH after incubation. Table 2 showed that addition of coastal sediment 14 Mg ha -1 in the Entisols had been able to increase the pH of the soil after incubation compared to control. Soil reaction (pH) increased significantly with the addition of coastal sediment 14 Mg ha -1 -100 Mg ha -1 . This was due to the coastal sediment containing alkaline cations, so the higher the addition of coastal sediment the soil pH would increase. This was due to the added ameliorant containing alkaline cations that could increase soil pH. Sabiham (1993) explained that the addition of coastal sediment to peat soil can raise the pH, due to neutralized reaction of ion H + from peat by ions OH of base cations contained on the coastal sediments. Application of coastal sediment significantly affected K soil availability after incubation. The provisions of coastal sediment of 14 Mg ha -1 in the Entisols soil could increase K soil availability compared to control ( Table 2). Provision of coastal sediment of 42 Mg ha -1 could still increase K soil availability significantly, but there was no difference than provision of coastal sediment at a higher dosage of up to a dosage of 100 Mg ha -1 . This was due to coastal sediment containing K, so that when it was added to the soil it could increase K soil availability. Besides of the coastal sediment as a K source nutrients, a high proportion of clay mineral at coastal sediment gave a great potential to soil K availability. Soil containing montmorillonite or mica had greater K availability than kaolinite (Havlin et al., 2005). The results of variance analysis of Ca soil availability after incubation showed that coastal sediment treatments significantly affected Ca soil availability. Table 2 showed that the application of coastal sediment of 14 Mg ha -1 could increase Ca availability compared to control and there was a difference with the addition of coastal sediment until the dosage of 86 Mg ha -1 . This was due to the coastal sediment containing high Ca, higher than the content of other base cations (K, Mg and Na), so it could act as a source of Ca. In addition, the coastal sediment was very important factor in determining the Ca availability including soil pH and CEC (Havlin et al., 2005). Table 2 showed that the addition of coastal sediment of 14 Mg ha-1 significantly increased the availability of Mg. Provision of coastal sediment until the dosage of 100 Mg ha-1 could increase the availability of Mg soil after incubation and it was significantly different from control. Mg availability increased by the addition of coastal sediment up to 100 Mg ha-1. This was due to the coastal sediment containing Mg so it could serve as a source of soil Mg. Provision of coastal sediment would reduce the availability of soil Mg, because Mg tend to accumulate in rich clay soil (Bohn et al., 2001). The results of variance analysis of the Na availability in soil after incubation showed that the coastal sediment treatments significantly affected Na availability. Table 2 showed that the addition of coastal sediment 14 Mg ha-1 increased Na availability. Provision of coastal sediment until the dosage of 86 Mg ha-1 could increase the availability of Na after incubation and it was significantly different from control. This was due to the coastal sediment containing high Na (Table 1) so it could serve as a source of Na. , whereas the other dosage ofs of coastal sediment (L 0 , L 1, L 2 , L 4 , L 5 , L 6 and L 7 ) only generated 7.59 -5.00 dry grain Mg ha -1 . The effect of coastal sediment to weight of dry grain per plant was quadratic with the regression equation Y =-0.0181x 2 +1.7633x + 79.186, as shown in Figure 1. Ameliorant treatment could improve dry grain weight per plant at a certain dosage; at dosage of 14, 28 and 42 Mg ha -1 of coastal sediment (L 1 , L 2 and L 3 ) and it had generated dry grain 90.88, 106.88 and 150.54 g, respectively. Applying coastal sediment at dosage of more than 42 Mg ha -1 caused reduction of dry grain weight per plant, though it was still higher than control (L 0 ). Dry grain weight per plant at dosage of 56, 72, 86 and 100 Mg ha -1 of coastal sediment (L 4 , L 5 , L 5 and L 7 ) was 123.76, 92.41, 88.86 and 86.35 g, whereas controlled produce 81.53 g of dry grain. There was a declining trend of dry grain weight per plant with addition of increasing coastal sediment due to the availability of nutrient conditions that role in increasing the weight of dry grain per plant. Remarks: Values followed by the same letter in the column and the same treatment groups did not differ by Duncan's test 5 %. CONCLUSION Application of coastal sediment on Entisols had a positive effect on all parameters analyzed, such as: pH, availability of K, Ca, Mg and Na in soil. Dosage of coastal sediment of 42 Mg ha -1 was the best treatment than other treatments. This treatment could increase the availability of base cations (K, Ca, Mg and Na) and maize productivity compared to control (without coastal sediment) and when converted per hectare then the result to 9.23 Mg ha -1 was higher than mean. SUGGESTION The results of this study showed that the increasing nutrient availability and maize productivity was caused by the addition of coastal sediment. It is necessary for the further researcher to do research in the field on the same soil type. This is to determine the effect of environmental factors influencing coastal sediment in increasing nutrient availability and productivity of maize. ACKNOWLEDGEMENT This research was supported by List of budget projects from University of Tanjungpura.

v3-fos

2019-04-01T13:15:07.575Z

2015-11-01T00:00:00.000Z

88557692

s2

Comparison of total phenolic contents and antioxidant capacities in mint genotypes used as spices : Objective: Mint ( Mentha spp.) genotypes used as spices are cultivated in Turkey and used for different pur-poses including herbal tea, spices, the cosmetics industry, and are used in folk medicine. While mint species have been used in traditional practices during humanity’s long history, there is limited research on the comparison of their antioxidant capacity and phenolic contents. This aim of the research is to compare antioxidant capacity and phenolic contents in mint clones to determine superior genotypes for herbal and spice usage and cultivation. Methods: Antioxidant capacity was evaluated by ferric reducing antioxidant power (FRAP) and trolox equivalent antioxidant capacity (TEAC). Total phenolic content in clones were determined by Folin-Ciocalteu method. Results: Maximum total phenolic content [28.27±3.95 µg gallic acid equivalent (GAE)/g dry weight (DW)] and FRAP activity 577.09±46.02 µmol Trolox equiv/g DW were obtained from Clone 13 ( M. spicata ), while M. piperita clones were higher for TEAC activity (800.02±1.10 µmol Trolox equiv/g DW). The results were first records for M. villoso nervata . FRAP and TEAC activities selected clones were correlated with total phenolic content (r=0.77; 0.73 respectively). Conclusion: According to the results, it can be suggested that Clone 13, Clone 5, ( M. spicata ) Clone 3 and Clone 8 ( M. piperita ) are more suitable for use as spice, herbal tea, and antioxidant agents. The clones must be selected for commercial cultivation by the grower and they can be used as spices and herbal teas. Introduction Edible plants contain biological active components having antioxidant activity in addition to their food values. Antioxidant activities of plants are a result of their phenolic contents [1][2][3]. Phenolics are functional compounds synthesized for defensive metabolites in plants. They have a critical role in the human diet for healthy living. Herbs such as mint are an important source of them [4]. Some mint (Mentha spp) genotypes have been used in the Mediterranean diet as herbal teas and spices [4][5][6][7]. Mentha spicata, M. canadensis, M. piperita etc, have economic importance due to their medicinal and aromatic value [8,9]. Besides essential oil composition, other secondary metabolites composition determines herbal tea and pharmacological properties of the plants [10,11]. While essential oil composition determines quality in cultivated mint, phenolic components and their biological activity (antioxidant, antimicrobial etc.) have vital importance for herbal tea, spice and medical uses of cultivated mint. As results of breeding and characterization research, superior varieties for herb yield and essential oil composition were selected by our group. However, there are no reports on phenol compounds of the selected clones. According to previous reports on Mentha species antioxidant activity of M. piperita and M. spicata species had been carried out by different methodology [12][13][14][15][16][17]. Four of the selected clones belong to M. villoso nervata. Contrary to limited essential oil composition, there are no records on antioxidant capacity of the species, M. villoso nervata. There are also limited reports on comparison of the correlation antioxidant capacity with total phenolic content in Mentha species. Therefore, the aim of the research is to compare antioxidant capacity and phenolic contents in mint clones to determine superior genotypes for herbal and spice uses and cultivation. Plant material The selected 13 mint clones of three Turkish cultivated species, Mentha piperita, Mentha spicata and Mentha villoso-nervata, were grown in experimental plot of Agriculture Faculty in Gaziosmanpasa University. Species and origin of selected clones were given Table 1. Rooted samples of each clone were planted experimental plots with three replications. Plants were harvested at the stage of flowering in Mid-July 2009. After, they are dried in drier cabin at 35 o C, the leaves were separated from the aerial parts of the plants. Voucher specimens were deposited at the plants herbarium of Science and Arts Faculty at Gaziosmanpasa University (GOPU). Herbarium numbers of voucher specimens are given in Table 1. Extraction procedures Plant material, (100 g) was ground into a fine powder in a mill after 10 minutes of storage in liquid nitrogen. The powdered samples were mixed with 500 mL methanol/ chloroform (3/1) at room temperatures for 24 hour. The residue was re-extracted under same condition until extraction solvents became colourless. The obtained extracts were filtered over Whatman No.1 paper and the filtrate was collected, then, by extraction the solvent mix was removed by a rotary evaporator at 50 o C. The extracts were used for antioxidant capacity and total phenolic compounds content analysis. Determination of total phenolic content The total phenolic contents were determined by Folin-Ciocalteu reagent [18]. 0.1 ml of the extract solution (contains 0.1 mg extract) was mixed with water (46 ml). 1 mL of Folin-Ciocalteu reagent was added and mixed thoroughly. 3 mL of Na 2 CO 3 (2%) was added to the mixture. The absorbance was measured at 760 nm. The calibration curve was prepa-red by 0-100 µg/ml solutions of gallic acid in ethanol. The concentration of total phenolic compounds in the extracts was determined as gallic acid equivalent and calculated as µg gallic acid equivalent/g dry weight (DW) of the plant material using the calibration curve (R 2 : 0.99). The data were given as the average of triplicate analyses. Ferric reducing antioxidant power (FRAP) Reducing power of mint samples was measured by method of Oyaizu [19] with a slight modification [20]. According to this method the reduction of Fe 3+ to Fe 2+ was determined by measuring absorbance of the Perl's Prussian blue complex. This method is based on the reduction of (Fe 3+ ) ferricyanide in stoichiometric excess relative to the antioxidants. For positive control Trolox was used. Absorbance of these mixtures was measured at 700 nm using a UV spectrophotometer (Hitachi U-2900). Increased absorbance indicates ferric reducing power capability of the samples [21]. Results were given as µmol trolox equiv/g DW. The data were given as the average of triplicate analyses. Trolox equivalent antioxidant capacity (TEAC) ABTS •+ radical scavenging activity was determined using a modified method of Re et al. [22]. The stock solutions included 5 mL of 7 mM ABTS •+ solution and 88 µL of 140 mM K 2 S 2 O 8 . The working solution was allowed to react for 16 hours at room temperature in the dark. The ABTS solution was adjusted with distilled water to an absorbance of 0.650±0.050 at 734 nm using the UV-visible spectrophotometer. Fresh ABTS •+ solution was prepared for each assay. 0.1 mL of Mentha species extract was allowed to react with 2.9 mL of the ABTS •+ solution in the dark at room temperature for 10 minutes then the absorbance was measured at 734 nm. The data were given as the average of triplicate analyses. Results were given as µmol trolox equiv/g DW [22]. Statistical analysis The numerical data of total phenolic content and antioxidant activity are presented as mean±standard deviation calculated from triplicate. The values were subject to analysis of variance (ANOVA) using randomized block design. The mean data being significant in variance analysis were grouped with Duncan multiple test. The relationship between total phenolic content and antioxidant activities were also determined by correlation values. The significance threshold was set at 0.01. All statistical calculation were performed using the SPSS 20 statistical software [23]. Results and Discussion Several methods have been developed to evaluate total antioxidant capacity of food and dietary supplements, herbal extracts, and pure compounds. Nevertheless, few of them have been used widely due to the difficulty of measuring total antioxidant capacity as result of limited methodological protocol and free radical sources [24]. In this study, the antioxidant capacities of the selected clones extracts were evaluated with TEAC and FRAP in vitro tests (Table 2). Total phenolic contents were also evaluated and a direct correlation was observed between the total phenolic content of the extracts and their antioxidant capacities ( Table 2 and Figure 1). Total phenolic contents The amount of total phenolic compound ranged from 9.39 (clone 15) to 28.27 (clone 13) µg GAE/g DW ( [13,15,[25][26][27]. The content in M. spicata was found as 334 mg CAE/g plant, 84 mg GAE/g plant, 80 mg GAE/g plant, 27 mg GAE/g plant and 22.43 mg GAE/g fresh plant by Husseinimehr et al. [28], Elmastas et al. [12], Naidu et al. [29], Conforti et al. [30] and Derakhshani et al. [31] respectively. No investigation of total phenolic content in M. villoso nervata has been included in the literature. Phenolic compounds that are synthesized for defending and protecting purpose and have an important role in the human diet varies according to genetic structure, growing, and climate conditions [32]. In the research the change in the phenolic content is due to genetic differences of the clones because the used clones are grown in the same ecologic and field conditions. The total phenolic content of M. piperita in this work was lower than that of some reports [13,15,26] and higher than that of others [27,33]. Total phenolic amounts of M. spicata clones were generally higher than that of Ninckavar's result [13] except two clones (Clone 15 and 11). Total phenolic contents in all clones of M. spicata in this study were lower than a previous study [12,28,34]. Total phenolic contents in M. spicata clones were in agreement with results reported by Derakhshani et al. except clone-11, clone-12 and clone-15 [31]. Those three clone phenolic contents were lower than results of Derakhshani et al. [31]. Total phenolic contents in M. spicata clones were higher than that of Naidu et al's result [29]. Although, there are no records on total phenolic amount of M. villoso nervata, total phenolic content of the species was similar to the records of M. spicata and lower than that of other mentha species such as M. piperita, M. longifolia, M. pulegium, M. roduntifolia [13]. Even though there was significant variation statistically (p<0.01) between clones of the spearmint (M. spicata and M. villoso nervata) species, M. piperita Clones (two clones) had similar data, statistically. Total phenolic of the clones was 23.99 (µg GAE/g DW) in Clone 3 and 21.56 (µg GAE/g DW) in Clone 8. There is dramatic variation in total phenolic amount of M. spicata and M. villoso nervata (Table 2). Antioxidant activities Ferric reducing antioxidant power (FRAP) FRAP activities varied between 280.73 and 577.09 µmol Trolox equiv/g DW in M. spicata. The lowest and the highest FRAP activity of M. piperita were 317.6 and 558.33 µmol Trolox equiv/g DW, while 314.76 and 374.83 µmol Trolox equiv/g DW in M. villoso nervata ( Table 2). Different studies have indicated that the electron donation capacity of bioactive compounds is associated with antioxidant activity [35,36]. The presence of reductants in the samples causes the reduction of the Fe 3+ / ferricyanide complex to the ferrous form. Therefore, Fe 2+ can be monitored by measuring the formation of Perl's Prussian blue at 700 nm [37]. There are a number of assays designed to measure overall antioxidant activity [38]. FRAP assay take advantage of an electron transfer reaction in which a ferric salt is used as an oxidant [39]. In this assay, the yellow colour of the test solution changes to various shades of green and blue depending on the reducing power of antioxidant samples. The reducing capacity of an extract may serve as a significant indicator of its potential antioxidant activity. The outcome of the reducing reaction is to terminate the radical chain reactions that may otherwise be very damaging [40]. Reduction power of Mentha species extracts ranged from 280.7 to 577.1 µmol Trolox equiv./g DW. The highest activity was seen in Mentha spicata Clone 13 and the lowest in Mentha spicata Clone 11 ( Table 2). The second highest FRAP activity of this study was observed M. piperita Clone 3. FRAP result of M. piperita Clone 3 was agreement with some previous reported results [13,15,16]. According to the results in this study, FRAP activity differences in Mentha clones were correlated (r=0.77) with total phenolic content (Figure 1a). Chrpova et al. [15] studied antioxidant activities of some herbs belongs Lamiaceae, including M. piperita, used for medicinal purposes, and they reported that M. piperita had strong free radical scavenging activity and good correlation (r=0.96) with total phenolic contents [15]. Studies conducted on free radical scavenging or antioxidant activities of medicinal plant reveal that the efficiency of plant species depends on assay methodology, reflecting the complexity of the mechanisms involved in total antioxidant capacity [41][42][43]. Trolox equivalent antioxidant capacity (TEAC) Results of cation radical scavenging activities were given in Table 2. As seen in Table 2, TEAC activity of Mentha species extracts ranged from 375.1 to 800.02 µmol Trolox equiv/g DW. The highest activity was seen in Mentha piperita clone 3 while the lowest in Mentha spicata Clone 15 (Table 2). Assays based upon the use of DPPH • and ABTS •+ radicals are among the most popular spectrophotometric methods for determination of the antioxidant capacity of food, beverages, plant extracts and pure compounds [44,45]. Both chromogen radical compounds can directly react with antioxidants. DPPH • and ABTS •+ scavenging methods are used to evaluate the antioxidant activity of compounds due to the simple, rapid, sensitive, and reproducible procedure [40]. The reaction of the preformed radical with free-radical scavengers can be easily monitored by following the decay of the sample absorbance at 734 nm. ABTS •+ radicals are more reactive than DPPH radicals and unlike the reactions with DPPH • radical which involve H atom transfer, the reactions with ABTS •+ radicals involve electron transfer process [46]. Lopez et al. studied antioxidant activities of 5 Mentha species. According to their report ABTS •+ radical scavenging activities of M. piperita showed the best activity when compared to other species [14]. Lopez et al's results also agree with our result ( Table 2). Previous antioxidant activity reports on Mentha species clearly showed antioxidant activities of M. piperita and M. spicata. [12][13][14][15][16]. TEAC activity differences in Mentha clones were correlated with total phenolic content (r=0.73). It can be said that phenolic compounds are responsible for TEAC activities of Mentha species. Conclusion Results obtained in the research is summarized as following: 1. Different methods and units in antioxidant activity caused difficulty in comparison of the results. So, a standard unit should be used by researchers for accurate evaluation of antioxidant results in Mentha species in future researches. 2. There is a positive correlation with total antioxidant capacity and total phenolic content in Mentha clones. In future studies the relationship between antioxidant capacity and phenolic contents on Mentha species should be based on antioxidant activities and individual phenolic compound contents in Mentha species. 3. According to antioxidant capacity and total phenolic content results it can be suggested that Clone 13, Clone 5 (M. spicata) Clone3 and Clone 8 (M. piperita) are more suitable for use as spice and herbal tea. The results contain valuable data for breeding studies too. 4. The result of M. villoso nervata is the first record on both antioxidant capacity and phenolic content. 5. In future studies the relationship between antioxidant capacity and specific phenolic components of Mentha species should be studied in Mentha species.

v3-fos

2019-03-21T13:11:21.545Z

2015-02-19T00:00:00.000Z

84344882

s2

Distribution of clustered regularly interspaced palindrome repeats CRISPR2 and CRISPR3 in Lactobacillus delbrueckii ssp. bulgaricus strains The function of several families of clustered regularly interspaced palindrome repeats (CRISPRs) in prokaryotic genomes was recently found to be related to the protection of bacterial cells against the expression of foreign DNA, originating from plasmids or bacteriophages. The present study was the first attempt to screen a broader number of Lactobacillus delbrueckii ssp. bulgaricus strains, widely used in yoghurt and cheese production, for the presence of CRISPRs. Database search of four completely sequenced L. delbrueckii ssp. bulgaricus genomes indicated the presence of CRISPR2 in three of them - ATCC 11842, ATCC BAA-365 and ND02, and the presence of CRISPR3 in strain 2038. In the first three strains, the CRISPR2 was invariably located between a 3′–5′ exonuclease gene and a gene for a ppGpp-synthetase. The location of CRISPR3 in strain 2038 was between a histidine-kinase gene and an acetyl-CoA acetyltransferase gene, 2 kbp downstream of the CRISPR2 locus in ATCC 11842. Specific primers were designed to amplify with polymerase chain reaction the target regions containing the potential CRISPR2 and / or CRISPR3 in a total of 33 L. delbrueckii ssp. bulgaricus strains. Thirteen strains yielded a high molecular mass product corresponding in size and location to CRISPR2 of the type strain ATCC 11842, while another 17 strains indicated the presence of potential CRISPR3, analogous to that of strain 2038. Three strains did not indicate the presence of CRISPRs. Interestingly, none of the tested strains carried both CRISPR2 and CRISPR3 simultaneously in its genome at the investigated region. Introduction Bacteria from the Lactobacillus delbrueckii ssp. bulgaricus species are a basic component of yoghurt and whitebrined cheese starters. Compared to the phage attack on Streptococcus thermophilus, phage infection on L. delbrueckii ssp. bulgaricus cultures occurs much more rarely. [1,2] Nevertheless, bacteriophages that attack the L. delbrueckii group of subspecies are well documented in the literature. [3À6] Recently, the first local isolate of L. delbrueckii ssp. bulgaricus bacteriophage was classified into group 'b' L. delbrueckii bacteriophages, based on its partial genome sequencing. [6] The clustered regularly interspaced palindrome repeats (CRISPRs) were first described in the genome of Escherichia coli K-12 and their presence was confirmed for a multitude of bacterial and archaeal genomes. [7,8] Recently, these palindrome repeats and the cas-genes, associated with them, have been found to function as a new defence mechanism in prokaryotic cells against invading phages and plasmids. [9,10] The regular repeats are interspaced with short sequences named 'spacers' which derive from foreign genetic elements. [9,11,12] When, for example, the bacterial host is under a bacteriophage attack, it acquires a new spacer sequence within its CRISPR locus that matches a DNA sequence in the phage genome, referred to as a 'protospacer'. [13,14] The complete set of repeats and spacers is then transcribed and processed into individual crRNAs that interact with the viral DNA, facilitating its cleavage and rendering the bacterial cell immune to infection by the particular phage. [14À16] Depending on the organization of the cas-genes and the sequence of the palindrome repeats, four classes of CRISPRs are described and numbered from 1 to 4. As an example, in S. thermophilus, all four classes of CRISPR/cas systems are described as CRISPR1 and CRISPR3, which are considered to be the most active ones. [14,16] As data on CRISPRs in the genome of L. delbrueckii sp. bulgaricus are still lacking, in the present study we reported the results from the database search of potential CRISPRs in this subspecies and from the screening of 33 strains for the presence of potential CRISPR2 and CRISPR3 loci in their genome. Materials and methods Bacterial strains, culture conditions and DNA isolation Thirty-three L. delbrueckii ssp. bulgaricus strains maintained in the LBB culture collection (LB Bulgaricum PLC, *Corresponding author. Email: [email protected] Sofia, Bulgaria) were included in the study. All cultures were grown in MRS medium (peptone À 10 g/L; meat extract À 8 g/L; yeast extract À 4 g/L; glucose À 20 g/L; sodium acetate trihydrate À 5 g/L; Tween-80 À 1 g/L; dipotassium hydrogen phosphate À 2 g/L; triammonium citrate À 2 g/L; magnesium sulfate heptahydrate À 0.2 g/L; manganese sulfate À 0.05 g/L; pH was adjusted to 6.2) [17] for 24 hours at 37 C. We used 5 mL of the cultures for DNA isolation with the GenElute Bacterial Genomic DNA Kit (Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer's instructions. A list of the tested L. delbrueckii ssp. bulgaricus strains is presented in Table 1. Database search for CRISPRs and primer design Four publicly available genomes of L. delbrueckii ssp. bulgaricus' strains ATCC 11842, ATCC BAA-365, ND02 and 2038 (GenBank Acc. Nos. NC_008054; NC_008529; NC_014727 and NC_017469) were searched for CRISPRs with the CRISPRFinder software. [18] Conservative regions upstream and downstream of CRISPR2 and CRISPR3 were identified by alignment with the CLC Sequence Viewer (www.clcbio.com) and suitable primers were designed with the PrimerBLAST tool. [19] A complete list of the primers, their sequence and location is presented in Table 2. Polymerase chain reaction (PCR) amplifications All amplifications were performed on a 9600 GeneAmp PCR System (Perkin-Elmer, Norwalk, Connecticut) in a 25 mL reaction mixture consisting of diluted VWR Taq DNA Polymerase Master Mix (VWR International, Haasrode, Belgium), 50 ng template DNA and 10 pmol of each primer. The PCR programme was as follows: one cycle of 3 min at 95 C, 30 cycles of 30 s at 93 C, 30 s at 60 C and 1 min at 72 C, and one cycle of 7 min at 72 C. The potential CRISPR2 and CRISPR3 regions were amplified with primer pairs pr21 / pr22 and pr35 / pr32, respectively. Additionally, primers pr31 / pr32 were used to amplify a Table 1. List of the tested bacterial strains with the presence (C) or absence (¡) of the respective products from a CRISPR2-specific and CRISPR3-specific PCR amplification. ÃÃÃ Primers amplifying a short sequence between the histidine-kinase gene and acetyl-CoA acetyltransferase gene, proving the absence of CRISPR3. Results and discussion The database search of the four completely sequenced L. delbrueckii ssp. bulgaricus genomes indicated the presence of CRISPR2 in three of them À ATCC 11842, ATCC BAA-365 and ND02 and the presence of CRISPR3 in strain 2038. In the first three strains the CRISPR2 was invariably located between a 3 0 À5 0 exonuclease gene and a gene for a ppGpp-synthetase ( Figure 1). In strain 2038, the region preceding the analogue of the ppGpp-synthetase gene contained completely different genes instead of CRISPR2. The location of CRISPR3 of strain 2038 was between a histidine-kinase gene and an acetyl-CoA acetyltransferase gene, 2 kbp downstream of the CRISPR2 locus in ATCC 11842 (Figure 1). In strains ATCC 11842, ATCC BAA-365 and ND02, which did not have the CRISPR3 region, the acetyl-CoA acetyltransferase gene followed immediately after the histidine-kinase gene. The CRISPRFinder software listed 40, 20 and 64 spacers within CRISPR2 of strains ATCC 11842, ATCC BAA-365 and ND02, respectively, and 19 spacers within CRISPR3 of strain 2038. A list of the spacers within CRISPR2 and CRISPR3 of strains ATCC 11842 and 2038 is presented in Table 3. Using the specific primers pr21/pr22 for the CRISPR2 region, 13 strains yielded a high molecular mass product, corresponding in size (>3 kbp) and location to CRISPR2 of the type strain ATCC 11842 ( Table 1). The separation of the CRISPR2-specific amplified product for strain ATCC 11842 and five representative 'CRISPR2-positive' strains is presented in Figure 2(A). Neither of the 'CRISPR2-positive' strains yielded an amplification product with the CRISPR3-specific primer pair pr35/pr32 (Table 1, Figure 2(A)). Additionally, for all 'CRISPR2positive' strains a short amplification product was obtained with primers pr31 / pr32 that proved that the histidine-kinase gene was followed immediately by the acetyl-CoA acetyltransferase gene with no insertion of CRISPR3 between them. A visualization of this short amplification product for strain ATCC 11842 and five representative 'CRISPR2-positive' strains is included in Figure 2(A). Another 17 strains gave positive amplification with primers pr35/pr32, specific for the CRISPR3 region of strain 2038, indicating the presence of potential CRISPR3 in these cultures ( Table 1). The separation of the CRISPR3-specific amplified product for strain 2038 and five representative 'CRISPR3-positive' strains is presented in Figure 2(B). All of the 'CRISPR3-positive' strains failed to produce amplification product with the CRISPR2-specific primer pair pr21/pr22 suggesting the Table 3. List of spacers within CRISPR2 and CRISPR3 of Lactobacillus delbrueckii ssp. bulgaricus strains ATCC 11842 and 2038, respectively, retrieved by CRISPRFinder software. [18] absence of CRISPR2 in these strains ( Table 1). The 'CRISPR3-positive' strains also did not yield the short amplification product of primer pairs pr31/pr32, possibly due to the large size of the targeted region, resulting from the insertion of CRISPR3 between the histidine-kinase and acetyl-CoA acetyltransferase genes. The absence of CRISPR2-specific products and the short amplification product of primers pr31/pr32 for strain 2038 and five representative 'CRISPR3-positive' strains is demonstrated in Figure 2(B). 1, 7, 13), B5/1S (2,8,14), B60/8S (3,9,15), B208/5S (4, 10, 16), B226/2S (5,11,17) and B278/2S (6,12,18) The indirect evidence that the amplified region contains CRISPRs was the formation of amplification subproducts with regularly increasing size, which formed a typical ladder-like electrophoretic pattern (Figure 2(A) and 2(B)). This phenomenon can be explained by the formation of loops in some of the template molecules due to internal annealing of the palindromic repeats that prematurely terminate the elongation process. Interestingly, none of the tested strains carried both CRISPR2 and CRISPR3 simultaneously at the investigated region of their genome. Three strains did not indicate the presence of CRISPRs, due to their absence in the genome of the strains, or a completely different location of the potential CRISPRs. A complete list of all strains with the respective amplification products is given in Table 1. Conclusions Thirty-three strains of L. delbrueckii ssp. bulgaricus were analysed for the presence of CRISPR2 and CRISPR3 in their genomes. For 13 and 17 of them it was found that CRISPR2 and CRISPR3, respectively, were present and had the same location as that in strains ATCC 11842 and 2038. Neither strain contained the two CRISPRs simultaneously. Although the tested strains may still have CRISPRs in different locations that were not analysed in this study, it seems an interesting finding that with respect to the presence and location of CRISPR2 and CRISPR3 L. delbrueckii ssp. bulgaricus strains could be divided into two lineages -that of strain ATCC 11842 and that of strain 2038. The actual work on determining the sequences of spacers within the CRISPRs of L. delbrueckii ssp. bulgaricus strains is still ahead and may reveal important evolutionary relations between different cultures and highlight the history of particular strains with respect to encounters with foreign DNA. Disclosure statement No potential conflict of interest was reported by the authors.

v3-fos

2019-03-28T13:41:13.143Z

2015-01-01T00:00:00.000Z

86485962

s2

Endophytic bacteria (Sphingomonas sp. LK11) and gibberellin can improve Solanum lycopersicum growth and oxidative stress under salinity This study aims to understand the effects of salinity on the growth and oxidative stress enzymes of endophytic bacteria (Sphingomonas sp. LK11) and tomato plants. In response to salinity and gibberellic acid (GA4), catalase (CAT), superoxide dismutase, and reduced glutathione were significantly regulated in LK11 as compared to peroxidase (POD) and polyphenol oxidase (PPO). Salinity stress to tomato plants caused significant cessation in growth and biomass, which was accompanied by threefold increase in lipid peroxidation and decrease in glutathione, CAT, POD, and PPO activities. In contrast, sole and combined treatment of LK11 and GA4 rescued plant growth and biomass production whilst exhibited lower lipid peroxidation and higher glutathione content under salinity stress. The activities of CAT, POD, and PPO were either lower or nonsignificant as compared to control. In conclusion, inoculation of bacterial endophytes offers a relative stress counteracting potentials as evidenced by the known plant growth regulators. Introduction Salinity has often known as a limiting factor for effecting crop growth and development. Exposure to salinity stress causes increase in water stress, ionic influx, oxidant imbalance, membrane disintegration, cell division impairment, and fruit development (Flowers 2004;Gill & Tuteja 2010;Shabala & Munns 2012). Plants respond to such stress conditions by transducing signaling pathways, which enable them to initiate defensive metabolism (Khan et al. 2012). This strategy includes the changes in the activities of ionic/osmotic carriers, stomatal closure, stress hormones, and secondary metabolites. Salinity induces ionic imbalances in cell and causes the production of reactive oxygen species (ROS) such as hydrogen peroxide, superoxide anion, hydroxyl radicals, etc., which can suppress the cellular functions (Manai & Gouia 2014). To counteract ROS production, plant recruits various antioxidants (like reduced glutathione) and enzymes (such as superoxide dismutase -SOD, peroxidase -POD, catalase -CAT, polyphenol oxidase -PPO, etc.), which buffer the cellular ionic efflux to minimize cellular toxicity (Mittler 2002;Türkan & Demiral 2009). Under stress conditions, superoxide anion (O 2 − ) and hydrogen peroxide (H 2 O 2 ) production are higher in plants, and self-defense mechanisms such as SOD and CAT are activated to prevent oxidative damage (Vranova et al. 2002). SOD converts O 2 − content to H 2 O 2 and water, whereas CAT dismutates H 2 O 2 to water and oxygen (Vranova et al. 1997;Mittler 2002). PPOs (EC 1.14.18.1) are involved in the oxidation of polyphenols into quinones using molecular oxygen as an electron acceptor, which can act as essential defense response arsenal. PODs (EC 1.11.1.7), on the other hand, oxidoreductive enzymes that participate in the wall-building processes such as oxidation of phenols, suberization, and lignification of host plant cells during the defense reaction against biotic and abiotic stresses (Mohammadi & Kazemi 2002). Similarly, glutathione involved in reducing H 2 O 2 by donating electrons to encounter the stress-induced ROS attack. The stress stimulates changes in plant growth activities, which drastically hinder crop yield. It is estimated that 45 million hectares of irrigated land have been damaged by salinity whilst approximately 1.5 million hectares are losing fertility for crop cultivation (Munns & Tester 2008). Though there are some physical and chemical ways to remediate arable land effects with salinity, whilst cultivation of varieties developed through conventional breading and transgenic approaches can also help in sustainable production. However, assessment and application of rhizospheric microbes can be more eco-friendly. Up till now there are numerous studies performed on using plant growth promoting rhizobacteria (PGPRs) for improving the adverse effects of salinity on crops as shown by Mayak et al. (2004), Kang et al. (2014), Bashan et al. (2014), Egamberdieva and Lugtenberg (2014), etc. However, very few plant growth promoting endophytic bacteria (PGPEB) are known to confer ameliorative impacts during salinity. PGPEB are colonizing the healthy plant tissues without causing any symptoms of disease to the host plants. Endophytes have been regarded the most prolific bioperspective class of microorganisms, which can help the host plants in counteracting negative impacts of biotic and abiotic stress conditions (Khan et al. 2013). Endophytic symbiosis with host plants especially in roots can regulate and change the uptake of mineral nutrients, balance of plant hormones, exudation of defensive metabolites from root (Khan et al. 2013;Bashan et al. 2014). Very few endophytic bacterial strains have been reported to condone such beneficial effects. In the present study, we isolated endophytic bacteria Sphingomonas sp. LK11 from a medicinal plant growing in arid environment. Sphingomonas belongs to a group of gram-negative bacteria producing yellow-pigmented colonies and exist in diverse range of environments. The bacterium is metabolically versatile, which means it can utilize a wide range of naturally occurring compounds as well as some types of environmental contaminants (Miyauchi et al. 1998;Aylward et al. 2013;Puskarova et al. 2013). Sphingomonas sp. has been recently shown to help in the degradation of persistent metabolites in environment (Puskarova et al. 2013). The genetic makeup of Sphingomonas sp. was shown to contain genes responsible for carbazole degradation. Additionally, it has the capability to regulate certain class of pesticides like dibenzo-p-dioxins and remediate heavy metals (Puskarova et al. 2013). It also possess the potential to accumulate the intracellular Zn 2+ , reduction of Cd 2+ uptake enhanced expression of the low-molecular weight cysteine rich protein metallothionein that sequesters heavy metals and their binding by associated proteins . Additionally, its whole genome has also been sequenced recently by Miller et al. (2010), Lee et al. (2012), Qu et al. (2013), and Tabata et al. (2013). However, its biological role in relation with salinity stress has not been explored. Therefore, present study was planned to further explore its potential for application in improving crop production especially in saline environment. The study aimed to assess the growth dynamics and responses of various oxidative stress enzymes in Sphingomonas sp. LK11 in varying saline environment. Upon positive results, we inoculated this strain to tomato plants to evaluate the effects of Sphingomonas sp. LK11 application during salinity stress. Since, previously, it was found that Sphingomonas sp. LK11 also produce GA 4 (2.97 ng ml −1 ; Khan et al. 2014) therefore, for comparative assessment, exogenous GA 4 was also applied to tomato plants. Endophytic bacterial potential in saline environment To know the potential of Sphingomonas sp. LK11 in saline environment, it was grown in varying concentrations of sodium chloride (NaCl; 100-500 mM) in nutrient broth on shaking incubator (200 rpm; temperature 28°C) for seven days. The potential of Sphingomonas sp. LK11 against exogenous gibberellin (GA 4 ) was also assessed to understand whether this is toxic or not to bacterial growth. Sphingomonas sp. LK11 was grown in GA 4 (10 and 100 µM) solely and in combination with NaCl in nutrient broth using same conditions. The bacterial growth was assessed by the bacterial cell density (OD 600 ) and biomass. The bacterial cells were harvested through centrifugation (10,000 × g at 2°C for 15 min). The bacterial cells were immediately stored at -80°C for further protein and antioxidant enzyme analysis. Sphingomonas sp. LK11, GA 4 , and NaCl application to tomato plants The bacteria culture suspension was grown in nutrient broth for five days at 30°C on shaking incubator to obtain an estimated cell density of 10 8 CFU/mL. All materials, including seeds, pots, soil, and distilled water were sterilized by autoclaving at 121°C for 20 min prior to the experiment. Tomato (Solanum lycopersicum) seeds were surface sterilized with NaOCl (5% v/v) for 10 min and thoroughly rinsed with autoclaved distilled water. Seeds were sown in plastic pots containing horticulture soil (peat moss [13-18%], perlite [7-11%], coco-peat [63-68%], and zeolite [6-8%], while the macro-nutrients present were as follows: NH 4 +~9 0 mg/ L, NO 3 -~2 05 mg/L, P 2 O 5~3 50 mg/L, and K 2 O~100 mg/L) under controlled greenhouse conditions at temperatures of 30 ± 2°C . Tomato seeds were primed with 50 mL of bacterial culture suspension at the time of sowing. Since the growth responses of Sphingomonas sp. LK11 were pronounced against salinity (NaCl; 250 mM) and GA 4 (100 µM), therefore, both the concentrations were applied to the tomato plants. Both, Sphingomonas sp. LK11 (50 mL with cell density of 10 8 CFU/mL) and GA 4 (50 mL of 100 µM solution) were applied to 14 days old tomato seedlings. NaCl induced salinity stress (50 ml of 250 mM solution) was applied to three weeks old tomato seedlings for seven days on daily basis. Upon completion of stress treatments, the representative tomato plants were harvested in liquid nitrogen and immediately stored at -80°C for enzymatic analysis. Nutrient broth in the same amount was used as controls. The growth parameters, i.e. shoot and root length, shoot and root biomass and leaf numbers, were recorded. The experiment comprised of treatments with three replications. Each replication comprised of 21 plants. Determination of antioxidant-related enzymes The LK11 was grown in different concentrations of sodium chloride (NaCl). After five to seven days of treatments, the bacterial cell proteins were extracted according to the method of Qin et al. (2006). Briefly, proteins were extracted with lysis buffer (0.5 M Tris-HCl, pH 8.3, 20 mM MgCl 2 , 2% (v/v)mercaptoethanol, and 1.0 mM phenylmethylsulfonyl fluoride) followed by sonication (and cooling on ice). The cell debris were removed by centrifugation (10,000 × g for 20 min at 2°C), while the supernatant was precipitated with icecold trichloro acetic acid (TCA, 10% w/v). The proteins were collected by centrifugation (10,000 × g for 20 min at 2°C) and washed three times with cold acetone to remove remaining TCA. The precipitate was finally solubilized in buffer (300 µl; 2 M thiourea, 7 M urea, 4% (w/v) 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (pH 8.0)) and either used immediately or stored at -80°C until use. Similarly, tomato plant's leaves were grinded in liquid nitrogen and powdered with pre-chilled mortar and pestle. The powder was homogenized in 50 mM Tris-HCl buffer (pH 7.0) containing 3 mM MgCl 2 , 1 mM EDTA, and PVP (1.0% w/v). Both the homogenates of bacterial cells and plant tissues were centrifuged at 10,000 × g for 15 min at 2°C. The supernatant was used for total protein quantification according to the methods of Bradford (1976). All the enzymes activities were expressed as unit per mg protein. CAT (E.C1.11.1.6) activity was assayed by the method of Aebi (1984). The crude enzyme extract was treated with 0.5 ml 0.2 M H 2 O 2 in 10 mM potassium phosphate buffer (pH 7.0). CAT activity was estimated by the decrease in absorbance of H 2 O 2 at 240 nm and one unit of CAT was defined as μg of H 2 O 2 released mg protein min −1 . POD (E.C 1.11.1.7) and PPO (E.C 1.10.3.1) activities were measured as described by Kar and Mishra (1976) with some modifications. The samples were homogenized with potassium phosphate buffer pH 6.8 (0.1 M) and centrifuged at 2°C for 15 min at 5000 × g in a refrigerated centrifuge. The assay mixture for the POD activity comprised 0.1 M potassium phosphate buffer (pH 6.8), 50 μl pyrogallol (50 µM), 50 μl H 2 O 2 (50 µM), and 0.1 ml enzyme extract. The mixture was incubated for 5 min at 25°C after which the reaction was stopped by adding 0.5 ml 5% (v/v) H 2 SO 4 . The amount of purpurogallin formed was determined by the absorbance at 420 nm. The same assay mixture as that of POD without H 2 O 2 was used to assay the activity of PPO. The absorbance of the purpurogallin formed was taken at 420 nm. One unit of POD and PPO was defined as an increase of 0.1 units of absorbance. Reduced glutathione (GSH) content was measured according to the method of Ellman (1959). Briefly, samples were ground in a chilled mortar and pestle in 3 mL of 5% (v/v) TCA. The homogenates were centrifuged at 10,000 × g for 15 min at 4°C. Supernatant (0.1 mL) was added to 3.0 mL of 150 mM NaH 2 PO 4 (pH 7.4). Five hundred microliters of 5,5′-dithio-bis(2nitrobenzoic) (DTNB; 75.3 mg of DTNB was dissolved in 30 mL of 100 mM phosphate buffer, pH 6.8) was then added. The mixture was incubated at 30°C for 5 min. Absorbance was determined at 412 nm and the GSH concentration was calculated by comparison to a standard curve. All the experiments were repeated thrice. The extent of MDA was determined by the method of Ohkawa et al. (1979). For this assay, 0.2 ml of 8.1% (w/v) sodium dodecyl sulfate, 1.5 ml of 20% acetic acid (v/v; pH 3.5), and 1.5 ml of 0.81% (w/v) thiobarbituric acid aqueous solution were added in succession in a reaction tube. To this reaction mixture, 0.2 ml of one tissue homogenate extracted from 10 mM potassium phosphate buffer (pH 7.0) was added. The mixture was then heated in boiling water for 60 min. After cooling to room temperature, 5 ml of butanol:pyridine (15:1 v/v) solution were added. The upper organic layer was separated and the intensity of the resulting pink color was read at 532 nm using a spectrophotometer. Tetramethoxypropane was used as an external standard. The level of lipid peroxides was expressed as micro moles of malondialdehyde (MDA) formed/g fresh weight. The experiments were repeated thrice. Statistical analysis All the experiments were repeated three times while each treatment had three replications (21 plants per replica). Endophytic bacterial growth dynamics in salinity environment Endophytic bacterial microbiome has been recently coined for their potential role in confronting abiotic stress situation (Brader et al. 2014). Although, rhizospheric bacteria are known regulators of soil ionic influx into the plant, but the potential of endophytic bacteria is least known. We have grown bacterial endophyte LK11 to understand the responses against the presence of sodium chloride (NaCl) ions in the medium. Assessing the adaptation to stress environment of a particular bacterial strain is essential, which can envisage the survival of endophyte in natural environment, where salinity is higher. We had grown LK11 in 100, 250, and 500 mM NaCl stress and assess its growth dynamics. Under normal growth conditions, the LK11 growth was significantly high with 12.5 × 10 8 CFU mL −1 . Addition of NaCl to the growth media influenced the bacterial cell growth. At low concentrations (100 mM NaCl), LK11 growth was 9.84 × 10 8 CFU mL −1 , which was reduced to 8.37 × 10 8 CFU mL −1 at higher concentrations (500 mM NaCl). However, at moderate concentration (250 mM NaCl), the bacterial cell growth was not significantly different from the LK11 growing under normal growth conditions. Its growth was 11.96 × 10 8 CFU mL −1 (Figure 1). This suggests that the viability and cellular structures of bacterial cells have not been disturbed during moderate concentration. This regulatory networking extends ameliorative responses to the plants growing in the same niche as previously shown by Hoper et al. (2006) and Tsuzuki et al. (2011). Co-synergism of salinity and gibberellin during bacterial growth Gibberellin A 4 (GA 4 ) has been described as a potent regulator of plant growth and development. Its application to economically important crops has also been suggested to improve fruit yield (Hamayun et al. 2010). Additionally, it also extends stress tolerance to the crops after drought and salinity exposure (Philipson 1983). However, the interaction of endophytic bacteria with exogenous GA 4 application has been least understood. Previously, we reported that LK11 has the potential to secrete GA 4 (2.56 ± 0.06 ng mL −1 ; Khan et al. 2014). To know the effects of exogenous GA 4 addition in bacterial growth medium, we have grown LK11 in different concentrations of NaCl and GA 4 (10 and 100 µM). The results showed that GA 4 application was inhibitory to the growth of LK11 as compared to control LK11 growing under normal growth conditions. The lower concentration (10 µM) was more inhibitory than higher (100 µM) one. However, when NaCl and GA 4 (10 µM) were applied together, the 100 and 250 mM NaCl showed improvement in the bacterial cell density as compared to 500 mM NaCl concentration. Although, the values were significantly different than the untreated LK11 whilst the addition of NaCl reduced the toxicity of GA 4 . This trend was more pronounced in higher concentration of GA 4 (100 µM) in 100 and 250 mM NaCl than 500 mM NaCl. This suggests that combination of NaCl (250 mM) and GA 4 (100 µM) treatments does not show inhibitory effects toward the growth of LK11, whilst such application might also be growth promoting for crops plants. This also shows that there are no or least damages to the bacterial cell structure and resultant oxidative stress develops by NaCl during growth. Effect of salinity and GA 4 on bacterial oxidative apparatus Excess of sodium chloride (NaCl) in any medium can be toxic to the living organism, since, it creates an ionic flux in cell by further invigorating the production of ROS such as hydrogen peroxide and superoxide anion (Vranova et al. 2002). To avoid massive disruption of cellular function due to ROS attack, the oxidative stress enzyme production is activated (Mittler 2002;Tsuzuki et al. 2011). Antioxidants and related enzymes such as CAT, POD, polyphenol peroxidase, superoxide dismutase, and glutathione are activated to convert the reactive species in harmless constituents (Vranova et al. 1997;Halverson et al. 2000;Torres-Barceló et al. 2013). Our preliminary results revealed that 250 mM NaCl and 100 µM GA 4 treatments in bacterial medium were not harmful for LK11 growth. We applied the same environmental conditions to bacterial growth to assess the regulation of various essential oxidative stress enzymes. The results showed that bacterial proteins were significantly (~1.2 to 1.8-fold) activated in sole NaCl and NaCl with GA 4 treatments as compared to LK11 without any treatment. The CAT activity was pronounced by the combined treatment of NaCl and GA 4 . This stimulation was in gradient manner but the level of CAT activated was not significantly different between sole NaCl and combined treatment with GA 4 (Figure 2). In response to NaCl (250 mM) and GA 4 applications, the POD and PPO activities were almost similar in sole and combined treatments. Only NaCl (250 mM) showed a little increase in POD activity as compared to other treatments; however, this was statistically not significant. The superoxide dismutase activity was significantly higher in LK11 + NaCl treatment as compared to normal growth conditions. Combined application of GA 4 and NaCl showed significant superoxide dismutase activity in comparison to LK11 and sole GA 4 growing in normal conditions (Figure 2). In case of bacterial glutathione contents, the combined treatment of GA 4 and NaCl has significantly increased its synthesis as compared to other treatments and LK11 growing under normal conditions. This suggests the active participation of glutathione in ionic influx resulted from salinity especially Na + . Excessive ionic flow can influence the equilibrium in the cell. Though bacterial cells have energy-dependent active transport system to maintain the influx of such ions, however, during such continued exposure the radical/ion scavenging enzymes can help in arresting the adverse effects (Halverson et al. 2000;Ali et al. 2014 Effect of LK11 inoculation on growth of tomato plants during salinity Regular exposure to salinity hinders crop biomass and production. In current experiments, the LK11 inoculated plants exhibited significantly higher shoot length (~40%) under normal growth conditions. Similarly, GA 4 application also showed significantly higher shoot length (~55%) than non-inoculated control tomato plants. Overall, GA 4 application showed pronounced shoot elongation as compared to LK11 and control (Table 1). Almost same beneficial effects were also observed for shoot biomass, root length, and biomass in GA 4 and LK11 application under normal growth conditions as compared to non-inoculated control plants. The number of leaves and internodes was significantly higher in GA 4 and LK11 plants than in control. However, when the tomato plants were exposed to NaCl (250 mM), the shoot/root length, shoot/root biomass, and leaf number were significantly retarded. The sole NaCl treated showed stunted and abnormally lower growth potential. Previously, such negative and harmful effects were also reported by Wang et al. (2003), Hasegawa et al. (2000), Munns and Tester (2008), Khan et al. (2011Khan et al. ( , 2012Khan et al. ( , 2013, and Kang et al. (2014) in various crop plants such as rice, soybean, cucumber, pepper, etc. These showed that the toxicity of NaCl caused reduced plant growth and biomass which was also noted in our experiments. Plant growth parameters were significantly ameliorated when tomato plants were treated with endophytic bacteria LK11 and GA 4 as compared to sole NaCl treatments. The LK11 inoculation significantly increased (~35 to 100%) the plant growth parameters (shoot/root length, shoot/root biomass, and leaf number) as compared to only NaCl application (Table 1). However, this increase in tomato plant growth was more pronounced when treated with GA 4 , which is a commercially known growth regulator for crops. In comparative assessment of GA 4 and LK11, only shoot length was significantly higher in GA 4 treatments but other parameters such as root length and biomass were either significantly higher or nonsignificant in LK11 inoculation under salinity stress (Table 1). This suggests the regulative role of endophytic bacteria in salinity stress. A combined application of both GA 4 and LK11 showed more vigor and tolerance to the salinity stress, resulting in significantly higher plant growth parameters among all sole treatments (Table 1). Tomato plants are very sensitive to salinity stress. Most of the previous studies showed an application of 50-200 mM (Zhang & Blumwald 2001;Santa-Cruz et al. 2002;Mayak et al. 2004;Tuna et al. 2008;Kang et al. 2014). However, current study shows that combine and sole application of endophytic bacteria and GA 4 can extend the salinity tolerance to tomato crops. On the other hand, exogenous GA 4 application to crops can not only increase crop growth and yield but also increase plant's fitness against abiotic limiting factors such as salinity as shown by Maggio et al. (2010), Tuna et al. (2008), Zhang et al. (2014), etc. Endophytic microbes residing in the host tissues can regulate the ionic influx Mercado-Blanco & Lugtenberg 2014) which enters through passive transport via roots (Sattelmacher 2001). Since the endophyte was growing proficiently in NaCl media with lesser damage to the cellular oxidative apparatus (Figure 2), therefore, during salinity stress, the tomato plants inoculated with endophyte resulted in increased biomass and growth. Similar regulatory and stress modulatory behavior of bacterial endophyte was also observed by Ali et al. (2014), Mercado-Blanco and, and Egamberdieva and Lugtenberg (2014). Regulation of oxidative stress in tomato plants NaCl induced ROS development damages the function of cellular organelles by peroxiding the essential lipid layer (Munns & Tester 2008). ROS are removed from the cell directly (CAT and POD) or indirectly (redox molecules like glutathione). In present study, the tomato plant grown under normal growth conditions did not showed any significant sign of lipid peroxidation as the level of MDA formation was not significant (Table 1) in LK11, GA 4 , and control plants. However, exposure to NaCl stress caused an increased rate of lipid peroxidation in sole NaCl treated plants. The sole LK11 and GA 4 application counteracted the NaCl toxicity by lesser effecting the lipid peroxidation. The combined application of LK11 + GA 4 resulted in significantly reduced level of lipid peroxidation (Table 1). Since membrane bounded lipid hydroperoxides are difficult to measure due to their instability, therefore we measured the degree of lipid peroxidation to quantify secondary breakdown products like MDA. Higher ROS, on the other hand, autocatalyze peroxidation of lipid membrane and affect membrane semi-permeability under high stress (Khan et al. 2013). Activation of antioxidant scavengers can enhance membrane stability against ROS attack while MDA content can be used to assess the stress injury of plants (Rivero et al. 2014). Thus our result suggests a lesser membrane injury to the LK11, GA 4 , and LK11 + GA 4 plants than sole NaCl plants. Both endophytic bacteria and fungi have been recently shown to ameliorate adverse effects of stress by reducing the level of lipid oxidation , which is in compliance with our findings as well. To minimize the responses of salinity induced stress, the plant cell recruits glutathione to mitigate the oxidative imbalance. The major role of glutathione is to protect cell from the toxic burst of H 2 O 2 and its pre-requisites such OH radicals (Ruiz & Blumwald 2002). Our results showed that endophyte and GA 4 treatment induced the production of glutathione as compared to control plants under normal growth conditions (Table 1). The glutathione levels were significantly higher in LK11 and GA 4 as compared to sole NaCl treatment under salinity stress. However, glutathione level in tomato plants got reduced when a combined treatment of LK11 and GA 4 was given under salinity. This suggests a stress aversion by endophytes during salinity. While in case of sole application, both LK11 and GA 4 treated plants has to activate the synthesis of glutathione to incinerate the toxic effects of NaCl. However, these levels were significantly lower in sole NaCl applied tomato plants (Table 1). To further regulate the NaCl induced oxidative stress, antioxidant enzymes such as CAT, POD, and PPO are also activated. Our results showed the CAT activity was significantly higher in LK11 + GA 4 and control treatments whilst significantly lower in LK11 + GA 4 + NaCl, GA 4 and LK11 (Figure 3). The activity of POD was significantly higher in LK11 inoculation while in other treatments, its activity was significantly lower. The control and other treatments had a similar response (nonsignificant) except LK11 inoculation. In case of PPO, the enzyme activity was significantly higher in LK11 and LK11 + NaCl treated plants. In rest of the other treatments, the activity was significantly lower and was similar to that of control plants growing under normal conditions. This inactivation behavior of enzymes against stress was also previously described by Cavalcanti et al. (2004) and Khan et al. (2012). Conclusion The current study supports the use of endophytic bacteria during saline environmental conditions. The endophyte had the potential to grow prolifically in sodium chloride induced salinity stress without compromising its vital cellular assets for production of antioxidant and related enzymes as was noted for the activities of PODs, PPO, and CAT. The bacterial cells were synergistically growing in 250 mM of NaCl and known growth regulator GA 4 , thus showing little or no signs of toxicity against growth. The comparison of sole and combine endophyte and GA 4 treatment on tomato plants showed a significant increase in plant growth and biomass. Additionally, the inoculation and PGR treatment also modulated the oxidative stress and lipid peroxidation during salinity stress. Continued NaCl toxicity becomes lethal to plants because it excites the generation of ROS inside tissues. These along with other species, such as superoxide anion, hydrogen peroxide, etc., imitate the role and function of cellular organelles by creating an imbalance of oxidation/reduction cycle. To rescue cellular function, plant cell synthesizes a wide array of antioxidants and related enzymes. Since, the endophytic bacterial inoculation helped the plants to restore the tissues from peroxidizing the essential lipid membrane, therefore, it is revealed that LK11 might also counteract ROS induced toxicity through regulation of antioxidants and related enzymes response. The present results conclude that application of endophytic bacteria shows similar growth-promoting effects, which are known for PGPR and exogenous gibberellic acid application to crop plants during salinity stress. Disclosure statement No potential conflict of interest was reported by the authors.

v3-fos

2016-05-04T20:20:58.661Z

2015-11-03T00:00:00.000Z

15436762

s2

Exocarp Properties and Transcriptomic Analysis of Cucumber (Cucumis sativus) Fruit Expressing Age-Related Resistance to Phytophthora capsici Very young cucumber (Cucumis sativus) fruit are highly susceptible to infection by the oomycete pathogen, Phytophthora capsici. As the fruit complete exponential growth, at approximately 10–12 days post pollination (dpp), they transition to resistance. The development of age-related resistance (ARR) is increasingly recognized as an important defense against pathogens, however, underlying mechanisms are largely unknown. Peel sections from cucumber fruit harvested at 8 dpp (susceptible) and 16 dpp (resistant) showed equivalent responses to inoculation as did whole fruit, indicating that the fruit surface plays an important role in defense against P. capsici. Exocarp from 16 dpp fruit had thicker cuticles, and methanolic extracts of peel tissue inhibited growth of P. capsici in vitro, suggesting physical or chemical components to the ARR. Transcripts specifically expressed in the peel vs. pericarp showed functional differentiation. Transcripts predominantly expressed in the peel were consistent with fruit surface associated functions including photosynthesis, cuticle production, response to the environment, and defense. Peel-specific transcripts that exhibited increased expression in 16 dpp fruit relative to 8 dpp fruit, were highly enriched (P<0.0001) for response to stress, signal transduction, and extracellular and transport functions. Specific transcripts included genes associated with potential physical barriers (i.e., cuticle), chemical defenses (flavonoid biosynthesis), oxidative stress, penetration defense, and molecular pattern (MAMP)-triggered or effector-triggered (R-gene mediated) pathways. The developmentally regulated changes in gene expression between peels from susceptible- and resistant- age fruits suggest programming for increased defense as the organ reaches full size. Introduction Fruit development is typified by a progression from fruit set, to exponential fruit growth, maturation, and ripening. Morphological and transcriptomic analyses of early cucumber fruit growth indicate that the period spanning anthesis through the end of exponential expansion is marked by two developmental transitions, one at the onset of exponential growth, the second at the end of exponential growth [1]. The first several days post-pollination (0-4 dpp), prior to exponential growth, are characterized by extensive cell division [1][2][3]. Transcripts nearly exclusively expressed during this time period include homologs of genes associated with cell cycle and DNA replication [1,4]. The first transition, as is typical of fruit development in general [5], is from cell division to cell expansion. Rapid fruit elongation in cucumber occurs from approximately 4-12 dpp [2,3,6,7]. Genes with peak expression at mid-exponential growth (8 dpp), included genes encoding cytoskeleton, cell wall, cuticle, and phloem-specific proteins [1,6]. A second shift in gene expression occurred at the end of exponential growth, 12-16 dpp, but well before the transition to maturity and ripening that occurs at about 25-30 dpp. The transition accompanying the late/post-exponential growth stage, which has received little attention in the literature, was marked by strong enrichment for abiotic and biotic-stress related genes and induction of stress-related and development-related transcription factor gene homologs [1]. In certain cultivars, this time period is also associated with transition from susceptibility to resistance to an important cucumber disease, fruit rot caused by Phytophthora capsici. The soil borne oomycete pathogen, P. capsici, causes severe yield and economic losses for a variety of important vegetable crops, including cucumber (Cucumis sativus) [8,9]. The primary infectious agents responsible for spread of disease during the growing season are motile zoospores, which are released from asexual sporangia upon contact with water. For cucumber plants, it is primarily the fruit, rather than the leaves and vines that become infected [10]. Our prior studies showed that that very young cucumber fruit are highly susceptible, but at 10-12 dpp transition to resistance, becoming fully resistant by 16 dpp [10,11]. The transition to resistance was observed in bee-pollinated field-grown fruit, hand-pollinated greenhousegrown fruit, and fruit that set parthenocarpically in the greenhouse, indicating that the resistance did not depend on the presence or development of seeds within the fruit [10,12]. An agerelated reduction in susceptibility to P. capsici also was observed in fruit of several other cucurbit crops, including pumpkin, butternut squash, and acorn squash, although the effect was most pronounced for cucumber [11,13]. Age-related, or ontogenic, resistance also has been observed in strawberry fruit and grape berries in response to powdery mildew (Podosphaera aphanis and Erysiphe nectator, respectively) [14,15] and for apple fruit resistance to scab (Venturia inaequalis) [16]. In addition to the above examples in fruit systems, age-related resistance (ARR) has been observed in a number of other host-pathogen systems in a variety of tissue types, thus ARR is becoming increasingly recognized as an important component of plant defenses against fungal, oomycete, bacterial and viral pathogens [17][18][19][20]. However, the specific mechanisms responsible for resistance are just beginning to be explored and appear to vary among systems. By definition, ARR results from developmentally regulated changes that occur prior to contact with the pathogen, possibly by physical or chemical barriers, or by priming the organ for more rapid detection or response to infection. In several cases, ARR coincides with a developmental transition such as transition to flowering or critical leaf number as occurs for resistance of tobacco (Nicotiana tabacum) to P. parasitica, Arabidopsis to Pseudomonas syringa pv. tomato, and maize to Puccinia sorghi [21][22][23][24]. As the fruit surface is the first point of contact between the host and pathogen, in this study we sought to examine the role of the cucumber fruit surface in ARR to P. capsici. Tests with cucumber peels at different ages indicated a critical role of the fruit exocarp in expression of ARR, including possible biochemical defenses. Transcriptomic analysis of fruit peels identified developmental changes in gene expression in the surface tissue of resistant-age fruit. Peels from resistant-age fruit exhibited specific increase in transcripts associated with potential physical barriers, chemical defenses, and pathogen recognition responses. Fruit peel experiments Cucumber plants (pickling type, cv. Vlaspik; Seminis Vegetable Seed Inc, Oxnard, CA) were grown in greenhouse facilities at Michigan State University. No permits were required. Plants were grown in 3.78 L plastic pots filled with BACCTO (Michigan Peat Co., Houston, TX) or Suremix Perlite (Michigan Grower Product, Inc., Galesburg, MI) soil medium and fertilized once per week. Temperature was maintained at 21-25°C; supplemental lights were used to provide an 18 h light period. Pest control was performed according to standard management practices. Sets of 20-25 flowers were hand-pollinated on two dates to provide 15-20 fruit of each age to be harvested on the same day. To avoid competition between fruits, only one fruit was set per plant. The experiment was repeated three times. The harvested fruit were washed, surface sterilized by brief immersion in a 5% sodium hypochlorite solution, rinsed with water several times, and allowed to air dry. Zoospore suspensions were prepared from 7-day old cultures of P. capsici isolate OP97 [10] grown on diluted V8 media and flooded with 6-10 ml sterile distilled water to release zoospores as described by Gevens et al. [10]. Preparation of zoospores of P. capsici isolate OP97 was performed according to Gevens et al. [10]. Concentration of the zoospore suspensions was determined by hemacytometer and diluted to 1 x 10 5 per ml. Exocarp sections (3 cm x 3 cm x 1-2 mm) from the middle part of the fruit were excised from both 8 and 15 dpp fruit with petit knife or razor blade without introducing nicks. A sterile plastic tube (0.6 cm height, 0.8 cm diameter) was placed on the exocarp section and anchored to underlying intact 8 or 16 dpp fruit using a strip of 1 cm wide parafilm. A twenty-two gauge sterile needle was used to deliver 30 μl zoospore suspension into the tube by penetrating the parafilm; the needles did not come in contact with the fruit tissue. Inoculated fruit were incubated under constant light at 23-25°C in plastic wrap covered trays lined with moist paper to maintain high humidity. Intact 8 and 15 dpp fruits were similarly inoculated and included in each tray as control. Each tray contained each treatment combination: 8 day peel/8 day fruit; 8 day peel/15 day fruit; 15 day peel/8 day fruit; 15 day peel/15 day fruit; intact 8 day fruit; intact 15 day fruit. The peel sections and underlying fruit were monitored daily for 10 dpi and scored for stage of disease progression (1 -no symptoms, 2 -water soaked, and 3 -sporulation). The experiment was repeated three times. Data were analyzed as a randomized complete block design by ANOVA using the SAS program 9.1 (SAS Institute Inc., Cary, NC) with mixed procedures. Each value is the mean of at least 9 peel sections or fruit ±SE. Bars marked with different letters indicate significant difference by LSD, P<0.05. redissolved in water and 10% methanol, respectively, to a final concentration of 25 μg ul -1 . A 96-well clear (Thermo Fischer Scientific Inc., Waltham MA) or black microtiter plate (Griener Bio-One, Orlando, FL) was prepared with 200 μl clarified V8 media (centrifuged at 10,000 rpm for 10 min) per well. Samples were treated with 10 μl crude extract solution or solvent controls, and inoculated with 20 μl of 1x10 5 zoospores ml -1 suspension of either P. capsici isolate OP97 or NY0664-1 expressing red fluorescent protein (RFP) ( [26]; kindly provided by C. Smart, Cornell University) as described above. The inoculated plates were incubated at 25°C with a 16h light/ 8h dark cycle for 72 hours. Visual ranking was performed on a 1-5 scale at 3 dpi as illustrated in Fig 2A. Fluorescence values of the RFP-expressing cultures were measured at 530nm (excitation) and 590nm (emission) using SpectraMax M2e (Molecular Devices, Sunnyville, CA) at 0, 24, 48 and 72hrs post inoculation. Mean fluorescence measurements from the control (media with aqueous/methanolic extracts) were subtracted from the mean fluorescence values for the corresponding treatments. Samples within the plate were arranged in a randomized complete block design. Data were analyzed by ANOVA, followed by means separation by LSD, P<0.05. Each experiment was repeated two or three times with five replicate samples per treatment. Imaging of fruit exocarp by SEM Sample preparation and imaging of cucumber fruit exocarp sections (2-3 mm) was performed by the Center for Advanced Microscopy of Michigan State University as briefly described here. Exocarp tissues were fixed in glutaraldehyde solution and dried in Balzers Model 010 critical point dryer (Balzers Union Ltd., balzers, Liechtenstein). After drying, the samples were mounted on aluminum stub using high vacuum carbon tabs (SPI supplies, West Chester, PA) and coated with osmium using a NEOC-AT osmium coater (Meiwafosis Co. Ltd., Osaka, Japan. Processed exocarp tissues were examined using a JEOL JSM-7500F scanning electron microscope (JEOL Ltd., Tokyo, Japan). Sample preparation for pyrosequencing Cucumber plants were grown in the greenhouse as described above and in Ando et al. [1]. All flowers for each experiment were hand pollinated on a single date (1-2 flowers per plant). The experiment was repeated three times. Randomly assigned groups of twenty fruit were harvested at 8 and 16 dpp and ranked by size; the middle ten fruits were used for RNA extraction. Peel sections (1-2 mm thick) were removed by razor blade, immediately frozen in liquid nitrogen, and stored at-70°C until RNA was isolated. Each biological replicate consisted of peel sections pooled from ten fruits; two biological replicates were prepared for each age. RNA extraction and oligo(dT)-primed cDNA sample preparation were based on the procedures of Schilmiller et al. [27] and Ando and Grumet [6]. Final concentration was assessed by the nanodrop ND-1000 Final concentration was assessed by the nanodrop ND-1000 (Thermo Scientific, Wilmington, DE) method and subsequent steps for 454 Titanium pyrosequencing analysis were performed by the Michigan State University Research Technology Support Facility (RTSF). Each sample was loaded on a 1/4 plate 454 Pico TiterPlate (454 Life Sciences, a Roche Corporation, CT). Pericarp samples consisting of exocarp, mesocarp, and placenta tissue but not seeds, from fruit grown at the same time in the greenhouse as those used for peel analysis, were sequenced previously [1]. Contig assembly, EST mapping, and gene annotation Contigs were assembled by the MSU RTSF Bioinformatics Group. Transcript assemblies were created from a collection of EST data sets from Cucumis sativus. An integrated pipeline was used to align individual reads to the C. sativus genome (ICuGI) using BLAT [28] and then to assemble clusters of overlapping alignments. The pipeline, Program to Assemble Spliced Alignments (PASA), is described in Hass et al. [29]. Prior to submitting the EST sequences to PASA they were cleaned using the TIGR SeqClean pipeline [http://compbio.dfci.harvard.edu/tgi/ software/]. This was used to remove and vector or primer sequences, poly(A) tails and other low quality or low complexity sequences. Input to the PASA pipeline was comprised of 1.65 million reads generated from the nine libraries of ESTs from fruit at various developmental stages; all libraries were from fruit grown at the same time in the greenhouse. 99.2% of the ESTs were mapped to the genome. PASA assembled 53,677 putative transcripts clustered at 32,000 loci on the genome. Read data for 8 day post pollination samples is available from the Sequence Read Archive (SRA), accessible through NCBI BioProject ID PRJNA79541. Read data for 0, 4, 12 and 16 dpp samples and the 8 and 16 dpp peel samples in SRA as well as assembled contig sequences deposited as Transcriptome Shotgun Assemblies (TSA) and expression profiling data in the Gene Expression Omnibus (GEO) are available through NCBI BioProject ID PRJNA169904 and DDBJ/EMBL/GenBank under the accession GDIL00000000. Putative transcripts were annotated by BLAST comparison to both the Arabidopsis proteome (TAIR9) and the NCBI RefSeq Plant database; 37,800 putative transcripts scored a significant (e-value 10 −10 ) hit to TAIR9 and 40,000 to RefSeq Plant. To estimate relative expression, the number of reads originating from each cDNA library were counted for each contig and reported relative to the total number of reads generated for that library as transcripts per hundred thousand (TPHT). Transcriptome analysis The Classification SuperViewer Tool w/Bootstrap web database [30] was used for GO categorization, determination of normalized frequencies relative to Arabidopsis, and calculation of bootstrap standard deviations, and P-values. Princomp procedure SAS 9.1 (SAS Institute, Cary, NC) was used for principal component analysis. The first two principal components, which explain nearly 90% of the total variation were extracted from the covariance matrix. To identify transcripts either preferentially or minimally expressed in peel tissue, the proportion of reads obtained from the peel samples was calculated for each transcript for which there were 30 reads [i.e., reads from peel samples at 8 and 16 dpp/total reads (peel + pericarp) at 8 and 16 dpp]. Transcripts with increased expression in 16 dpp peel were identified by the ratio of reads from 16 dpp peel vs. 8 dpp peel. Putative cucumber homologs of the Arabidopsis SYP121/ SNP33 regulon [31] were identified within the cucumber fruit transcriptome set and tested for co-expression with cucumber SYP121 and SNP33 by correlation analysis of transcript frequency in peel and pericarp across fruit age. qRT-PCR Cucumber fruit used for qRT-PCR analysis were grown as described above for fruit peel experiments and pollinated on two dates, 8 days apart. Five fruits of each age (8 and 16 dpp) were harvested and quickly processed for RNA isolation. Peduncle and blossom ends were removed and peels separated from pericarp of the middle 5-10 cm of fruit tissue using a razor blade. Samples were quickly frozen in liquid nitrogen and stored at -80°C. RNA extraction and oligo (dT)-primed cDNA sample preparation were as described above. qRT-PCR primers were designed using NCBI Primer-BLAST [http://www.ncbi.nlm.nih.gov/tools/primer-blast] and tested for product specificity and reaction efficiency (S1 Table). qRT-PCR reactions were performed with the ABI Prism 7900HT Sequence Detection System (Life Technologies, Inc., Gaithersburg, MD). Samples were prepared using the rEVAlution Master Mix (Syzygy Biotech, Grand Rapids, MI) with ROX reference dye (Syzygy Biotech, Grand Rapids, MI) according to manufacturer's instructions. Three technical replicates were prepared for each of the five peel and pericarp samples at each age. C. sativus Ubiquitin 3 (CsUBQ3) was used as an endogenous control. For standard curve dilutions a pool of 2 μl from each of the cDNA samples was collected and diluted to 20, 4, 0.8 and 0.16 ng/μl. PCR conditions were 50°C for two minutes, 95°C for 10 minutes enzyme activation, then 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Samples were quantified using the relative standard curve method for each gene. Relative quantification values (RQ) were normalized to the concentration of CsUBQ3 in each sample. Results Cucumber fruit surface is an important determinant of ARR to P. capsici With the exception of pathogens that enter through wounds or are delivered by a vector, the outer surface of a plant organ is typically the first point of contact with the host. We therefore sought to determine whether the ARR of cucumber fruit to infection by P. capsici was influenced by the fruit surface. Preliminary tests showed that when 16 dpp fruits were peeled prior to inoculation, 100% formed sporulating lesions, whereas none of the intact control fruit at 16 dpp become infected. While these results suggest that the fruit surface plays an important role in the resistance of older fruits to infection by P. capsici, it is also possible that the observed infection was facilitated by wounding as has been observed in other systems. To eliminate possible confounding effects of injury, exocarp sections from fruits at 8 dpp or 15 dpp were placed on top of a second, intact fruit, and then inoculated with P. capsici. The peel sections responded equivalently to intact fruit (Fig 1). Like whole 8 dpp fruit, the 8 dpp peel pieces exhibited either water-soaking or sporulation, regardless of the age of the fruit underneath. Similarly, peel sections from 15 dpp fruit responded like intact 15 dpp fruit, regardless of fruit age underneath. In addition, even when subjected to the additional disease pressure of contact with an infected 8 dpp fruit peel, the underlying 15 dpp fruit did not typically become infected (8d/15d treatment). Finally, when 15 dpp fruit surface pieces were inoculated, the underlying 8 dpp fruit also maintained a very low disease score (15d/8d treatment), indicating that the 15 dpp fruit surface sections protected the underlying 8 dpp fruit. These results suggest that the surface of 15-16 dpp fruit possesses properties that inhibit P. capsici infection. Surface factors influencing resistance may include biochemical or structural components. Cucumber leaves have been found to produce methanol-soluble phenolic and flavonoid compounds with antimicrobial properties [32][33][34]. Therefore we sought to test whether cucumber peels might also produce compounds with antimicrobial activity. Testing of aqueous and methanol extracts from peels of fruit at different ages showed that methanolic extracts from cucumber fruit peels inhibited growth of two P. capsici isolates in vitro (Fig 2A and 2B). Methanolic extracts from 16 dpp peels provided greater inhibition than from 8 dpp peels. Structural changes in the 16 dpp peels included thicker epidermal cell walls, increased cuticle thickness, and increased intercalation of cutin and waxes between adjacent cells in the epidermal layer ( Fig 2C). Gene expression in cucumber fruit peel To examine changes in gene expression specifically occurring in cucumber peels between fruit at susceptible and resistant ages, cDNA libraries were prepared from peel samples from 8 and 16 dpp fruit. Each biological replicate consisted of peel sections pooled from ten fruits; two biological replicates were prepared for each age. Pyrosequencing analysis yielded 814,250 ESTs from peel samples, which were combined with ESTs obtained from pericarp samples from 0, 4, 8, 12 and 16 dpp fruit that had been grown in the greenhouse at the same time [1], providing a data set of 1.65 million reads. Of those, 99.2% were mapped to the cucumber draft genome [35] at 38,318 loci. The number of ESTs per assembled transcript ranged from 2-16,817 with a mean of 76 reads/transcript and a median of 9 reads/transcript. As was observed by Ando et al. Principal component analysis indicated that pericarp or peel samples at the different ages were more closely associated with each other than were pericarp and peel at the same age, indicating more commonality based on tissue type than fruit age (Fig 3A). To identify transcripts either preferentially or minimally expressed in peel tissue, the proportion of reads obtained from the peel samples relative to pericarp was calculated for each transcript as: reads from peel samples 8 and 16 dpp / total reads (peel + pericarp) at 8 and 16 dpp (Fig 3B). The proportion of reads obtained from the peel samples was approximately normally distributed over the population of transcripts. Genes present in the tails of the distribution, i.e., the top or bottom 5% (Fig 3C and 3D) showed functional differentiation. The 5% of transcripts with highest proportion of expression in the peel relative to the pericarp (a set of 813 transcripts for which >72% of the reads for each transcript were obtained from one or both of the peel samples), were specifically significantly enriched for cellular component categories of extracellular, ER, cell wall and plastid-related genes (S2 Fig). For example, cuticle-related transcripts, such as GDSL motif lipase gene homologs, were primarily expressed in the peel while phloem and aquaporin related gene homologs had minimal expression in the peel (Fig 3E and 3F) Putative transcription factor genes that were primarily expressed in the peel (>80% reads from peel samples) included many that have been annotated to be involved in biotic and abiotic stress responses, including: R2R3 MYB domain proteins 30 and 96; WRKY 15 and 40; ethylene response factors (ERF6); heat shock factors (HSFA3); and salt tolerance zinc finger (STZ) factors ( Table 1). The peel-expressed MYB factor genes all belong to R2R3 subgroup 1, which in Arabidopsis, is involved in drought stress and disease resistance functions including the ABA signal cascade, regulation of stomatal movement, and hypersensitive cell death response [36]. In contrast, expression of many development-related transcription factors observed in the pericarp, such as homologs of AGAMOUS, Auxin response factor 4, FLOWERING LOCUS T, Collectively, these observations reflect functional differentiation between the peel and pericarp. Transcripts predominantly expressed in the peel were consistent with fruit surface associated functions including photosynthesis, cuticle production, response to the environment, and defense. Transcripts expressed specifically in peel from 16 dpp fruit We next sought to identify transcripts that were primarily expressed in the peel, and also showed increased expression in 16 dpp fruit relative to 8 dpp fruit, i.e., specifically expressed at 16 dpp rather than 8 dpp. The 105 transcripts that met both criteria (top 5% for expression in peel, and top 5% for increase in 16 dpp peel vs. 8 dpp peel) exhibited 8-800 fold enrichment in 16 dpp peel relative to 8 dpp peel (S2 Table). Of those transcripts, 9 did not have putative homologs in the NSCI RefSeq Plant gene database, and an additional 12 with homologs did not have functional annotation. Those with annotation showed strong enrichment for GO categories of response to stress, extracellular, response to abiotic or biotic stimulus, signal transduction, and transport functions ( Table 2). The greatest reductions in expression in 16 dpp peel relative to 8 dpp peel were observed for the categories of chloroplast, and plastids. This observation is developmentally consistent with the reduced chlorophyll content observed at these ages [1]. Greater than 40% of the 16 dpp peel-enriched genes were potentially associated with pathogen defense based on annotations from other systems (Table 3). A subset of 20 putative defense-related genes whose transcriptome patterns showed elevated expression in 16 dpp peel samples was selected for verification of expression by qRT-PCR analysis of pericarp and peel samples from 8 dpp and 16 dpp fruit (Fig 4A). The qRT-PCR results mirrored those of the 454 analysis, substantiating the 454 analysis, and also demonstrating reproducibility of gene expression patterns across experiments grown at different times in the greenhouse. Predominant expression was observed in 16 dpp peel tissue, although for a few genes (EDS1, NUDT7, SYP121), the difference between 8 dpp peel and 16 dpp peel was less pronounced in the qRT-PCR analysis than from the transcriptome data. Age-related, peel-specific, gene expression potentially associated with biochemical and structural defenses. The 16 dpp peel samples were specifically enriched for expression of several groups of genes potentially associated with biochemical or structural defenses. These include putative homologs of genes encoding enzymes associated with flavonoid biosynthesis such as: phenylalanine amonia-lyase (PAL), flavanone-3-hydroxylase (F3H) and flavonoid 3'- Cucumber Fruit Peel Transcriptome monooxygenase (also flavonoid 3' hydroxylase; TT7) ( Table 3). A putative homolog of flavonol synthase At5g08640 (1.0E-120) was also highly expressed in the peel (92% of transcript reads) with 4.7-fold higher expression in 16 dpp peel than 8 dpp peel. Another group of genes with strong expression in the 16 dpp peel samples included members of the glutathione S-transferase (GST) gene family (Table 3). GSTs, which are highly expressed in plants and can comprise 1-2% of the soluble proteins, have been associated with a range of biotic stress response related functions, including increased resistance to several fungal or oomycete pathogens [37]. The GST family is classified into six groups (phi, tau, theta, zeta, lambda, and dehydroascorbate reductase) that exhibit tissue-and developmental-specific expression. The cucumber fruit transcriptome included twelve putative GST family members representing five of the six groups (Additional File 4). Three showed strong expression specifically in the 16 dpp peel samples (Table 3); all three were members of the tau group, which has been associated with resistance to biotic stresses [38,39]. One of the genes with the strongest 16 dpp peel-specific expression was a putative member of the fungal and plant-specific, pleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporter sub-family. CsPDR12 (PEN3) showed greater than 70-fold elevated expression in 16 dpp peel relative to 8 dpp peels, and essentially exclusive expression in 16 dpp peel as assessed by qRT-PCR analysis ( Table 3, Fig 4A). ABC transporters have been implicated in transport of a wide variety of structurally unrelated molecules, including flavonoid related compounds [40][41][42]. The 16 dpp peel sample also showed peak expression of putative homologs of several cuticle associated genes [43,44] including: two GDSL motif genes; two GDSL-like extracellular lipases; a long chain fatty acid-CoA ligase family protein gene; and two very long chain fatty acid synthesis related genes [KCS6 and 20 (3-ketoacyl-CoA synthase 6 and 20)] (Table 3; S2 Table; Fig 4A). Age-related, peel-specific, gene expression of defense pathway associated genes. Several of the genes strongly represented in the 16 dpp peel samples are putative homologs of genes that have been specifically associated with microbial associated molecular pattern (MAMP)triggered defense, suggesting possible priming for defense (Table 3). A central feature of MAMP defense is an oxidative burst resulting from rapid production of reactive oxygen species (ROS), including peroxidase-mediated production of hydrogen peroxide [45,46]. The oxidative burst can inhibit pathogen growth, signal induction of host defense responses, and promote hypersensitive response. The genes showing increased expression in the 16 dpp peel included putative homologs of three members of the peroxidase gene family with 20-200-fold up-regulation relative to 8 dpp peel. Peroxidases also have been implicated in additional roles that may contribute to plant defense, including strengthening of cell walls (e.g., protein cross linking, lignification), potentially inhibiting pathogen penetration [46]. Notably, two of the genes specifically highly expressed in the 16 dpp peel were putative homologs of genes that confer resistance to penetration of fungal and oomycete pathogens in Arabidopsis and barley, SYP121 (SYNTAXIN OF PLANTS)/PEN1(PENETRATION1) and SNAP33 [47]. SYN121/PEN1 function is associated with more rapid formation of penetrationresistant papillae structures containing callose, phenolic compounds, lignin, and reactive oxygen species [48]. As was observed for Arabidopsis and barley, the cucumber SYP121 and SNAP33 homologs were highly co-expressed (R = 0.976, P<0.001). In silico analysis of Arabidopsis genes identified a set of 107 genes co-expressed with PEN1, SNAP33 and MLO2 [31]. Putative homologs for 37 of these genes were observed in the cucumber fruit data set, of which 17 showed patterns of expression correlated with expression of the cucumber SYP121/PEN1 and SNAP33 homologs (Table 4; Fig 4B). These genes included putative homologs of the elicitor response genes, CERK1 (chitin elicitor receptor kinase 1), and MKK9 (MAP kinase kinase 9) [48]; SA-mediated and hypersenstitive response genes, SARD1 (SAR deficient 1) and CPB60G [49]; the SA receptor, NPR3 (NPR1-like gene 3; non-expressor of PR genes 1-like protein 3) [50], and a defense response regulator, NSL1 (necrotic spotted lesions 1) [51]. Putative homologs of genes associated with effector triggered-defense such as EDS1 (ENHANCED DISEASE SUSCEPTIBILITY 1) and NUDT7 (NUDIX HYDROLASE HOMOLOG 7) (Table 3) were also enriched in the 16 dpp peel samples. EDS1 in Arabidopsis regulates R gene-mediated and systemic resistance, acting in combination with several other factors including AtNUDT7 and the flavin-dependent monooxygenase, FMO1, to regulate cell death responses [52,53]. A homolog of FMO1 (At1g19250, 1.0E-116) was minimally expressed in cucumber fruit samples at 0, 4 or 8 dpp, but then increased 50-and 100-fold at 12 and 16 dpp, respectively. The transcripts, however, were almost exclusively located in the pericarp (97%) samples, rather than peel (Fig 4C), suggesting that if FMO1 plays a role in resistance to P. capsici, it likely occurs at an infection step post-penetration. The set of 16 dpp peel-enriched genes also included putative homologs of R2R3 subgroup 1 MYB transcription factor MYB 96 and a vacuolar processing cysteine protease enzyme, Gamma-VPE, which is a critical component of the hypersensitive programmed cell death Table 4. Co-expression analysis of putative cucumber homologs of MLO2/SYP121/SNAP33-co-expressed genes from Arabidopsis. Putative homologs of MLO2/SYP121/SNAP33-co-expressed genes from Arabidopsis (as identified by Humphrey et al. [31] were tested for co-expression with cucumber SYP121 and SNAP33 genes in cucumber pericarp (0, 4, 8, 12, and 16 dpp) and peel ( response (Table 3) [36,54]. The peel-expressed putative homologs of abiotic and biotic stress MYB factor 96 was 18 fold up-regulated in the 16 dpp peel relative to 8 dpp peel (Table 3), while MYB30, which in Arabidopsis has been found to encode an activator of the hypersensitive cell death response via regulation of 402 biosynthesis of very long chain fatty acids [55], exhibited 4.1-fold increase in 16 dpp peel relative to 8 dpp peel (Table 1, S2 Table). Discussion These studies showed that ARR of cucumber fruit to infection by P. capsici is associated with the fruit surface. Peel sections from 16 dpp fruit exhibited resistance, while peel sections from 8 dpp fruit were highly susceptible. We therefore sought to examine changes in the fruit peel that might contribute to increased resistance. Peel sections from 16 dpp had obvious differences in surface morphology and produced increased levels and/or types of methanol-soluble compounds capable of inhibiting growth of P. capsici in vitro. Transcriptome analysis reflected functional differentiation for gene expression between the peel and pericarp with increased expression of fruit surface associated functions such as photosynthesis, cuticle production, response to the environment, and defense in the peel tissue. Gene expression that was specifically associated with peel sections from resistant age fruit showed strong enrichment for transcripts annotated to be associated with response to stress or abiotic or biotic stimuli, signal transduction, and transport and extracellular functions. Greater than 40% of transcripts of the 16 dpp peel-enriched genes were potentially associated with pathogen defense. Consistent with methanol-soluble compounds capable of inhibiting growth of P. capsici, was increased expression of homologs of several genes associated with flavonoid and phenylpropanoid biosynthesis, PAL, F3H, TT7 and FLS. Previous studies have observed phenylpropanoid-derived phenolics, C-glycosyl flavonoids, and aglycones associated with resistance to powdery mildew (Podosphaera xanthii) in cucumber leaves [32][33][34] and inhibitory glycosidelinked phenolic compounds that increase with leaf age, have been found to localize to cucumber leaf cells beneath penetrating appressoria of Colletotrichum orbiculare [56]. Such defensive compounds may serve roles as phytoanticipins, accumulating prior to infection, or may exhibit increased resistance in response to infection. The developmental regulation of expression of phenylpropanoid-associated genes, and the presence of pathogen-inhibitory, methanol-soluble compounds in the 16 dpp peel, suggests synthesis of preformed chemical barriers. However, induced resistance in cucumber leaves to powdery mildew has been found to be dependent on elevated activity of flavonoid pathway enzymes [32], suggesting potential for induced response as well. Increased expression of PAL, a key upstream enzyme for flavonoids, as well as salicylic acid and lignin biosynthesis, has been observed in a wide range of systems, including cucumber and melon, in response to treatment with pathogens or chemical inducers (e.g., [57,58]). The 16 dpp fruit peel sections also had specific elevation of expression of several GST genes and the pleiotropic drug resistance gene family member homolog, CsPDR12. Members of both GST and PDR gene families can facilitate export of flavonoid and terpenoid related compounds to the cell wall where they can accumulate in the cuticle [59,60]. The PDR12 homolog from Nicotiana plumbaginifolia (NpPDR1), shows age-related and epidermal-specific transcription and exports an anti-fungal/oomycete terpenoid to the leaf surface [41,61]. Several members of the Arabidopsis PDR family show age-specific expression in developing seedlings [62]. Other types of defense related genes that were specifically, highly expressed in the peel of 16 dpp fruit were homologs of genes that have been associated with resistance to fungal and oomycete penetration by more rapid formation of papillae, as well as numerous putative elicitor-, effector-, and SA-response genes which may play roles in MAMP or R-gene mediated resistance. While at this time, we cannot determine whether these genes are specifically associated with ARR to P. capsici, the transcriptomic analysis indicating increased expression of a large number of putative-defense related genes, raises the possibility that ARR results from systematic, developmental reprogramming for defense. In summary, analysis of cucumber fruit indicated importance of the fruit surface for ARR to P. capsici and a potential role for methanol-soluble inhibitory compounds. Transcriptomic studies of the fruit peel suggest developmentally-regulated expression of defense genes potentially associated with structural, biochemical (flavonoid pathway and transporters), MAMP response, and effector-triggered or R-gene mediated resistances.

v3-fos

2018-12-20T20:28:19.520Z

2015-11-05T00:00:00.000Z

89989572

s2

Geographical distribution of Puccinia triticina physiologic races in Egypt during 2012-2014 growing seasons Wheat leaf rust caused by Puccinia triticina Eriks. is a common disease in Egypt and worldwide. Survey of wheat leaf rust samples and identification of physiological races using twenty single Lr genes are very important in describing virulence pattern variation, geographical distribution of leaf rust pathotypes and how its change in response to host selection. Variability in population of the causal organism is annually determined using samples collected from wheat growing areas in Egypt for three growing seasons that is, 2011/2012, 2012/2013 and 2013/2014. The results obtained showed a significant variability in pathotypes which are different from season to season. In the course of this study a total of 50, 65 and 33 leaf rust samples were collected in 2011/2012, 2012/2013 and 2013/2014, respectively from different wheat growing areas in eight governorates of Egypt that is, Beheira, Dakahlia, Gharbiya, Minufiya, Sharqiya, Domiatta, Qalyubiya and BaniSweif. A total of 118, 166 and 61 physiologic races were identified in 2011/2012, 2012/2013 and 2013/2014, respectively. The most frequent races included STTST and TKTTT (each with 2.54%) in 2011/2012; PKTST (6.63%), TTTTT (7.83%) and TTTST (10.24%) in 2012/2013 as well as FKTTT (4.92%) and PTTTT (11.47%) in 2013/2014. Race groups PT--and TK--were common at eight locations during the three growing seasons. Cluster analysis based on percentage frequency of virulence of P. triticina race groups in different location showed that in 2011/2012 and 2012/2013 growing seasons two main clusters were formed. While, in 2013/2014 growing season the cluster analysis was divided into six main clusters. Lines with Lr 1, Lr 2c, Lr 3, Lr 16, Lr 24 and Lr 26 were susceptible against most race groups, while, the leaf rust monogenic lines Lr 2a and Lr 9 showed different reactions against the tested race groups. INTRODUCTION Leaf rust of wheat caused by Puccinia triticina Eriks. (syn. Puccinia recondita Roberge ex Desmaz. f. sp. tritici E. Henn.) is found wherever wheat is grown (Samborski, 1985). In Egypt, leaf rust is the most common and important wheat diseases. It causes sever losses in grain yield which reached 23% on some varieties depending on *Corresponding author. E-mail: [email protected]. Author(s)agree that this article remain permanently open access under the terms of the Creative Commons Attribution License4.0InternationalLicense environmental conditions, level of resistance, dominant physiologic races and the stage of crop development when intial infection occurs (Nazim et al., 1983). Each 1% increase in leaf rust severity decreases yield weight by 40.07 kg/ha and 1000 kernel weight 0.13 g (Leonard et al., 2005). Using resistant genotypes are the most economic and effective method to control plant diseases in general and particularly obligate parasite including leaf rust of wheat (Elyasi-Gomari and Lesovaya, 2009). The causal orgainsm of leaf rust, is highly variable, consisting of different physiological races or virulence phenotypes (Long et al., 1992;Kolmer and Liu, 1997). Effective of leaf rust resistance of wheat cultivars in a region is dependent on the virulence of the regional populations of P. triticina. The development of resistant varieties, requires a knowledge about the virulence diversity, race distribution in particular region, and which resistance genes are effective against these races. In addition, virulence surveys are important for studying the evolution of new races and forecasting the virulence shifts in a physiologic races population (Admassu et al., 2009). Rust disease surveys are conducted in many wheat growing areas of the world to studying the evolution of new races and forecasting or detect virulence phenotypes that may have been introduced to a region. In breeding for rust resistance, the survey provide essential information not only to determine the direction of the breeding program, but also to detect new virulent pathogen phenotypes that threat the currently grown wheat cultivars. Moreover, survey plays an effective role in determining the geographical sources of disease pathotypes and providing information about the effectiveness of currently used resistance genes (McIntosh et al., 1995;Park, 2008). The main objectives of this study were to study the geographical distribution of P. triticina in eight Egyptian governorates during three successive growing seasons (2012 -2014) to assist in the development of wheat cultivars with high levels of leaf rust resistance. In addition to characterize the virulence phenotypes of P. triticina collected in Egypt over the three growing seasons. MATERIALS AND METHODS Samples of wheat leaf rust urediniospores were collected during 2011/2012, 2012/2013 and 2013/2014 growing seasons from some commericial fields and wheat rust trap nurseries grown in eight governorates of Egypt that is,Beheira, Dakahliya, Gharbiya, Minufiya, Sharqiya, Domiatta, Qalubiya and Bani Sweif (Table 2). Samples were kept at room temperature (18 to 24°C) overnight in order to dry the moisture content associated with samples. Samples were kept in glassine envelopes (8 x 15 cm) and stored in the refrigerator at 2 to 5°C until using in isolation. Isolation, purification and spore multiplication The susceptible cultivar Morocco was planted as ten seeds per 10 cm diameter plastic pots in the greenhouse of Wheat Diseases Research Department, Plant Research Pathology Institute, ARC, Egypt. When first leaf fully expanded in seven days old seedlings, it rubbed gently between moist fingers with tap water then infected samples were scraped using sterile spatula and transferred to these seedlings and sprayed gently again with water in order to form a film of free water which is essential to initiate spore germination and establishment of infection. Finally, the inoculated seedlings were incubated in moist chambers for 24 h at 18 to 20°C and 100% RH then moved onto the benches in a greenhouse and kept for 14 days at approximately 20 ± 2°C. After pustules rupture, three single pustules were isolated separately from each specimen for multiplication on the highly susceptible variety Morocco to obtain enough urediniospores for identification as described by Stakman et al. (1962). Race identification The North American race nomenclature system used to design the leaf rust races in a letter code was adopted by Long and Kolmer (1989) and McVey et al. (2004) including five sets with 20 differential monogenic lines each with single gene for leaf rust resistance (Table 1). These lines were grown in 6 cm square plastic pots each with seeds of four lines, planted in the corners of each pot in clockwise order. After seven-days growing seedlings were inoculated with the previously isolated single pustule isolates of P. triticina, by shaking. The inoculated seedlings were incubated in the humid chamber overnight (100% RH), as described above. The inoculated seedlings were transferred also, onto the greenhouse benches. Infection type data for all monogenic lines were assessed 14 days after inoculation using standard disease scoring scale 0-4 (Stakman et al., 1962). Entries which showed low infection types (LITs) (scores = 0, 0, 1, and 2) were considered host resistant and a virulent isolate, while those with scores = 3 and 4 were susceptible (high infection types, HITs) and virulent isolate. Each single isolate was assigned five letter virulence phenotype description based on high or low infection type to the differentials lines (Stakman et al., 1962;Kolmer, 1989 andMcIntosh et al., 1995). Virulence frequency Percentage of virulence frequency was calculated as a number of virulent isolates to the total number of the tested isolates. Cluster analysis A similarity matrix of virulence phenotypes based on the simple matching coefficient was used to construct a dendogram using the unweighted pair group method with arithmetic means clustering method in numerical taxonomy system (NTSYS-pc version 2.1) according to Rohlf, (2000). RESULTS During the three successive growing seasons that is, 2011/2012, 2012/2013 and 2013/2014 The race code consists of the letter designation for the pattern of ITs for the Puccinia triticina isolate on differential set 1 followed by set 2, then set 3, set 4 and set 5. For example, race MGBLQ is virulent on the Lr 1 and Lr 3a differntials in set 1, Lr 16 in set 2, none in set 3, Lr 10 in set 4 and Lr 14b and Lr 15 in set 5. b L = low infection type (avirulent isolate); H = high infection type (virulent isolate). 2013/2014. Moreover, the highest number of isolates was during season 2012/2013 followed by seasons 2011/2012 and 2013/2014. Therefore, the highest total of collected leaf rust samples and isolates were in Sharqiya (36 and 91, respectively) followed by Beheira (33 and 73, respectively), while Domiatta (3 and 6, respectively) was the lowest one (Table 2). Race identification A total of 234 physiologic races were identified during the three growing seasons 2011/2012 to 2013/2014 from 345 isolates based on infection types on the 20 differential monogenic lines (Table 3). No of the identified physiologic races were found in the three seasons. Moreover, in 2011/2012, the most common races were STTST (2.54%) and TKTTT (2.54%). While in 2012/2013, races PKTST (6.63%), TTTTT (7.83%) and TTTST (10.24%) showed high frequencies. On the other hand, in 2013/2014 the most dominant and common races were FKTTT (4.92%) and PTTTT (11.47%). Most identified races during the three growing seasons were rare and represented by a single isolate. A total of 100 and 49 races in 2011/2012 and 2013/2014, respectively represented by a singleisolate comprising 89.28 and 96.07%, respectively of the total races. While, in 2012/2013 a total of 67 races appeared and showed single isolate and comprised 81.70% of the total races. Similarity of identified race groups in different locations To display the relationships between leaf rust populations in different geographic wheat locations and similarity based on percentage frequency of virulence race groups in eight locations were illustrated in Figure 1. As indicated in cluster analysis of similarities, the studied locations in 2011/2012 growing season formed two main clusters. The first divided into two additional subclusters. The first included three locations that is, Dakahlia, Gharbiya and Qalyubia. Meanwhile, the second included only one location that is Sharqiya. On the other hand, the second cluster included two sub-clusters; the first included Minufiya, BaniSweif and Beheira and the second included only Domiatta ( Figure 1A). In 2012/2013 growing season, the similarity of the studied locations divided into two main groups. The first group included seven locations from a total of eight locations. This cluster divided into two sub-clusters; the first included Beheira, BaniSweif, Minufiya, Qalyubia, Gharbiya and Sharqiya and the second contained only Dakahlia. Moreover, the second group cluster included only Domiatta ( Figure 1B). In 2013/2014 growing season, dendrogram of similarity showed differences in race groups frequency. The cluster analysis in this growing season divided into six clusters; the first included Beheira, Qalyubia and Minufiya, second included Bani Swear. The third included Sharqiya. The fourth included Domiatta. The fifth included Dakahlia and the sixth included Gharbiya ( Figure 1C). Virulence frequencies: Tests for virulence of identified race groups indicated that the leaf rust resistance genes Lr 1, Lr 2c,Lr 3,Lr 16,Lr 24 and Lr 26 were susceptible against most race groups tested during the studied three seasons of study (Table 5). While, the leaf rust monogenic lines Lr 2a and Lr 9 showed different reactions against the tested race groups. They proved to be active against some identified race groups and inactive against others. On the other hand, race group TT---was the most virulent pathotype to eight leaf rust monogenic lines that is, Lr 1, Lr 2a,Lr 2c,Lr 3,Lr 9,Lr 16,Lr 24 and Lr 26 followed by race groups ST---and PT--. While, race group BB---was the least virulent one (avirulent to all tested leaf rust monogenic lines) followed by race group LB---which was virulent to Lr 1 only. ( were FKTTT and PTTTT. Similar results were obtained by Kolmer (1999) who reported that races MCRK, MBRJ, MBRK and TLGF were the most common phenotypes in 1997. While, in 1996 MBRJ was the most common phenotype. Moreover, Negm et al. (2013) found that the two races PTTT and TTTT were the most common in the two growing seasons 2009/2010 and 2010/2011. These two races were comprised 12. 21 and 15.84%, respectively in 2009and 10.90 and 12.80%, respectively in 2010/2011. Moreover, McVey et al. (2004 found that race MCDLQ was the most common race in Egypt, also races MCDLL and TCDML were found in Israel. While, races BBBLL, MBBLL and MBDLQ were found in Turkey as well as in Egypt but the frequencies of these races in Egypt never exceeded 1.2%. Also, race MBBLL was found in Sudan in 1998. In the present study, a total of 112, 82 and 51 pathotypes were appeared in Egypt during 2011/2012, 2012/2013 and 2013/2014 growing seasons, respectively. This is likely due to the differences in number of sampling between the three seasons. In 2011/2012 growing season, the collected samples were fifty, in 2012/2013, sixty five and thirty three in 2013/2014. It is likely a greater number of identified races will be detected in 2013/2014 if the collected samples had been obtained from a larger number of sites. McCallum et al. (2011) found that the most common races in Canada during 2008 growing season were TDBJ (23.6%), TDBG (23.1%) and MLDS (18.9%). Moreover, Hanzalova and Bartos (2014) found that the leaf rust races 14, 77, 61, 53 and 2 were dominated in Czechoslovakia/Czech Republic in the years 1966-2001. Finally, Anjum et al. (2014) identified the two leaf rust races 12-9 (93R37-1) and 77-11 (125R28) in India during 2009 growing season. DISCUSSION Frequencies of race groups based on IT's of the first two sets of leaf rust North American race nomenclature system (Long and Kolmer, 1989) of wheat leaf rust differential monogenic lines were compared in eight governorates in Egypt. Race groups PT---and TK---were the most dominant race groups, which were found at all eight governorates during the studied three growing seasons. In contrast, race group LB---was found at only Sharqiya governorate, followed by race group FK---found in also two locations that is, Minufiya and BaniSweif and race group LS---found in two locations that is ,Beheira and Minufiya. Regardless of the presence of leaf rust races during the three seasons, which they appeared in some governorates and disappeared in others were mainly due to variations over locations and weather conditions. Moreover, as the prevalence of races in a specific season and region depends on the type of wheat cultivars grown and to some extent on the predominant environmental conditions, especially temperature . Also, due to the long-distance dispersal of leaf rust races (intercontinental migration) (Burdon and Silk, 1997;Kolmer, 2005;Hanzalova and Bartos, 2014). This process led to the shifts in the genetic structure of the recipient pathogen population, especially when such pathogen cannot survive the summer in the country (Saari, 1976;Burdon and Silk, 1997). Nazim et al.(2003Nazim et al.( , 2010 suggested that wheat leaf rust urediniospores cannot survive the summer in Egypt because the summer temperatures are very high and cannot permit such spores to survive. Similar results were obtained by McVeyet al. (2004) they suggested that the common races in Egypt also found in Romania and Israel indicated that windborne urediniospores of P. triticina frequently move between Egypt and Israel and some inoculum may come to Egypt from Eastern Europe. On the other hand, the contribution of races from either Sudan or Turkey to the population of P. triticinain Egypt in 1998 to 2000 is very small. Negm et al. (2013) found that race groups TT--and PT--were common in ten governorates of Egypt that is, Domiatta, El-Beheira, El-Dakhlia, El-Nubaria, El-Sharkia, El-Suez, Kafr El-Sheikh, BaniSweif, El-Qalubia and Sohag. Also, Soliman et al. (2012) found that race groups BB--and PK--were the most common race groups in five governorates of Egypt that is, Dakhlia, Kafr El-Shekh, Beheira, Sharqia and Sohag. Similarity between leaf rust populations in different locations under study showed that in 2013/2014 growing season there are great differences between race groups frequency so the dendrogram of the tested race groups was divided into six clusters. This probably caused by the low number of collected field samples in this growing season.These results are in agreement with Negm et al. (2013) found that in 2009/2010 growing season dendrogram of similarities based on frequency of virulence of race groups divided into two clusters; the first included ten locations that is Gharbiya, Minufiya, Sohag, Nubariya, Sharqiya, Suez, Domiatta, Beheira, Dakahlia and Kafer El-Sheikh. Meanwhile, the second cluster included the two locations BaniSweif and Qalyubia. On the other hand, in 2010/11 growing season the tested locations clustered divided into two main clusters. The firs cluster included BaniSweif, Sohag, Nubariya, Gharbiya, Minufiya, Qalyubia, Sharqiya, Kafer El-Sheikh, Domiatta, Beheira and Dakahlia. While, the second main cluster included Suez only. Virulence against leaf rust resistance genes showed that Lr 1, Lr 2c, Lr 3,Lr 16, Lr 24 and Lr 26 genes were susceptible, while, Lr 2a and Lr 9 showed different reactions against the tested race susceptible to most tested race groups. Also, Negm et al. (2013) found that Lr 3, Lr 16,Lr 24 and Lr 26 were ineffective against most race groups tested during 2009/2010 and 2010/2011 growing seasons. While, Lr 1,Lr 2a,Lr 2c and Lr 9 showed different infection types (IT) against the tested race groups. Conclusion Survey of wheat leaf rust pathotypes using leaf rust North American differential lines is very important in describing virulence variation, geographical distribution of virulence pathotypes and how leaf rust pathotypes change in response to host selection. This activity should be execute in all wheat growing seasons using rust survey and planting of wheat rust trap nurseries at all Egyptian governorates including rust hot spot locations. This will ultimately provide timely warning to wheat breeders about the change in virulence of P. triticina pathotypes. Thus, it will be very important to avoid future leaf rust epidemics and reduced annual losses of the commercial wheat cultivars grown in Egypt.

v3-fos

2018-12-18T09:05:32.943Z

2015-11-09T00:00:00.000Z

58902742

s2

The Application of Polymerase Chain Reaction – Restriction Fragment Polymorphisms (PCR-RFLP) to Determine Genetic Diversity of Madura Cattle in Sapudi Island The aim of this study was to determine genetic diversity of Madura cattle using Polymerase Chain Reaction – Restriction Fragment Length Polymorphisms (PCR-RFLP) analysis of the cytochrome b (cytb) gene. Samples used for the experiments were blood of 43 cattle that consist of 15 cattle obtained from Madura Island, 23 cattle from Sapudi Island, and 5 Limousin-Madura (Limura) cattle. A fragment of 464 base pair of cytb gene was amplifi ed by forward primer L14735 and reverse primer H15149. The PCR product was digested with TaqI and HinfI restriction enzymes to identify genetic patterns. Data of PCR-RFLP showed two haplotypes, that were A and B, in cattle obtained from both Madura Island and Sapudi Island. The frequencies of haplotype A and B of cattle from Sapudi Island were 69.57% and 30.47%, respectively. More diverse frequencies were observed in cattle obtained from Madura Island, where haplotype A and B were 86.67% and 13.33%, respectively. In this experiment, Limura cattle had only haplotype A. As a conclusion, PCR-RFLP of the cytb gene had been able to determine a genetic diversity of Madura cattle. Introduction Madura cattle are local cattle used to be work animals, bull race (Karapan), art contest (Sonok) and beef cattle. Madura cattle have uniform in shape and are stable inbred hybrid between Zebu (Bos indicus) and Banteng (Bos javanicus). The uniformity of the breed was developed through selection for many years by local people in Madura. Physically, the color of the cattle is reddish brown with nonspecifi c white pattern on the back and rump. In 2002, the population was estimated of around 900,000 cattle. Efforts are now being made to conserve cattle breed on Sapudi Island, as this is a conservation place for Madura cattle, where only the original Madura cattle are allowed to live in that area. Non-Madura cattle from outside Sapudi Island are not permitted to entry and live in this area (Umar et al, 2011;Widi et al, 2010) Cattle variety is configurated from native cattle with that of genetically-imported resources. Madura cattle are a combination of Bali-Ongole-Java native composite breed, developed from Bali (Bibos banteng), Javanative (Bos javanicus) and Ongole (Bos indicus). Nijman et al. (2003) reported that DNA of Madura cattle are mixture of DNA from both Zebu cattle and Banteng. Meanwhile, crossbreeding between Limousin bull and Madura cows by artifi cial insemination (AI) has produced Limura cattle. The exterior characteristic of Limura cattle show diversity on phenotypes as this was produced by a combination of both parental breed. Mitochondrial DNA (mtDNA) is an extra-nuclear DNA that has been proven to be a useful molecular marker for evolutionary studies in animal populations due to its predominantly maternal inheritance, relatively rapid base substitution rate, and lack of recombination (Avise et al. 1987). Mitochondrial DNA contains more sequence diversity compared to that of nuclear DNA (Brown et al., 1996). Analysis of Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) has been successfully applied to amplify the target sequence. Nijman et al. (2003) employed PCR-RFLP technology to analyze hybridization between banteng (Bos javanicus) and Zebu (Bos indicus) in Southeast Asia using mitochondrial DNA and to identify the nucleotide polymorphisms in the 359 base pair of mitochondrial gene cytochrome b (Cytb) Iberian cattle (Bos taurus) (Prado et al., 2005). Cytochrome b gene of several vertebrates, including mammals, had been used to investigate evolutionary and genetic diversity and molecular phylogenetic studies (Wolf et al., 1999). In this study, Cytb gene was employed to evaluate genetic diversity of Madura cattle in Sapudi Island. Blood samples and DNA extraction Blood samples was collected from 43 cattle, consist of 15 Madura cattle from Madura Island, 23 Madura cattle from Sapudi Island, and 5 Limura cattle. Samples were prepared for DNA isolation by using a KIT high pure PCR template preparation (ROCHE) according to the protocol. Ear tissues were prepared by using standard SDS/ProteinaseK method according to Sambrook et al. (1989). Polymerase Chain Reaction (PCR) A fragment of 464 base pair of cytochrome b gene was amplifi ed using both forward primer of L14735 (5'-AAA AAC CAC CGT TGT TAT TCA ACT A-3') and reverse primer of H15149 (5'-GCC CCT CAG AAT GAT ATT TGT CCT CA-3') (Wolf et al., 1999). PCR was carried out in a fi nal volume of 20 μl reaction mixture containing 1 μl sample DNA (10-100 ng), 1 μl each primer, 10 μl PCR KIT (Fastart, Roche), and 7 μl aquabidest. The amplifi cation process was performed using Thermocycler (Infi nigen, TC-25/H). Amplifi cations were performed under the following conditions: initial denaturation at 94°C for 2 min, followed by 35 thermal cycles of denaturation at 95°C for 36 sec, annealing at 51°C for 73 sec, and extension at 72°C for 84 sec. The fi nal extension was performed at 72°C for 3 min (Prado et al., 2005). PCR products were visualized on 1% agarose gels buffered with 1X Tris-Boric-EDTA (1X TBE), stained with ethidium bromide, and visualized under UV light. To get the DNA sequence of PCR product, we performed DNA sequencing by Macrogen. Restriction Fragment Length Polymorphism (RFLP) PCR products was digested by restriction enzymes as described previously (Verkaar et al., 2002). The 464 bp amplicon was digested with TaqI and HinfI restriction enzymes to identify genetic patterns. A volume of 3 μl PCR product was digested with 1U HinfI enzyme in Tango buffer at 37ºC for 3 h (Fermentas) and followed by digestion with 1U Taq I enzyme at 65ºC for 2 h. The digestion product was separated on 12% polyacrylamide gels at 50 V for 3 h. The gel was stained gently by ethidium bromide and visualized under UV light. The size of DNA fragments were compared to the DNA marker ΦX174 DNA/BsuRI (HaeIII), (Fermentas). Results and Discussion To obtain genetic patterns, the amplifi ed 464 base pair of DNA fragment of the Cytb gene from all of samples collected from 43 cattle was subjected to RFLP analysis. The data showed that there were two haplotypes observed in cattle obtained either from Madura Island and Sapudi Island, while the Limura cattle had only haplotype A (Figure 1 and 2). The amplifi cation of both haplotypes with mitochondrial-specifi c primers L14735 and H15149 revealed the presence or absence of restriction sites for HinfI and TaqI enzymes. Using HinfI as restriction enzyme, haplotype A was observed as shown by two bands of 305 and 159 base pairs and haplotype B was observed as shown by three bands of 198, 149 and 117 base pairs (Figure 1). Meanwhile, using Taq1 as restriction enzyme, haplotype A was observed as shown by two bands, namely 225 and 191 base pairs, and haplotype B was observed by one band of 416 base pair (Figure 2). The specific haplotype of Madura cattle was shown in Table 1.The frequencies of haplotype A and haplotype B of cattle obtained from Sapudi Island were 69.57% and 30.47%, respectively. Those frequencies were less diverse then that obtained from cattle of Madura Island where haplotype A and B were 86.67% and 13.33%, respectively. This data suggests that cattle from Sapudi Island were genetically closer compared with that in Madura Island. We also observed that 100% of Limura cattle had only haplotype A ( Table 1). The RFLP data reported here were similar with that of previous experiment (Wolf et al., 1999), suggesting the accuracy of PCR-RFLP in determining genetic patterns. Wolf et al (1999) suggested that to distinguish between species, amplifi ed bands of specifi c PCR products were digested with different restriction endonucleases enzymes, and his could resulting in a species-specifi c RFLP. They proved that even closely related animal species was able to be distinguished by application of one or two restriction endonucleases enzymes. The differences on haplotype frequencies indicated a genetic diversity. In this study, cattle in Sapudi Island had only a single species that were divided into two haplotype patterns. Interestingly, PCR-RFLP of Cytb gene of mtDNA using Hinf I and TaqI enzymes could potentially be used as molekuler (Figure 3 and 4). Of the nucleotide polymorphisms observed, included were differences from T to C (12 sites), from C to T (12 sites), from A to G (5 sites), from C to A (2 sites), and from G to A (1 site) ( Figure 3). Meanwhile, Figure 4 showed amino acid differences as a consequence of differences on nucleotide sequences. As can be seen in Figure 4, amino acid sequence number 18 had changed from Serine (TCA) to Proline (CCA), meanwhile amino acid number 22 had also changed from stop codon (TGA) to Tryptophan (TCC). Another amino acid differences were also observed in sequence number 31 from Isoleucine (ATC) to Valine (GTA), and sequence number 50 from Serine (TCC) to Proline (CCC) (Figure 4). Differences on both nucleotides and amino acid sequence strongly suggesting any genetic diversity in cattle from Sapudi Island. As this island is restricted area for any other cattle breeds, the less genetic diversity of cattle observed is of understandable. In this case, Sapudi Island is one of best practice in conserving genetic of Madura cattle in Indonesia.

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Identification , in vitro establishment and preliminary phytochemical analysis of wild yam ( Dioscorea spp . ) used for medicinal purposes Wild tubers of the genus Dioscorea sold for medicinal use were collected for the purpose of achieving its establishment under in vitro conditions. First we taxonomically identified the species and through phytochemical analysis demonstrated pharmaceutical potential. The material collected was identified as Dioscorea coriacea, D. lehmannii, D. meridensis, D. polygonoides and the edible species D. trifida. Tubers collected from wholesale distributors and from the field were washed, disinfected, sprayed with Gibberellic Acid (GA3) and planted in substrate BM-2®, in a greenhouse at 18 ° C during the day and 10 ° C overnight. Whole tubers or sections thereof were stored in sealed bags at room temperature. Subsequently plant material of the species D. coriacea, D. lehmannii, D. meridensis and D. polygonoides was disinfected and healthy buds (D. coriacea / laboratory) were selected for in vitro establishment. Three different culture media were evaluated for establishment; that which presented the best results was the Murashige & Skoog (1962) medium, supplemented with BAP 1 mL / L, GA3 1 mL / L and Putrescin 2 mL / L. For the collection and analysis of secondary metabolites, tubers of D. coriacea, D. lehmannii and D. polygonoides were used, using methanol as the extraction solvent. The highest concentration of plant ex* Grupo de Investigación sobre el Cultivo de Ñame. Instituto de Biotecnología Universidad Nacional de Colombia – Sede Bogotá. [email protected], [email protected], [email protected] ** Grupo de Investigación en Fitoquímica y Farmacognosia. Facultad de Farmacia. Universidad Nacional de Colombia – Sede Bogotá. [email protected], [email protected] *** Instituto de Ciencias Naturales. Facultad de Ciencias. Universidad Nacional de Colombia – Sede Bogotá. [email protected] ARTÍCULO DE INVESTIGACIÓN Rev. Colomb. Biotecnol. Vol. XVII No. 1 Junio 2015 9-17 9 10 Rev. Colomb. Biotecnol. Vol. XVII No. 1 Junio 2015 9-17 Introducción El género Dioscorea agrupa a 600 especies de plantas herbáceas trepadoras, lianas o bejucos, distribuidas mayormente alrededor de los trópicos. Numerosas especies silvestres sirven de alimento en diferentes países en África, Asia, Latinoamérica y Australia, sobre todo en tiempos de carestía o pobreza. Otras son usadas en Asia, Europa, Norte y Centroamérica con fines medicinales, para tratar el reumatismo, cólicos y espasmos intestinales, dolores o como abortivas. Algunas se emplean en fitoterapia y en homeopatía, y popularmente como piscicidas, pediculicidas, insecticidas, para elaborar champús, jabones, o para envenenar flechas para la cacería (Waizel, 2009). Los ñames en los Estados Unidos son en realidad batatas, por esta razón, el Departamento de Agricultura de los EEUU requiere que la etiqueta “yam” siempre sea acompañada por la de “sweet potato”, los ñames verdaderos sí son especies de Dioscorea (USDA, 2012). Los ñames verdaderos han sido comúnmente usados en la industria farmacéutica, para tratar condiciones tan diversas como la inflamación, dolor en articulaciones, diabetes, infecciones y la dismenorrea. Los componentes farmacológicamente activos de las especies de Dioscorea incluyen diosgenina, que es una saponina esteroidal, y dioscina, una forma de diosgenina con azúcares. Entre las diversas metodologías utilizadas para micropropagación de ñame se encuentra el cultivo de meristemos, efectivo para la eliminación de infecciones virales y la conservación del germoplasma. Otro de los mecanismos utilizados para la propagación es la microtuberización, que permite la conservación de material genético básico de clones madres libres de enfermedades en un espacio reducido sin la necesidad de hacer operaciones costosas para recolectar ñame en el campo. (Perea & Buitrago, 2000) El Grupo de Investigación sobre el cultivo de ñame del Instituto de Biotecnología (IBUN) de la Universidad Nacional de Colombia, enfoca sus investigaciones tanto a ñames silvestres como a especies y variedades cultivadas, trabajos que han desarrollado metodologías de cultivo en condiciones in vitro, generando conocimiento sobre un cultivo considerados huérfano (FAO 2006), a nivel nacional e internacional, bajo un enfoque claro de incidir favorablemente en la calidad de vida de los productores de ñame de Colombia. Este trabajo buscó establecer las condiciones de cultivo in vitro de especies de ñame, silvestres y comercializadas en plazas de mercado de Bogotá, contribuyendo de esta manera a futuros trabajos con estas especies al garantizar material vegetal disponible. Como complemento, se propuso identificarlas taxonómicamente y realizar un primer análisis fitoquímico para identificar su potencial fitoterapéutico. Materiales y métodos Selección del material Tubérculos conocidos como zarzaparrilla fueron adquiridos en centros de acopio de la ciudad de Bogotá, por secciones y completos, adicionalmente, se contó con material donado (tubérculos con tallos, hojas y raíces) por el Instituto de Ciencias Naturales y el Grupo de Investigación en Fitoquímica y Farmacognosia de la Universidad Nacional, y material colectado en diferentes municipios del departamento de Cundinamarca. Estudio y determinación de la especie Para la determinación de la especie de los tubérculos recolectados en su hábitat natural provenientes de campo, se tuvo en cuenta caracteres morfológicos que definen las características del género y la especie, tomando como referencia las colecciones en el Herbario Nacional Colombiano (COL) y la literatura botánica (Knuth, 1924). Se registró altitud, localidad, descripción de la planta, y especie. Para los demás tubérculos, se reportaron los caracteres morfológicos que definen las características de las especies del género Dioscorea spp., color y diámetro del tallo tanto joven como maduro, presencia o no de espinas en la base o en todo el tallo, presencia o ausencia de tubérculos aéreos, forma del tubérculo subterráneo así como su anchura, color de la epidermis, color de la parénquima, presencia o ausencia de grietas en su superficie y propiedades organolépticas del tubérculo (IPGR/IITA, 1997). Adaptación de ñame silvestre en invernadero Los tubérculos adquiridos en centros de acopio fueron previamente lavados con detergente y agua y sembrados en macetas, previamente se les aplicó Ácido Giberélico (AG3) por aspersión para romper la dormancia. Tubérculos donados en secciones que se encontraban en bolsas herméticas con desarrollo de brotes, tract, 54%, was found in D. coriacea, a higher value than that of D. polygonoides, which had been reported previously; the presence of saponins was confirmed by thin layer chromatography (TLC). These results will enable more advanced analysis of the present compounds and enhance their mass propagation under in vitro conditions. Introduction The Dioscorea genus groups together 600 herbaceous, climbing, liana or reed plant species, mostly distributed around the tropics. Numerous wild species of this genus serve as food for different countries in Africa, Asia, Latin America and Australia, primarily in times of shortage or poverty. Others are used in Asia, Europe and North and Central America for medicinal purposes to treat rheumatism, intestinal spasms and cramps, and pains, or as abortifacients. Some are used in phytotherapy and homeopathy, and popularly as piscicides, pediculicides and insecticides; to make shampoos and soaps; or to poison arrows for hunting (Waizel, 2009). Yams in the United States are actually sweet potatoes and therefore, the US Department of Agriculture requires that the "yam" label is always accompanied by the "sweet potato" label. Real yams are a species of Dioscorea (USDA, 2012). Real yams have been commonly used in the pharmaceutical industry to treat greatly diverse conditions, such as inflammation, joint pain, diabetes, infections and dysmenorrhea. The pharmacologically active components of the Dioscorea species include diosgenin, which is a steroid saponin, and dioscin, a form of diosgenin with sugars. The diverse methods for yam micropropagation include the cultivation of meristems, effective for the elimination of viral infections and the conservation of germoplasm. Another one of the mechanisms used for propagation is microtuberization, which allows the conservation of basic genetic material of mother clones free from disease in a reduced space without the need for costly operations of collecting yam in the field (Perea & Buitrago, 2000). The Research Group on Yam Cultivation of the Biotechnology Institute of the Universidad Nacional de Colombia (IBUN, for the Spanish original) focuses its research on wild yam, as well as on farmed species and varieties. This work has developed methodologies for in vitro cultivation, generating knowledge on crops considered to be orphans (FAO 2006), nationally and internationally, with a clear approach to have a positive impact on the quality of life of yam producers in Colombia. This work aimed to establish the conditions of in vitro cultivation of wild and commercialized yam species in Bogotá marketplaces, contributing to future work with these species to ensure availability of vegetable ma-terial. Additionally, their taxonomic identification was proposed, as well as a first phytochemical analysis to identify their phytotherapeutic potential. Selection of Material Tubers known as sarsaparrille were acquired at collection centers in Bogotá in sections and as complete tubers. Additionally, there was material donated (tubers with stems, leaves and roots) by the Natural Sciences Institute and Research Group on Phytochemistry and Pharmacognosy of the Universidad Nacional, and material collected in different municipalities of the Cundinamarca department. Study and Determination of the Species To determine the species of tubers collected in their natural habitat from the field, the morphological features that define the characteristics of the genus and the species were considered, using the Herbario Nacional Colombiano (National Colombian Herbarium -COL) and botanical literature as a reference (Knuth, 1924). The altitude, location and description of the plant and species were recorded. For the other tubers, the morphological features that define the characteristics of the species of the Dioscorea spp. genus; the color and diameter of the young and mature stem; the presence or absence of thorns at the base or throughout the stem; the presence or absence of stem tubers and the shape of the root tuber, as well as the width, epidermis color, parenchyma color, presence or absence of cracks in their surface and organoleptic properties of the tubers were reported (IPGR/IITA, 1997). Adaption of Wild Yam in the Greenhouse The tubers acquired in collection centers were previously washed with detergent and water and planted in flowerpots. Gibberellic Acid (GA3) was previously applied to them by spraying to break dormancy. Tubers donated in sections that were found in airtight bags with the development of shoots were washed and disinfected for establishment. Tubers from the field were washed and stored in airtight bags for one month to break dormancy and then planted in the greenhouse. Statistical Analysis To establish the environment with the best results and to verify the significant differences, an analysis of variance, ANOVA, was conducted with a level of significance of α = 0.05, using the Windows® Statistix program. Selection of Material The material collected in the marketplaces was taxonomically identified as D. lehmannii, and the tubers donated in sections were identified as D. coriacea and D. meridensis. Tubers collected from the field were identified as D. coriacea, D. polygonoides and D. lehmannii. D. trifida tubers came from the Chocó Region, from a marketplace in Quibdó. D. polygonoides tubers were only used to obtain an extract for reference. The D. coriacea material corresponded to the tuber donated by the laboratory of the Research Group on Phytochemistry and Pharmacognosy of the Universidad Nacional. The Dioscorea spp. tubers were purchased at marketplaces. Prevention and relief of illnesses related to the blood system, by purification or reconstruction, are attributed to them. However, different species of tuber are offered under the same name of sarsaparrille, which generates uncertainty about the attributed therapeutic effect. The collected material is presented in Figure 1 and Table 2. Pinzón (2011) recorded 72 medicinal plant stands in 26 points of sale distributed among 12 of the 20 locali-Once washed with plenty of soap and water, the tubers were planted in flowerpots with BM2® (mix for germination) and sand (2:1), taken to the greenhouse, watered every two days, and kept at a temperature of 20 °C daytime, 10 °C nighttime. The planted tubers were checked daily and the shoots and roots were checked weekly, recording at 8, 15, 30 and 60 days. At 30 days, they were kept at room temperature (approximately 18 °C daytime and 10 °C nighttime). Introduction of Vegetable Material to in Vitro Conditions Nodal cuttings were each cut at 2 cm and washed with plenty of water and Extran®. Then they were submerged in a solution of Tween 20.8 ml / 100 ml of sterilized distilled water, Isodine® 3.5% for 30 minutes, which has the active ingredient of povidone-iodine, and in sodium hypochlorite 2.5% for 15 minutes. They were rinsed three times with sterile distilled water between each step for 5 minutes. In Vitro Explant Culture Single-node, 15 mm-long segments without leaves from the stems of the donor plants were used. The explants were planted individually in 20 ml of medium per container, 12 containers per treatment were used, and the contamination was assessed at 3, 5, 8 and 15 days from planting. The following media were used. Extraction and Analysis of Secondary Metabolites The collected tubers were washed, cut and dried in a heater with forced air circulation for 48 hours. Subsequently, they were ground in a hammermill and the extraction was carried out with methanol by exhaustive percolation. The solvent was withdrawn from the extract using a rotary evaporator connected to a vacuum pump at 38 o C. Once the dry extract was obtained, each one of the extracts was characterized by the thinlayer chromatography (TLC) technique on silica gel 60 GF254 using the following mobile phases: i. Methanol: Chloroform: Acetic acid. 6:6:1, ii. Ethyl acetate: Formic acid: Glacial acetic acid: Water. 100:11:11:27, ties of Bogotá D.C.; primary and secondary markets and individual stands, with D. coriacea and D. meridensis (62-60%) being the species with the greatest supply in Bogotá. D. lehmannii was also acquired in several of the city's points of sale. When the tubers were put into plastic bags, they abundantly sprouted, with the D. coriacea species ( Figure 1f) being the one that developed the greatest number of shoots. Hata et al. (2003) reported similar results, recording dormancy in the period between harvest of the tubers and storage of the D. rotundata tuber material in airtight bags. A greater content of sapogenins and presence of diosgenin was obtained from this material, inferring that the dormancy phenomenon is related to an increase in the content of some sapogenins, possibly because when consuming the tuber's starch to form stems, the proportion of sapogenins increases with respect to the amount of dry material. The tubers previously placed in airtight bags presented nodal shoots (T1, T5, T6 and T7). As was to be expected, the tubers planted in BM2® and sprayed with GA3 produced abundant roots (T2, T3 and T4), due to the action of the gibberellins in activating the metabolism, protein synthesis and mobilization of reserve substances to generate the elongation of the germ or in this case, the formation of roots. Tubers of the D. lehmannii and D. trifida species (T2, T3 and T4), which only produced roots and no shoots, were not used in the trials because the material, disinfection designs and establishment were for nodal shoots. The action of GA3 is non-toxic for humans or animals and it is easy to manage. The techniques may vary from immersion of the tuber or spraying. This depends on the variety and state of rest in which the tubers are found, causing sprouting in many varieties when the rest period has almost finished. For immersion, Marca (1997) suggests that the recently harvested tubers be washed and infected, air dried and later submerged in a solution of GA3 (5 ppm) for 10 minutes. After the treatment, the tubers must be dried and placed in a chamber or warm environment between 18 °C and 25 °C to induce sprouting. After 15 days, there is abundant sprouting. Study and Determination of the Species Identified species from the field were recorded according to their characteristics (see Materials and Methods), and compared with specimens of the Dioscorea genus in the National Colombian Herbarium (See Table 3). Information about the distribution of the species was consulted in the herbarium's database (www.biovirtual.unal.edu.co). The majority of the Dioscorea species is from tropical latitudes with a few exceptions in North America, Europe and East Asia. Wilson (1977) includes the Asiatic species D. japonica and D. opposita as tolerant to frost. However, for D. alata and edible tropical species, temperatures under 20 °C restrict growth, which is promoted with temperatures between 25 °C and 30 °C, limited by the range of latitudes between 20 ° N and 20 ° S and maximum altitudes of 1,000 m.a.s.l. It can be inferred that wild Dioscorea species bear significant differences to edible species in their conditions of adaptation, with the content of diosgenin or saponins derived from it being an important factor. This is proportional to the consumption of starch by the tuber, which is higher in agroecological conditions of forests, subpáramos and páramos, where the wild species that generally grow without much upkeep from farmers are found. Adaption of Wild Yam All of the D. lehmannii tubers (T2 and T3), wild species of Dioscorea brought from the field (T1) and D. trifida tubers (T4) were planted in the same conditions, presenting slower sprouting of roots. In the planting conditions, the tubers simultaneously presented shoots and roots. The material donated by the laboratory was planted in the greenhouse just like the sections of different species donated by the Natural Sciences Institute (T5 and T6). Figure 2 shows that just the tubers previously placed in airtight bags produced shoots, and those sprayed with GA3 produced roots, which was not favorable for planting. It is possible that the humidity generated in the bags favors the growth of foliage because of the high respiratory rates, and that the absence of this humidity phase with the addition of GA3 generates roots and not shoots because of the yam phases designated for rest, vegetable growth and reproductive growth. The sprouting of tubers indicates the end of the rest period and the start of the vegetative phase, characterized by rapid growth of the stems and leaves. Haynes et al. (1967) indicated that for D. alata, the reproductive phase starts when tuberization reaches an exponential rate of growth, which coincides with a reduction of the growth of stems and leaves. The yam's reproductive phase coincides with flowering followed by the maturity of the tubers. The tubers treated with GA3 produced roots. Taking into account that the type of explant required was shoots or buds, they were not used for the planting stage. Additionally, the tubers cut into sections and stored in bags produced better results, reflected in greater sprouting. This coincides with Fergusson (1977), who used sections of D. alata tubers with less time required for the growth of the tubers. These results permit the interpretation that this phase is characterized by accelerated cellular division and expansion of cells that will be later involved in microtuberization. Extraction and Analysis of Secondary Metabolites The Petri dishes revealed that all the samples contain sapogenins, the color and formation of foam was a qualitative indicator, and the strips revealed greater concentration in the samples that presented a stronger color. The extraction solvent was methanol, which presented a greater proportion of polar substances. The mobile phase that allowed a greater profile of the extracts to be obtained is formed from a mixture of ethyl acetate: Methanol: Formic acid: Glacial acetic acid 100: 27: 11:11, a mobile phase frequently used to detect flavonoids. (See Figure 4). With the different chromatographic Petri dishes, it could be established that there is a group of high polarity organic compounds present in the extracts of D. coriacea, D. lehmannii and D. meridensis, and it could be confirmed that sapogenins are present in all the evaluated species, due to the formation of foam as a qualitative indicator. The thin-layer chromatography showed more polar sapogenin compounds than those present in D. polygonoides, which allowed a high degree of glycosylation to be inferred. As Hata and collaborators (2003) had already observed, when analyzing the metabolites present in D. rotundata, the concentration of sapogenins obtained for each sample is related to the number of sapogenins detected, concluding that there is a correlation between both results. In other words D. rotundata presented a greater number of sapogenins and a greater concentration of these compounds. The results of the extraction (Table 4) showed that the greatest yield was obtained from D. coriacea with a yield of 54% and 21% for each sample processed, followed by D. polygonoides and D. lehmannii. The Petri dishes developed polar substances (Figure 4e and 4f). However, groups of metabolites other than saponins were detected in the different species, which developed a yellow color with Godin's reagent. These results lead to the proposal of analysis by high-performance liquid chromatography (HPLC), or isolation and identification of the secondary metabolites to determine their chemical nature. Different metabolite groups are detected in different species, not only sapogenins, and other metabolites are detected that present a yellow color and also become a subject of study through more detailed analysis, such as HPLC, or isolation of the metabolites for identification by analysis including nuclear magnetic resonance and infrared. Studies by Flores (2010), recorded by Reina (2012) in the work document on the regional yam economy, assessed the saponin content in eight species in the Caribbean region, including D. polygonoides, to determine their potential use for medicinal and pharmaceutical purposes. The species analyzed were D. alata, D. bulbifera, D. cayenensis, D. dodecaneura, D. esculenta, D. polygonoides, D. rotundata and D. trifida. The re- Disinfection of Vegetable Material The material used came from conditions with a high presence of microorganisms or contaminating agents. Only the protocol that included Isodine® (PVP Iodine) as a disinfectant had favorable results. The nature of the explant (greenhouse, field or laboratory) and the type of explant (root or stem) were also important factors in planting. Bonafont (2011) states that the solution of Isodine®, which is not active itself, but slowly releases iodine, is the one that possesses bactericidal activity, penetrating the cell wall and combining with different organic substrates through reduction-oxidation reactions. The best results were obtained from young buds from the tips of the stems where possible endogenous contaminants had not invaded the plant's vascular bundles. Planting Explants Nodal explants of Medium iii reported shoots 30 days after being planted (0.2 -0.5 cm). At 60 days, they still were not ready for multiplication (1 -1.5 cm). In Medium i, growth was reported at 45 days after being planted due to the absence of regulators, and Medium ii did not present greater development of shoots either (0.8 -1 cm). Nodal segments of D. coriacea donated by the laboratory presented better results in Medium iii (1 -1.5 cm), which contained BAP and GA3 as growth regulators. The results were favorable in the medium to which GA3 was added, which is used for the growth of stems and especially for breaking the dormancy in some species (See Figure 3). The polyamine putrescine is considered to be a plant growth and development regulator because of its demonstrated effect on cellular growth, division and differentiation in low concentrations. Putrescine was included in the design of the environment because of the antioxidant and stabilizing capacity of its membranes (Perea, 2010). As a result of the statistical analyses, it could be determined that the best treatment was T3 (iii), finding a significant difference of 0.0578 at 30 and 60 days. Significant differences were not found in T1 and T2. Roots 160 140 120 100 80 60 40 20 0 T1 T2 T3 T4 T5 Similarly, Hata and collaborators (2003) found sapogenins in D. alata and D. rotundata. These results were based on the hydrolysis of the saponins to obtain the free sapogenins. The study presented a practical way of detecting saponins by thin-layer chromatography of di-osgenin/yamogenin and tigogenin/neotigogenin types in the majority of the accessions, using D. polygonoides as a reference considering that this species has been widely studied and the polarity and structure of the saponins and terpenes is known. The D. coriacea species presented an extract of 54% dry base and therefore, constitutes a species with an apparent high potential of saponin content. If we consider the phytotherapeutic value that it is recognized for in its traditional and popular use as treatment for different health conditions such as hypercholesterolemia and diabetes, its potential increases. As already mentioned by Hata and collaborators (2003), "un mejoramiento de estas especies puede convertirlas en especies promisorias para la obtención de sapogeninas esteroidales" [an improvement in these species could make them promising species for obtaining steroid sapogenins]. Conclusions The best conditions for sprouting were achieved by placing the material in temperature conditions of 18 °C daytime and 10 °C nighttime for 30 days. The culture environments with a dosage of 1 ml/L of GA3 were more suitable for the formation of shoots in D. coriacea under the experiment conditions. Culture environments that only contained BAP induced the formation of shoots. Similarly, the poylyamine putrescine was effective as an antioxidant. The wild species of Dioscorea that were collected presented a high content of saponins, qualitative indicators such as the surfactant activity developed by the formation of foam, and the presence of bands by TLC that these metabolites manifest as positive. As a result of this work, a procedure is proposed for the in vitro establishment of the D. coriacea species, which could be implemented for other species and will also permit the design of culture media for the multiplication, pretransplant and microtuberization stages.

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Relationship between Liver Mitochondrial Respiration and Proton Leak in Low and High RFI Steers from Two Lineages of RFI Angus Bulls The objective of this research is to evaluate liver mitochondrial oxygen consumption and proton leak kinetics in progeny from two lineages of Angus bulls with high and low residual feed intake (RFI). Two Angus bulls were selected based on results from a genetic test for RFI and were used as sires. Eight offspring at 10-11 months of age from each sire were housed in individual pens for 70–105 days following a diet adaptation period of 14 days. Progeny of the low RFI sire had 0.57 kg/d (P = 0.05) lower average RFI than progeny of the high RFI sire. There was no difference in dry matter intake between low and high RFI steers, but low RFI steers gained more body weight (P = 0.02) and tended to have higher average daily gains (P = 0.07). State 3 and State 4 respiration, RCR, and proton leak did not differ between high and low RFI steers (P = 0.96, P = 0.81, P = 0.93, and P = 0.88, resp.). Therefore, the increase in bodyweight gain which distinguished the low RFI steers from the high RFI steers may be associated with other metabolic mechanisms that are not associated with liver mitochondrial respiration and proton leak kinetics. Introduction Residual feed intake (RFI) is defined as the difference between actual dry matter intake and dry matter intake regressed on average daily gain and midtest metabolic body weight [1]. Residual feed intake is a commonly used measure of feed efficiency in cattle and has been used to select more feed efficient bulls. However, a link between RFI and mitochondrial oxygen consumption or proton leak has not been shown and currently gene chips do not include any sequences for mitochondrial DNA that could be used for selection purposes. An understanding of the role of mitochondria in feed efficiency (RFI) and growth in cattle would aid in determining its importance in sire selection. Mitochondria produce most of the ATPs and so inefficiencies in mitochondrial energy conversion will profoundly impact energy production and efficiency. To date, only 3 research publications have explored the relationship between production, feed efficiency (RFI), and mitochondrial respiration in cattle. Brown et al. [2] compared liver mitochondrial respiration to estimated differences in heritability of milk production in Holsteins and growth and milk production in beef breeds. They found no correlation for beef breeds. Kolath et al. [3] were the first to measure skeletal muscle mitochondrial respiration in high and low RFI steers and found that low RFI steers had higher rates of mitochondrial respiration but mitochondrial function was not different. Proton leak kinetics could account for differences in mitochondrial respiration rates but was not measured in either study. However, Lancaster et al. [4] also found higher liver mitochondrial respiration rates in low RFI cattle in one of the two experiments but no difference in proton leak kinetics. Proton leak kinetics are used to represent the uncoupling of hydrogen ion passage with ATP production and to assess changes in mitochondrial 2 International Scholarly Research Notices respiration. For example, an increase in proton leak kinetics could increase mitochondrial oxygen consumption without increasing ATP production. Liver is a highly active metabolic tissue and is the central organ of metabolism in the ruminant. Proton leak accounts for approximately 20% of total resting energy expenditure [5,6] and is an important contributor to basal energy expenditure and net energy for maintenance. Therefore, it would be expected that liver mitochondrial respiration rates and lower proton leak kinetics would contribute to improved feed efficiency. The objective of this research is to evaluate liver mitochondrial respiration from progeny of two Angus bulls with high and low RFI to examine the association between RFI, liver mitochondrial respiration rates, and liver mitochondrial proton leak kinetics. Steers and Management. This experiment was approved by the University of California, Davis Animal Care and Use Committee. Two popular commercial Angus bulls were selected based on an observed difference in their genomic breeding values for RFI of 0.32 kg/d (Zoetis Inc., Kalamazoo, MI), placing the low RFI bull in the top 1% and the high RFI bull in the bottom 10% for the breed. These bulls were used to artificially inseminate a group of predominantly Angus cows at Sierra Foothill Research and Extension Center (Browns Valley, CA). From progeny produced in September 2011, eight steers per sire were selected for participation in the study. One low RFI steer was removed from the trial because it did not adapt to the feedlot environment. When steers were at the age of 10-11 months, they were shipped to UC Davis feedlot and, following 14 days of diet adaptation, were housed in individual pens. Pens were randomly reassigned every 14 days to avoid social effects on feeding behavior. Diet consisted of 62.6% rolled corn, 17.2% dry distillers grain, 7.83% alfalfa, 4.74% molasses, 3.91% oat hay, 1.96% fat, 1.28% limestone, 0.26% salt, 0.13% magnesium oxide, and 0.01% rumensin. The diet contained 1.76 MJ/kg NEm, 1.18 MJ/kg NEg, and 12.2% CP. Feed was individually weighed and provided 4 times daily. Body weights, hip heights, rectal temperatures for health assessment, and ultrasound backfat thicknesses were taken every 14 days. Slaughter criterion was established as a minimum of 11 mm of backfat thickness assessed by ultrasound [7]. Steers were fed for 70-105 d (September-January, 2013) and were slaughtered at the UC Davis slaughter facility where euthanasia was performed via captive bolt. Immediately after euthanasia, liver samples were collected and transported to the laboratory for mitochondrial isolation. Isolated mitochondria were immediately used for oxygen consumption and proton leak kinetics assays. Mitochondrial Isolation. Approximately, 1 g of liver tissue was used for mitochondria isolation according to Cawthon [8,9]. The tissues were minced in isolation media (220 mM mannitol, 70 mM sucrose, 20 mM Tris, 1 mM EDTA and 0.1% (w/v) BSA, pH 7.4 at 4 ∘ C). The minced tissue was homogenized in a Potter-Elvehjem vessel with a Teflon pestle of 0.16 mm clearance maintained on ice. The homogenate was centrifuged at 1,800 ×g for 10 min, and the resulting supernatant was centrifuged at 8,100 ×g for 10 min to obtain the mitochondrial pellet. Fatty acid free BSA was used in the isolation of mitochondria to scavenge free fatty acids that can induce or cause proton leak in the inner mitochondrial membrane and to act as a moderate free radical scavenger to prevent oxidation of lipids and proteins during the study. The pellet was resuspended and washed twice in 10 mL isolation solution with and without BSA at 8,100 ×g for 10 min each. The resulting mitochondrial pellet was suspended in 200 L of isolation medium and placed on ice for oxygen consumption and proton leak kinetics assays as described below. Protein concentration was determined using the Bradford protein assay with BSA as the standard. Measurement of Mitochondrial Oxygen Consumption. Mitochondrial oxygen consumption was measured using a Hansatech Clark-type oxygen electrode (Norfolk, UK) [10,11]. Mitochondria (1.0 mg protein/mL final concentration) were incubated in 1 mL of oxygen consumption medium (120 mM KCl, 5 mM KH 2 PO 4 , 5 mM MgCl 2 , 5 mM Hepes, and 1 mM EGTA) in a magnetically stirred incubation chamber maintained at 30 ∘ C. Rotenone (5 M) was used to block electron transport chain at Complex I and State 4 respiration (nonphosphorylating respiration) was determined in mitochondria following the addition of 5 mM succinate. State 3 respiration was measured in mitochondria incubated in the presence of 5 mM succinate and 100 M ADP. Respiratory control ratio (RCR) was determined by dividing State 3 by State 4 [12]. Measurement of Mitochondrial Proton Motive Force (Δ ). Mitochondrial proton motive force (Δ ) [13] was assessed using a methyltriphenylphosphonium (TPMP + ) sensitive electrode. All measurements were completed in duplicate and simultaneous to determinations of mitochondrial oxygen consumption. Rotenone (5 M) and oligomycin (8 g/mg protein) were used to respectively block electron transport chain at Complex I and ATP synthase. These chemicals raise membrane potential and ensure that all changes in oxygen consumption and membrane potential in response to sequential additions of malonate are due to proton leak. Nigericin (0.4 g/mg protein) was added to convert the pH component of Δ to membrane potential units (mV), allowing Δ to be measured in mV units [11]. Data from the two electrodes (oxygen and TPMP + ) were collected by data acquisition software (Hansatech Oxygraph System, Norfolk, UK) allowing real-time simultaneous measurements of mitochondrial oxygen consumption and Δ . Mitochondrial membrane potential (MMP) in millivolts was calculated based on the Nernst equation [14] as follows: Statistical Analysis. All statistical analyses were performed using R Project for Statistical Computing (version 2.15.1). Data are presented as the mean ± SEM, and differences in means were detected using -tests. Initial body weights, final body weights (BW), dry matter intake (DMI), average daily gain (ADG), liver weight, age at slaughter, days on feed (DOF), and average rectal temperature were tested as covariates and if significant were included in following model: where = oxygen consumed (nmol/min) per mitochondrial protein (mg), is the overall mean, is RFI group ( = 1, 2), is the covariate effect ( = 1, 2, . . . , 7), and are the residuals which follow a normal distribution (0, 2 ). A probability level of ≤ 0.05 was considered statistically significant. For the analysis of proton leak kinetics, curves were estimated using the log function of Excel (Microsoft, 2007) and rates of oxygen consumption at a membrane potential of 150 mV were compared for high and low RFI steers using analysis of variance. Results and Discussion Mitochondrial DNA (mtDNA) is maternally inherited, explains 25-48% of variation in milk yield [2] and encodes 13 polypeptides involved in ATP synthesis [15]. However, the metabolic properties of mitochondria make them highly mutagenic environments. This mutational pressure introduces mtDNA variation (i.e., heteroplasmy) into the cytoplasmic population of cell lineages which can be influenced by sire genetics [16]. Thus selecting sires rather than dams for traits such as efficiency may be more beneficial to improve genetics associated with mitochondria metabolic activity. In this study, progeny of the low RFI sire had 0.57 kg/d ( = 0.05) lower average RFI than progeny of the high RFI sire. Therefore, progeny from both of the sires expressed a much greater difference in RFI than expected based on genomic predictions of 0.16 kg/d. Only performance parameters relating to weight gain were different between low and high RFI steers (Table 1). Rectal temperatures were collected every 14 days and were not different between high and low RFI sires ( = 0.99). Age at slaughter, liver weight, and DMI also did not differ among progenies from high and low RFI sires ( = 0.69, = 0.97, and = 0.43, resp.). Initial body weights of steers entering the feedlot were not different, but final body weights at slaughter were greater in low RFI steers. Average daily gain also tended to be greater for the low RFI steers ( = 0.07). Therefore, the difference in RFI was due to increased gain and not changes in DMI. Unlike this study, Kolath et al. [3], Lancaster et al. [4], and Castro Bulle et al. [17] did not find differences in ADG or final body weight among high and low RFI steers but did observe that low RFI steers had smaller DMI compared to high RFI steers. Therefore, RFI from previous studies was based on differences in DMI. It would be expected that differences in DMI would be more likely to result in differences in mitochondrial oxygen consumption [3]. Differences in RFI due to gain may involve more post mitochondrial metabolic functions which would explain why mitochondrial oxygen consumption and proton leak kinetics were not different in high and low RFI steers in this study. No differences in liver mitochondrial respiratory rates were observed between high and low RFI steers (Table 2). Oxygen consumption rates during State 3 respiration (maximum ADP stimulated respiration), State 4 respiration (leakdependent respiration), and RCR did not differ between high and low RFI steers ( = 0.96, = 0.81, and = 0.93 resp.). Unfortunately only three other studies have been published relating production efficiency to mitochondrial respiration and function in cattle. Brown et al. [2] examined variability in mitochondrial respiration rates by measuring State 3, State 4, and RCR in livers from both Holstein lactating cows and beef cows (Angus, Brangus, and Hereford). Similar to this study, they did not find any correlation in mitochondrial respiration for beef cattle with growth or milking traits but did for Holstein milking traits. Kolath et al. [3] compared RFI in 16 Angus steers (9 low RFI steers and 8 high RFI International Scholarly Research Notices steers) and State 2, State 3, and State 4 respiration rates and RCR in muscle mitochondria. But proton leak kinetics were not measured. Daily gain was not different between RFI groups, but DMI was greater and State 2 and State 3 respiration rates and RCR were lower for the high RFI steers. Similar to results from this study, mitochondrial function was not different between RFI groups, but in the Kolath et al. study [3] mitochondrial respiration rate was higher with low RFI steers. Therefore, mitochondrial respiration rate may be related to level of intake. The third study [4] examined liver mitochondrial respiration and proton leak kinetics and RFI in Angus heifers and Santa Gertrudis steers. Low RFI cattle had lower DMI but similar bodyweights and the same State 2 and State 4 respiration rates, proton leak kinetics, and RCR. State 3 respiration rates were higher for the low RFI Angus cows but were not different for the Santa Gertrudis steers. Therefore, it is unclear if differences among mitochondrial respiration rates, proton leak, and RCR exist for different species or tissues or are different with differences in feed efficiency [3,4]. However, Bottje et al. [12] did report differences in RCR for leg and breast muscle in chickens, suggesting that mitochondrial respiration may differ among tissues. Differences in feed efficiency were observed between low and high RFI steers in the present study. But, since no differences were found in liver mitochondrial respiration rates at State 3 and State 4 or in RCR values, the higher gain per kg of DMI of low RFI steers was not associated with a decrease in liver mitochondrial proton leak or the capacity for liver mitochondrial respiration. This may suggest that the size of the effect was too small to observe with this number of animals. However, Kolath et al. [3] did detect differences in respiratory rates with similar numbers of steers as in this study. Moreover, these results suggest that RFI of those two sire groups was not associated with liver mitochondrial respiration and proton leak kinetics and was instead driven by other cellular and physiological processes not measured in this study. Mitochondrial proton leak is a process that dissipates proton motive force through the movement of protons across the mitochondrial inner membrane without production of ATP [18]. In the present study, there were no differences ( = 0.88) in hepatic mitochondrial proton leak, assessed by calculating rates of oxygen consumption at common membrane potential of 150 mV, in Angus steers with high and low RFI (Figure 1). Furthermore, State 4 respiration (leak-dependent respiration represented by the points on the far right of each curve) and membrane potential at State 4 respiration were the same for both RFI groups. Thus, liver mitochondrial proton leak was not different between the two groups of steers. However, correlation coefficients for regression analyses of log transformations using the Log Function of Excel were 0.82 and 0.63 for high and low RFI steers, respectively. These results agree with those of Lancaster et al. [4] and Bottje et al. [19] in which no differences in liver proton leak kinetics between high and low RFI steers and muscle basal mitochondrial proton leak were observed among high and low feed efficient broilers. The lack of differences in liver mitochondrial proton leak observed in this study indicates that feed efficiency is not associated with mitochondrial proton permeability, a major contributor to mitochondrial efficiency. Whether mitochondrial proton leak in other tissues is related to feed efficiency in beef cattle remains to be determined. It was expected that differences in RFI between Angus steers were correlated with hepatic mitochondrial proton leak because proton leak is a major contributor to mitochondrial efficiency and resting energy expenditure [5,6]. Thus, it was expected that less proton leak would be observed in low RFI steers due to increased energy partitioning towards gain. However, results of this study showed that energy partitioning between the two RFI groups was not the same. Energy intake was not different between low and high RFI steers, but differences were found for ADG. Therefore, we can conclude that low RFI steers were partitioning more energy towards gain, while high RFI steers were partitioning more energy towards other physiological processes. Conclusions Differences in low and high RFI beef steers were not associated with liver mitochondrial proton leak kinetics, a contributor to mitochondrial efficiency, or mitochondrial respiration rates (State 3 and State 4 respiration and RCR). Therefore, which tissues and biochemical processes are primarily responsible for differences in RFI in beef cattle remains to be determined.

v3-fos

2016-05-12T22:15:10.714Z

2015-03-03T00:00:00.000Z

18453084

s2

Functional Analysis of GmCPDs and Investigation of Their Roles in Flowering The onset of floral development is a pivotal switch in the life of soybean. Brassinosteroids (BRs), a group of steroidal phytohormones with essential roles in plant growth and development, are associated with flowering induction. Genes involved in BR biosynthesis have been studied to a great extent in Arabidopsis, but the study of these genes has been limited in soybean. In this study, four CPD homologs (GmCPDs) catalyzing BR synthesis were isolated from soybean. Transcripts were mainly confined to cotyledons and leaves and were down-regulated in response to exogenous BR. Bioinformatic analysis showed strong sequence and structure similarity between GmCPDs and AtCPD as well as CPDs of other species. Overexpression of GmCPDs in an Arabidopsis BR-deficient mutant rescued the phenotype by restoring the biosynthesis pathway, revealing the functional roles of each GmCPDs in. Except for the rescue of root development, leaf expansion and plant type architecture, GmCPDs in expression also complemented the late flowering phenotype of Arabidopsis mutants deficient in CPD. Further evidence in soybean plants is that the expression levels of GmCPDs in are under photoperiod control in Zigongdongdou, a photoperiod-sensitive variety, and show a sudden peak upon floral meristem initiation. Together with increased GmCPDs in expression in the leaves and cotyledons of photoperiod-insensitive early-maturity soybean, it is clear that GmCPDs in contribute to flowering development and are essential in the early stages of flowering regulation. Introduction Flowering is one of the most important events in the life cycle of plants, with optimal timing being especially crucial. Therefore, flowering is controlled by numerous interacting endogenous and environmental cues to ensure appropriate conditions for seed production. At least four signaling pathways have been demonstrated to regulate flowering in concert, involving length of day (photoperiodism), winter cold (vernalization), regulation by gibberellins (GAs), and autonomous floral initiation occurring in the absence of any effective environmental signals [1]. In addition, other factors such as ascorbic acid, ethylene, ambient temperature and and CCA1 may hold clues to the causes of the late-flowering phenotype of cpd mutants [12,34]. Furthermore, it has been reported that BR can modulate circadian rhythms and promote the periodicity of the circadian clock genes CHLOROPHYLL A/B BINDING PROTEIN (CAB2), COLD AND CIRCADIAN-REGULATED 2 (CCR2) and CCA1 [35]. This interaction is consistent with the observation that the period of CCR2 is prolonged in cpd mutants [35]. Thus, the above findings suggest that BR regulates flowering time through the circadian clock system, a crucial mechanism in photoperiod pathway. Soybean is a short-day crop of agricultural and economic importance. Soybean flowering is largely regulated by photoperiod, with many varieties highly photoperiod-sensitive. Typically, Zigongdongdou will not initiate flowering until short-day induction; this variety even undergoes flowering reversion in which the floral meristem developing the floral organs reverts to produce leaves when the photoperiod is altered from a short day to a long day [36][37][38][39][40]. This high sensitivity restricts the adaptability of soybean to diverse environmental conditions, limiting the season and region available to many high yield varieties, negatively impacting soybean production [41]. In addition, the photoperiod sensitivity is diverse among soybean varieties, leading to multiple maturity periods. Consequently, many varieties with good behavior cannot be hybridized with each other as a result of asynchronous florescence. Therefore, it has been long recognized by breeders that controlling flowering time is crucial to ensuring soybean yield [42]. In the current study, four soybean CPD homologous genes belonging to the BR biosynthesis pathway are found to be associated with soybean flowering. These GmCPDs are extremely similar with AtCPD in sequence and structure and can complement the AtCPD function in Arabidopsis mutants deficient in AtCPD. The expression levels of these GmCPDs all exhibit a sudden peak upon floral meristem initiation in soybean and are increased in a photoperiod-insensitive soybean variety, suggesting a relationship between BR biosynthesis genes and floral transition. Plant Growth Conditions Soybean varieties Williams 82 and Zigongdongdou were grown in a chamber at day/night temperatures of 26/24°C. Zigongdongdou and Heihe27 used for the analysis of GmCPDs expression in soybean varieties with different photoperiod sensitivities were grown at the temperature of constant 25°C. Williams 82 plants were cultivated under a short-day condition (12/12 h day/ night cycle). Zigongdongdou and Heihe27 plants were cultivated under either short-day or long-day (16/8 h day/night cycle) conditions depending on the experiment. Arabidopsis accessions Col-0 and cpd-91 were grown at 22°C under a long-day condition (16/8 h day/night cycle) in potting soil or in half strength MS agar plates with 1% (w/v) sucrose. All plates were axenically cultured and packed with silver papers in the dark treatment. Brassinosteroid Treatment In the BR response assay, 5-day-old Williams 82 seedlings were cultivated in Hoagland solution after germination in soil. BR treatment was undertaken 10 days later by adding 1 μM 2,4-epibrassinolide (C28H48O6; TCR, Toronto, ON, Canada) to the solution. The treatment lasted for 2 hours, and the samples were collected every half hour. In the root inhibition assay, Arabidopsis seeds were planted on vertically oriented plates containing half-strength MS medium supplemented with 1% sucrose in the absence or presence of 100 nM 2,4-epibrassinolide . Root lengths were measured after seedlings were grown for 10 days. Sampling and RNA Isolation The entire Williams 82 plant was sampled for GmCPDs gene cloning. For tissue-specific expression analysis, hypocotyls, cotyledons and roots of Williams 82 were collected from 7-dayold seedlings, and the leaves, stems and shoot apices were collected from 20-day-old adult plants. The flowers were tagged on the day of anthesis, and the pods were harvested when 0.5-2 cm long. After BR treatment, the leaves of Williams 82 were collected every half hour and labeled 0.5 h (0.5 hour after treatment), 1 h, 1.5 h and 2 h. When the cotyledons of Zigongdongdou and Heihe27 opened, SD (short-day), LD (long-day) photoperiod treatments were carried out. The SD13d-LD (transfer to an LD condition after a 13-day SD treatment) condition was applied only to Zigongdongdou. Plant leaves were collected every other day until the 25 th day following photoperiod treatment. Cotyledons were obtained at 3 d (3 days after photoperiod treatment), 6 d, and 9 d with leaves removed after cotyledon opening. All samples were a mixture of more than five individual plants and were ground into powder in liquid nitrogen. Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). The RNA from a whole Arabidopsis plant sample was isolated using the same method. Analysis of mRNA Expression Level by Real-Time PCR cDNA for PCR was prepared using 1 μg of total RNA with a mixture of random primers. RT-qPCR analysis was performed on an ABI7900 instrument (Applied Biosystems, Foster City, CA, USA) using Takara SYBR Premix ExTaq (Takara, Shiga, Japan) for 40 cycles (95°C for 5 s; 60°C for 30 s; 72°C for 30 s). All reactions were carried out at least three times. Quantification of mRNA level was based on Ct (threshold cycle) values using a comparative Ct (2−ΔΔCt) method [43]. Data are presented as the mean±SD. The specific primers for each gene are shown in S1 Table. Vector Construction and Arabidopsis Transformation The coding regions of GmCPD1, GmCPD2, GmCPD3 and GmCPD4 with additional XbaI and SacI restriction sites were PCR-amplified. The XbaI-SacI flanked GmCPDs fragments were cloned into the XbaI-SacI sites of pTF101.1-GFP vector, replacing GFP and generating pTF101.1-GmCPD1, pTF101.1-GmCPD2, pTF101.1-GmCPD3 and pTF101.1-GmCPD4. These resulting constructs were verified by sequencing and restriction analysis and transformed into Agrobacterium tumefaciens strain GV3101. The Agrobacterium-mediated flower infiltration transformation method [44] was used to introduce GmCPDs into cpd-91 Arabidopsis mutant plants. T1 generation seeds were harvested and selected on antibiotic-containing MS plates with 10 mg/L glufosinate ammonium (Sigma, St. Louis, MO, USA). Positive plants were confirmed by PCR analysis and propagated to obtain the T3 generation. Measurements and Statistical Analysis All seedlings were axenically cultured on medium for light/dark analysis, BR treatment assays and leaf morphology analysis were scanned using an Epson perfection V700 photo scanner (Epson, Nagano, Japan). The images were analyzed using WinRHIZO Pro v.2009c software (Regent Instruments, Montreal, QC, Canada). For light/dark analysis, the hypocotyl lengths of 6-day-old seedlings were measured. Similarly, when the seedlings grown on medium with or without 24-epiBL in the BL treatment assay, the total root lengths of 10-day-old seedlings, hypocotyl length of 6-day-old seedlings and petiole length of 13-day-old seedlings were measured; the number of lateral roots of 10-day-old seedlings was also counted. For leaf morphology analysis, the first true leaves of 13-day-old seedlings were cut off at the bottom of the petioles and flattened on agar plates for scanning. Traits including petiole length, leaf area, length and width of the leaf blade were examined. Silique length and plant height were measured using a millimeter-graduated ruler. All measurements were repeated three times independently, and 30-50 seedlings were measured each time. Data are presented as the mean±SD and were subjected to Student's t test with a sample size of 30 to determine differences among the groups. Cloning and Sequence Analysis of GmCPD genes in Glycine max Four soybean CPD homologs (GmCPDs) were obtained from the soybean translated NCBI nucleotide database by a BLAST search using the amino acid sequence of Arabidopsis CPD (Gen-Bank accession No. XP_002873219) as a query. These predicted genes were then assigned names based on their correspondence with AtCPD. The four potential homologous proteins, GmCPD1 (GenBank accession No. XP_003545232.1), GmCPD2 (GenBank accession No. XP_003519393.1), GmCPD3 (GenBank accession No. XP_003552845.1) and GmCPD4 (Gen-Bank accession No. XP_003538460.1), are predicted to be between 473 and 480 amino acids in length and all belong to the cytochrome P450 (CYP) family. The deduced amino acid sequences of the GmCPDs share 82-97% identity with each other and exhibit high similarity to the Arabidopsis CPD protein, with identities between 79% and 81% ( Fig. 1). GmCPD1 has the highest identity of 81% while GmCPD2 has the lowest. An alignment of GmCPDs with known CPDs from other species reveals identities of 81-87% for MtCPD1 of Medicago truncatula, 76-80% for PtCPD of Populus trichocarpa, 75-77% for CsCPD of Cucumis sativus, and 59-63% for OsCPD1 of monocot Oryza sativa (Fig. 1). It is suggested that the amino acid sequences of CPDs are highly homologous across all species. There are generally four structural domains in CPD proteins that exhibit catalytic features. The proline-rich region was thought to ensure the correct folding and proper orientation of the CPD protein. Domain A and domain B are involved in the dioxygen and steroid binding required for catalytic activity. The most characteristic P450 consensus sequence, the heme binding domain, is responsible for carbon monoxide binding ability [45,46]. As shown in Fig. 1, all CPDs contain these characteristic domains, and their amino acid sequences are highly conserved. There are only two amino acid differences between AtCPD and GmCPDs in the proline-rich region, one amino acid difference in domain B and at most two amino acid differences in the heme-binding domain. As for domain A, AtCPD and GmCPDs share 100% amino acid sequence identity (Fig. 1). Based on these findings, GmCPDs bear a striking similarity to AtCPD in sequence and structure, a trait that might imply functional similarity. Phylogenetic analysis was performed using the deduced amino acid sequences of GmCPD and a range of putative CPDs from higher plants. The tree is clearly divided into two major clades: one clade corresponds to monocots, while the other clade corresponds to dicots (Fig. 2). The four GmCPDs all fall in the latter clade (Fig. 2). GmCPD1 and GmCPD2 are clustered together with Medicago truncatula and Cicer arietinum, while GmCPD3 and GmCPD4 branch off from the legume sub-clade (Fig. 2). The four GmCPDs all cluster relatively closely with AtCPD ( Fig. 2), indicating that these proteins may have inherited more ancestral characteristics. Genomic location of each GmCPD was targeted on physical map of soybean (Glycine max) genome based on the information on SoyBase (http://www.soybase.org) and Phytozome database (http://phytozome.jgi.doe.gov). They are all located in separate chromosome: GmCPD1 (Glyma.14g059900), GmCPD2 (Glyma.02g256800), GmCPD3 (Glyma.18g028300) and GmCPD4 (Glyma.11g228900) are located in Gm14 (B2), Gm02 (D1b), Gm18 (G) and Gm11 (B1), respectively (Fig. 3). There were not many SSR markers around GmCPDs. Around GmCPD1, Sat_177 and Sat_264 are associated with the QTLs of flower number; Satt126 is associated with lodging and Sat_287 also relates to seed coat color (Fig. 3). As for GmCPD2, Satt189, Satt350 and Satt546 are associated with the QTLs of first flower; Satt189 and Satt350 are associated with leaflet shape and leaf area respectively; Satt546 is associated with internode length; Sat_139, Satt546 and Satt172 are associated with the seed quality trait (Fig. 3). Satt309, Satt356 and Satt570 locate closely to GmCPD3: Satt309 and Satt356 associated with the QTLs of pod maturity; Satt356 is linked with internode length; Satt570 is associated with seed protein, lateral root density and root width (Fig. 3). Around the location of GmCPD4, Satt415 is associated with the internode length; Satt583 is associated with the length of reproductive stage; Sat_123 is associated with pod maturity and lodging; Sat_123, Satt583 and Sat_095 are all associated with seed weight (Fig. 3). Above all, the four GmCPD homologous are associated with the QTLs related to main aspects of soybean development. Expression Patterns of GmCPDs in Soybean Tissue-specific expression patterns of GmCPDs in soybean were systematically determined using RT-qPCR. These four GmCPDs are widely expressed in plant tissues but display different patterns. Although GmCPD1, GmCPD2 and GmCPD4 all showed higher expression levels in cotyledons and leaves, GmCPD2 and GmCPD4 had the highest level in cotyledons while GmCPD1 had the highest level in leaves (Fig. 4A). These results are consistent with the expression pattern of CPD in Arabidopsis [47]. However, GmCPD3 is an exception, exhibiting the highest mRNA accumulation in young pods but very low concentrations in other tissues (Fig. 4A). In addition, GmCPD4 as well as GmCPD1 and GmCPD2 showed relatively high levels in young pods (Fig. 4A). These results are consistent with the important roles proposed for BRs in processes such as fruit development and ripening [48,49]. The BR sensitivity of GmCPDs was also tested in soybean. William 82 adult plants were treated with 24-epiBL, and the leaf samples were collected every half hour. As shown in the RT-qPCR results, the expression levels of the four GmCPDs fluctuated, but overall the expression levels tended to decrease. The expression patterns of GmCPD1 and GmCPD2 were nearly equivalent but distinct from GmCPD3 and GmCPD4 (Fig. 4B). Following BR treatment, a sudden increase of GmCPD1 and GmCPD2 expression reached a maximum 0.5 h after treatment was initiated. GmCPD1 and GmCPD2 expression then sharply decreased, reaching a minimum approximately 1 h after treatment was started and subsequently increasing slightly to a plateau (Fig. 4B). In contrast, the expression levels of GmCPD3 and GmCPD4 rapidly decreased following BR treatment, reaching a minimum at 1 h and then leveling off (Fig. 4B). The above results indicate a highly sensitive response of GmCPDs to exogenous BR. Taken together with previous studies that show that CPD is feedback-inhibited by BR [13,47,50], our results corroborate the importance of GmCPDs in BR biosynthesis. Complementation of an Arabidopsis CPD-Deficient Mutant phenotype by GmCPDs Expression To test whether the GmCPDs can function in BR biosynthesis, the coding sequences of GmCPD1, GmCPD2, GmCPD3 and GmCPD4 were placed under the control of a 35S constitutive promoter and introduced into a cpd-91 mutant [13] of Arabidopsis. The goal was to evaluate whether the transgenes complement the mutant phenotype. Adult plants of cpd-91, a CYP90A1/CPD-deficient mutant, are small and dwarfed with rounded curled leaves. In contrast, the transgenic GmCPD1, GmCPD2, GmCPD3 and GmCPD4 cpd-91 mutant lines were all similar to the wild type in size, showing a rescue of the cpd-91 mutant adult phenotypes (Fig. 5A). The RT-PCR results revealed that GmCPD genes can only be detected in the corresponding transgenic plants, indicating complementation by GmCPDs overexpression (Fig. 5B). The leaf phenotypes of the transgenic lines all bear little resemblance to the cpd-91 mutant, instead resembling the wild type phenotype (Fig. 6A). In a quantitative comparison, the mutant retained minimum values of petiole length (Fig. 6B), leaf area (Fig. 6C) and length-width ratio (Fig. 6D). The transgenic GmCPD1, GmCPD2, GmCPD3 and GmCPD4 cpd-91 lines were all similar to the wild type and were significantly different (P < 0.01) from the mutant (Fig. 6B-D). Without the CYP90A/CPD gene, the morphological change in the roots was quite significant in the cpd-91 mutant, with a small root length and undeveloped lateral roots ( Fig. 7A-C). Conversely, all transgenic lines exhibited developed root systems that were similar to the wild type plants (Fig. 7A). Student's t-tests indicated a significant difference (P < 0.01) between the transgenic lines and the mutant plants in root length and lateral root number ( Fig. 7B-C). The most obvious complementation of the mutant is the rescue of dwarfness. Similar to other BR mutants, cpd-91 mutant is severely dwarfed. In contrast, the plant height of every transgenic line was remarkably higher (P < 0.01) than that of the mutant and very similar to that of the wild type (Fig. 7F). The silique size of mutants is very small, only an average of 0.2 cm long and about twenty percent the length of Col-0 siliques (Fig. 7D, E). Each transgenic line was extremely distinct (P < 0.01) from the non-transformed mutant in silique size and resembled the wild type (Fig. 7E). In conclusion, all four GmCPDs are functional and essential in leaf, root and plant type development. GmCPD Homologs Restore BR Biosynthesis in Arabidopsis cpd-91 mutants To further confirm that the rescue of the cpd-91 mutant phenotype is due to a restored BR biosynthesis pathway via GmCPDs transformation, we tested the BR responses of complemented Arabidopsis compared with untransformed cpd-91 and wild type Col-0. (B) RT-PCR analysis to detect GmCPD genes using specific primers described in S1 6-day-old seedlings grown in light and darkness were screened for hypocotyl elongation during skotomorphogenesis and photomorphogenesis. In the dark, cpd-91 mutant seedlings underwent constitutive photomorphogenesis, exhibiting short hypocotyls and open cotyledons (Fig. 8A, C). In contrast, the transgenic lines and wild type exhibited longer hypocotyls and closed apical hooks (Fig. 8A, C). When grown in the light, cpd-91 mutant seedlings exhibited shorter hypocotyls than the wild type (Fig. 8B, D). This mutant phenotype was complemented by all four transgenes (Fig. 8B, D). Student's t-tests indicate a significant difference (P < 0.01) in hypocotyl length between the transgenic lines and the mutant plants in both the light and darkness (Fig. 8C, D). In the root growth inhibition assay, 10-day-old complemented Arabidopsis, cpd-91 mutants and the wild type were grown on 1/2 MS medium containing 100 nM 2,4-epibrassinolide (24-epiBL). All seedlings showed shortened roots in response to 24-epiBL but behaved differently in root shortening (Fig. 9A-D). The transgenic lines and the wild type displayed greater shortening than cpd-91 plants (Fig. 9D), indicating a stronger response to BR. Additionally, under the treatment of 100 nM 24-epiBL, all the seedlings exhibited elongated hypocotyl and shortened petiole (Fig. 9E-H). Compared to transgenic lines and Col-0 Arabidopsis, cpd-91 mutant showed the shortest length of hypocotyl and petiole both in BL treatment and normal conditions (Fig. 9E, G). The transgenic lines resembled the wild type and displayed greater hypocotyl elongation and petiole shortening than cpd-91 mutant (Fig. 9F, H). Therefore, physiological response phenotypes of mutant plants are complemented by GmCPDs expression, suggesting a restored BR biosynthesis pathway in transgenic lines. This result further demonstrates that the CPD homologous genes in soybean, GmCPD1, GmCPD2, GmCPD3 and GmCPD4, are functional in the BR pathway. GmCPDs Are Involved in Floral Regulation of Arabidopsis The above results show the phenotypic rescue of Arabidopsis CPD-deficient mutant by GmCPDs expression. In addition to rescuing morphology, overexpression of GmCPDs also complemented the delayed flowering of the cpd-91 mutant. The observation that the cpd-91 mutant flowered approximately 10 days later than the Col-0 wild type is in agreement with previous observations (Fig. 10A, B). The transgenic plants transformed with any of the four GmCPD homologs all bloomed simultaneously with the wild type plant, much earlier than the cpd-91 plants (Fig. 10A, B). In order to investigate the roles of CPD in flowering regulation, the expression pattern of flowering integrating gene, Flowering Locus T (FT), was examined in the transgenic plants compared with non-transformed cpd-91 and Col-0 wild-types (Fig. 10C). AtFT acts as floral integrator of all four flowering pathways [51]. The AtFT product, which can move in long distance through the phloem to initiate flowering at the shoot apex, is a main determinant of the timing of flowering [52]. In our results, all the groups exhibited similar expression pattern that AtFT gene maintained at a very low level in the vegetative stage and expressed highly when flowered (Fig. 10C). Except for that the 35S::GmCPD2 transgenic line showed similar level of AtFT expression to the wild type, the other three transgenic lines expressed diversely but all higher than cpd-91 mutants (Fig. 10C). In addition, the expression pattern of AtCPD gene during flowering was also examined in Arabidopsis leaves that were collected in three developmental stages: vegetative growth (twoweek-old), flowering initiation and flowering period (one week after beginning flowering). It is showed that the AtCPD transcripts were more abundant in vegetative stage, but decreased during flowering (Fig. 10D). The Potential Roles of GmCPDs in Soybean Flowering Regulation To further study the roles of GmCPDs in flowering, GmCPDs transcript levels were tested in soybean, a typical short-day plant that can undergo flowering reversion. In a previous study by our lab, 13 days of SD treatment before transfer to an LD condition are enough for flowering reversion to occur in soybean var. Zigongdongdou. Based on this observation, an effective flowering reversion system was established. In this system, three developmental states, flowering, continuous vegetative growth and flowering reversion, can be observed in Zigongdongdou plants under different photoperiods (SD, LD, 13SD-LD). Genes related to photoperiodism and flower development are preferentially studied in this system. Accordingly, leaf samples were collected in each photoperiod, and the relative expression levels of GmCPDs were analyzed to investigate the potential roles of GmCPDs in flowering. As shown in the results, all GmCPDs have the same expression pattern: expression was maintained at a much lower level in the LD condition (Fig. 11A-D). Conversely, when treated in SD, GmCPDs expression levels were gradually elevated at first. Once SD treatment reached the 13 th day, GmCPDs levels sharply increased to a maximum, then decreased suddenly under both the SD and LD conditions (Fig. 11A-D). Obviously, these results suggest that GmCPDs expression is under photoperiod control and is upregulated by SD, a day length that induces flowering. Interestingly, the expression quantity of GmCPDs on the 13 th day is around tenfold that of the 9 th day and from nineteen to forty-five times that of the 19 th day (Fig. 11A-D). The peak on the 13 th day is so sharp that we cannot help but wonder what happens on this day. It was found in a previous study that the apical meristem of Zigongdongdou begins to initiate floral primordia on the 13 th day of SD treatment [39]. The above results suggest a certain relationship between GmCPDs and floral initiation through the photoperiod pathway. When plants were grown in LD after SD induction, the expression of GmCPDs all decreased but still higher than that of plants grown in either continuous LD or SD (Fig. 11A-D). This result was not consistent with the expression pattern of GmFT2a, an integrator in photoperiod pathway. The expression of GmFT2a maintained in a rather low level in either LD or vegetative stage and raised around the 13 th day in SD when flowering initiated (Fig. 11E). When returned to the LD condition, GmFT2a expression decreased to the same level of that in the continuous LD treatment (Fig. 11E). Unlike GmFT2a, the expression of GmCPDs had additive effect that the SD effects could be accumulated when turned into LD condition, suggesting the distinct roles of GmCPDs in flowering regulation. Since there was no obvious effect on the pattern of AtFT expression in the absence of CPD (Fig. 10C) and expression patterns between GmFT2a and GmCPDs in flowering reversion were different (Fig. 11A-E), it might imply that no direct interaction between GmFT2a and GmCPDs. To text this, the expression of GmCPDs expression was examined in GmFT2a transgenic soybean and compared with the non-transformed Zigongdongdou (Fig. 11F). The published data by our lab [53] have showed that one line of GmFT2a transgenic Zigongdongdou flowered approximately 20 days after emergence under non-inductive LD conditions. The expression level of GmCPDs in this line was found to be maintained in a quite low level and even decreased compared to the wild type on the occasion that GmFT2a expressed extremely high (Fig. 11F). Therefore, the involvement of GmCPDs in flowering regulation may not be linked to the direct interaction with GmFT2a. GmCPDs Expression in Soybean Varieties with Different Photoperiod Sensitivities Soybean varieties are diverse in photoperiod sensitivity. Zigongdongdou is a photoperiod-sensitive late-flowering variety that only flowers under the SD condition. In contrast, the photoperiod-insensitive early-flowering variety Heihe27 blooms approximately 25-27 days after emergence under both LD and SD conditions [54]. As it is shown in Fig. 12A, at the 36 th days after emergence, Heihe 27 had already set pods while Zigongdongdou still underwent vegetative growth under the LD condition. These two typical varieties were chosen to evaluate the expression pattern of GmCPDs in soybean varieties with different photoperiod sensitivities. We screened leaf samples from Zigongdongdou and Heihe27 after various days of LD treatment. In the 5 th , 7 th and 9 th day after LD treatment (5 d, 7 d and 9 d), all GmCPD genes were expressed at very low levels in Zigongdongdou but at extremely high levels in Heihe27 (Fig. 12). After the 11 th day, the transcript levels of GmCPDs were remarkably upregulated in Zigongdongdou but slightly decreased and maintained in Heihe27 (Fig. 12). The expression patterns of GmCPD1, GmCPD2 and GmCPD4 were nearly the same; the expression levels of these GmCPDs were obviously higher in Heihe27 than Zigongdongdou from day 5 to day 11 d. From day 13 to day 19, the expression levels in Zigongdongdou were increased and higher than Heihe27, in which the levels were downregulated. At day 25, Heihe27 had higher expression levels compared to Zigongdongdou (Fig. 12A, B and D). As for the GmCPD3 gene, the expression levels in Heihe27 were always higher than Zigongdongdou except for day 15. GmCPD3 was most highly expressed in Heihe27 at day 19 (Fig. 12C). However, the expressions of GmCPDs in Zigongdongdou under the LD condition in this experiment (Fig. 12) have differences with the results shown in Fig. 11A-D. This may due to the different culture temperature (described in section of Material and Methods) and sampling time. The leaf samples in this experiment were collected in the morning, while the samples in Fig. 11 were collected in the afternoon. Since genes usually have different expression levels during the day, the results in the two experiments are not comparable. We only analyzed the expression differences of GmCPDs among Zigongdongdou and Heihe 27 in this experiment that carried out in the same condition and sampled at the same time every day. Leaves and cotyledons are the two main tissues in which GmCPDs are expressed (Fig. 4A). Therefore, cotyledons were also collected from Zigongdongdou and Heihe27 on the 3 rd , 6 th and 9 th days after LD treatment. GmCPD1, GmCPD2 and GmCPD4 had similar expression patterns: their expression levels tended to decreased with time in Zigongdongdou but increased in Heihe27. Although levels in Zigongdongdou were higher on the 3 rd day compared to Heihe27, the levels were much lower on the 6 th and 9 th days (Fig. 13A, B and D). The expression pattern of GmCPD3 was rather special: the expression levels of GmCPD3 in both Zigongdongdou and Heihe27 decreased each day, but the gene was still expressed more highly in Heihe27 compared to Zigongdongdou (Fig. 13C). Regardless of whether leaves or cotyledons were measured, all GmCPDs exhibited more vigorous expression (much higher level) in Heihe27 compared to Zigongdongdou, especially in the early days of LD treatment. The flowering of Heihe27 is less regulated by photoperiod and can be initiated by the LD non-inducible day length. Compared to the strict short day flowering variety Zigongdongdou, the expression of GmCPDs is increased in Heihe27. In our opinion, differences in florescence between varieties is not only determined by the flowering regulation pathway but also by how each variety has been prepared for flowering; such preparation is affected by many factors, which may include GmCPDs. BR Intermediate Products Catalyzed by GmCPD Move Long Distances In the tissue-specific expression assay, the observation that GmCPDs are expressed mainly in cotyledons and leaves (Fig. 4A) is consistent with the expression pattern of CPD in Arabidopsis [47] but does not coincide with the distribution pattern of bioactive BR [55,56]. The bioactive BR levels in vegetative tissues are much lower [30,56,57], with the highest levels generally occurring in reproductive organs [48,49,56,58], where BR can easily perform its intended function due to its lack of transport [30,55]. It has also been reported that the transcript levels of most BR biosynthesis genes are generally higher in tissues with high BR levels [48,49,57,59]; CPD obviously is an exception. Because CYP90A/CPD encoded by the CPD gene catalyzes an early step of BR synthesis [19], long-distance movements are required for BR intermediate products to finish synthesis where untransported bioactive BR are accumulated. With this assumption, the paradox that GmCPDs transcript levels are not higher in tissues with high BR levels is readily explained. However, long-distance transformation is so costly that we wonder if the higher expression of GmCPDs in vegetative tissues holds further meanings. One possibility is because CPD is under light-dependent diurnal regulation primarily mediated by phytochrome signaling [23], leaves and cotyledons, where phytochrome collects, are preferred. In addition, recent work has revealed that BR plays a controlling role in the assembly and function of the photosynthetic apparatus. Moreover, severe thermal instability of oxygen yields has been observed in cpd mutants [60], suggesting the potential role of CPD in photosynthesis. All of these intriguing hypotheses are worthy of further investigation. Universality and Characteristics of GmCPDs Compared with AtCPD The homologous sequences of CPD in soybean have not been isolated until the current study. Strong similarities were found between GmCPDs and AtCPD in many aspects. First, GmCPDs and AtCPD bear high identities in amino acid sequence and structure. Second, GmCPD1, GmCPD2 and GmCPD4 were most highly expressed in leaves and cotyledons, consistent with the AtCPD expression pattern. Most importantly, transformation of GmCPD genes into an Arabidopsis CPD-deficient mutant restored the BR biosynthesis pathway and complemented the mutant phenotype with respect to root development, leaf expansion, plant type architecture and flowering regulation, suggesting functional similarity between GmCPDs and AtCPD. In addition, GmCPDs exhibit some special characteristics in soybean. One is that GmCPD3 only expresses highly in the young pods of soybean plants. The other is the potential role of GmCPDs in soybean flowering regulation. We scanned the entire developmental stage of soybean in a flowering reversion system and found that GmCPDs were under photoperiod control. The highest GmCPD transcript levels were observed on the 13 th day under SD treatment, when the floral meristem initiated. Additionally, GmCPDs expressed distinctly in soybean varieties with different photoperiod sensitivities, with insensitive varieties exhibiting higher expression levels especially in the early stages of development. The late flowering phenotype of the cpd mutant indicated an essential role of CPD in flowering regulation, but the expression patterns of GmCPDs in soybean suggested a contributing role of GmCPDs in the early stages of flowering development. Furthermore, all the four GmCPDs may perform individual roles and cooperate to regulate flowering. The genomic locations of GmCPD1 and GmCPD2 were associated to the QTLs of flower number and the time of the first flower (Fig. 3). Taken into account that GmCPD2 with the lowest identity of AtCPD was not influenced in the transcription level by highly expressed GmFT2a while other GmCPD homologs decreased in expression (Fig. 11F), GmCPD1 with the highest identity to AtCPD is more likely to play the major role in flowering regulation. Additionally, analysis of SSR markers around GmCPD3 and GmCPD4 suggested their association with QTLs of pod maturity and seed quality traits (Fig. 3). This result, taken together with GmCPD3 specifically expressing in young pods (Fig. 4A), was rather indicated that GmCPD3 and GmCPD4 may involve in post-flowering development and fruit ripping. Considering their behavior in flowering regulation, GmCPD3 and GmCPD4 are possible to contribute in the whole reproductive stage. Especially GmCPD4, bearing similar pattern with GmCPD1 and GmCPD2 in flowering regulation, may be the most versatile among this GmCPD genes. GmCPDs Act as Participants in Flowering Regulation Our study confirmed previous observations that cpd mutants exhibit a prolonged vegetative phase and delayed flowering (Fig. 10A, B) [12,34]. This phenotype can be rescued by overexpression of any of the GmCPDs we isolated (Fig. 10 A, B). It is therefore clear that GmCPDs are associated with flowering. CPD has been reported to interact with genes involved in the circadian clock [23,35], the upstream of FT in photoperiod pathway. However, in the analysis of AtFT expression in wild type, cpd-91 mutant and mutant with GmCPDs transformation, no obvious difference in expression pattern was found (Fig. 10C). In Col-0 Arabidopsis, the expression level of AtCPD was higher in vegetative stages and decreased after flowering (Fig. 10D). Therefore, GmCPDs may participate in flowering induction. Considering that there was no evidence of changes in flowering time when exogenous BR was applied, thus, GmCPD is not the trigger of flowering, acting as a participant rather than a decider. This hypothesis was illustrated by our analysis of GmCPD expression patterns in a flowering reversion system (Fig. 11). The striking observations were that expression of GmCPDs is under photoperiod control and is upregulated sharply on the 13 th day of SD treatment. The 13 th day of SD treatment (13SD) is rather special. In a previous study by our lab, Xiaomei Li et al investigated the morphological and anatomical changes that occur during flowering reversion of Zigongdongdou [39]. At day 13 under SD condition, the apical meristem began to initiate floral primordia inside the newly formed bracts. Before day 13, the apical meristem retained its vegetative status, and the floral primordia only appeared in the axils of newly formed trifoliolates. The same result also shown by Cunxiang Wu et al (Fig. 9) and Hongbo Sun et al (Fig. 6) [40,53]; although the lateral floral meristems appeared at SD7, inflorescence differentiation was initiated at the shoot apices at SD13, indicated by the formation of floral meristems and primordia. Logically, these results highlight the potential role of GmCPDs in the floral transition of apical meristem. One possible explanation of the delayed flowering in cpd mutants is that floral meristem formation is retarded in the absence of the CPD gene, resulting in prolonged flower development manifested as a flowering time delay. How CPD participates in floral meristem initiation has not been reported up to now, but the highest level of endogenous BRs and the highest expression of the BR-biosynthesis genes, DWF4, BR6ox1 and BR6ox2, have been observed in the apical shoots of Arabidopsis [57]. The effects of BR in cell elongation and cell wall modification is reported to be of vital importance for shoot apical meristem (SAM) function and inflorescence architecture in rice [61]. Further study on the relationship between CPD and the shoot apex meristem switch is needed. The new roles of CPD in plant development await uncovering.

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2019-04-20T13:14:14.793Z

2015-01-23T00:00:00.000Z

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Present and the future of axiomatic theory of boxed pigs Boxed pigs or rational pigs are well-known stories with many editions of game theory. In order to promote these to a systematic and scientific theory, an axiomatic theory, called L-system of boxed pigs, is established and some special subsystems are deduced from it. In this article, we introduce the background, the main results and the future research plan of axiomatic theory of boxed pigs. This introduction is divided into the following five aspects: appearance of boxed pigs, development and actualities of boxed pigs or rational pigs, boxed pigs in China, L-system of axiomatic theory of boxed pigs, and future research------non L-systems of axiomatic theory of boxed pigs. Appearance of Boxed Pigs The scholars who study animal psychology and behavior think that the equilibrium situation formed by the game amounts to the animals tending to the results calculated by game theory. However, the facts do not show that the animals know game theory. In fact, the reasons for the animals' behavior lie in genetic and natural selection (e.g., Dawkins, 1976;Smith, 1982;and Dawkins, 1986). Two psychologists, Baldwin and Meese (1979), did a famous experiment. They put two pigs, a big one and a small one, into a pigsty with a lever at one end and a trough at the other end. When the lever was pressed, a serving of pig feed would appear in the trough. They then observed the behaviors of the pigs. The final result was that the big one pressed the lever and the small one waited by the trough. Rasmusen (1989) introduced the example in his book on game theory called Boxed Pigs. He refined the model, the data set of which is according to Brockmann (1979), as follows. Two pigs are put in a Skinner box with a special panel at one end and a food dispenser at the other. When a pig presses the panel at a utility cost of 2 units, 10 units of food are dispensed. One pig is "dominant" (let us assume he is bigger), and if he gets to the dispenser first, the other pig will only get his leavings, worth 1 unit. If, instead, the small pig arrives first, he eats 4 units, and even if they arrive at the same time, the small pig gets 3 units. The following game summarizes the payoffs for the strategies "press the panel" and "wait by the dispenser." Development and Actualities of Boxed Pigs or Rational Pigs John McMillan (1992, pp. [13][14][15] rewrote Boxed Pigs as Rational Pigs. He rewrote the story into the following form. Two pigs, one dominant and the other subordinate, are put in a box. There is a lever at one end of the box which, when pressed, dispenses food at the other end. Thus, the pig that presses the lever must run to the other end; by the time it gets there, the other pig has eaten most, but not all, of the food. The dominant pig is able to prevent the subordinate pig from getting any of the food when both are at the trough. Assuming the pigs can reason, which pig will press the lever? For the sake of definiteness, let us attach some hypothetical numbers to this game. Suppose 6 units of grain are delivered whenever the lever is pushed. If the subordinate pig presses the lever, the dominant pig eats all 6 units, but if the dominant pig pushes the lever, the subordinate pig eats 5 of the 6 units before the dominant pig pushes it away. To fill in all the possibilities, we must make some assumption about what happens in the unlikely event that both press the lever simultaneously. Suppose the subordinate pig can run faster, so if both press, it gets 2 units of the food before the dominant pig arrives. Finally, suppose pressing the lever and running to the other end requires some effort, the equivalent of one-half unit of food. Our game, then, is represented by the following matrix (with the first number in each pair being the subordinate pig's amount of food and the second the dominant pig's): Mudambi (1996) said that "The big pig can run faster and eat faster than the little pig, so the little pig knows it will do better if it gets to the dispenser before the big pig" (p. 701). She said also, applying the pigs to a retail scenario, "The Boxed Pigs game predicts that if the innovation costs are high, the small retailer's best strategy is to monitor innovation investments by the large retailer, modify its own operation accordingly, and receive a small residual benefit" (p. 702). This shows that she realized that the big pig can run faster and eat faster than the little pig and that the two pigs' strategy choices involve costs. However, Mudambi's analysis is qualitative. Maxwell B. Stinchcombe (2002) wrote in a footnote that he first read about the rational pigs game in John McMillan's book (1992). Perhaps Maxwell thought that McMillan's model had unreasonable places (in fact, McMillan said that "we must make some assumption about what happens in the unlikely event"). For example, McMillan said, "Suppose the subordinate pig can run faster so, if both press, it gets 2 units of the food before the dominant pig arrives." Thus, Maxwell (2002, pp. 20-21) improved McMillan's model as follows. There are two pigs, one big and one little, and each has two actions. Little pig is player 1, big pig is player 2, and the convention has options with 1's being the rows, 2's the columns, and payoffs (x, y) meaning "x to 1, y to 2." The story is of two pigs in a long room; a lever at one end, when pushed, gives food at the other end; the big pig can move the little pig out of the way and take all the food if they are both at the food output together; the two pigs are equally fast getting across the room, but when they both rush, some of the food, e, is pushed out of the trough and onto the floor where the little pig can eat it, and during the time that it takes the big pig to cross the room, the little pig can eat α of the food. This story is interesting In order to facilitate comparing the symbols with those used by the book, we rewrite b as q and take the transposition of the above matrix. Then the payoff matrix becomes This model we will call the Maxwell Model. Boxed Pigs in China Rasmusen's model has economics background but is boring in mathematics. Before 1996, every scholar interested in game theory in China was a mathematician. Therefore at that time almost nobody in China knows boxed game. Zhang(1996) introduces Rasmusen's model. However he used the name "Rational pigs" which was given by McMillan. Since the simple story without mathematical formulas can be easily understood by humanists, the story "game of rational pigs" immediately attracted the humanists' attention in China and so the literatures in Chinese to use the story have increase sharply. However the grades of those literatures are low. L-system of Axiomatic Theory of Boxed Pigs In internet, Rasmusen's story is said to be "Game Theory on Rational Pigs". However, in fact, all these models are only "stories", instead of "theories" because they do not have any basic element of logic, such as concept, judgment, reasoning and argument, such as "big pig", "small pig", and the difference degree between the two pigs. In order to improve the stories into a systemic and scientific theory, we must use axiomatic methods, instead of stories. General Situation Jiang (2005) (L-system of Boxed Pigs and its Deductive Subsystems, 0≤L≤1) established L-system of axiomatic theory of boxed pigs and some deductive sub-systems were obtained from it, such as simple K-systems, instant K-systems and timing K-systems, K=0,1. Finally, the method to change a game among many pigs (can be infinitely many) into a game between a big pig and a small pig and applicable degree of the method are given. Basic Concepts Instant system: The pedals are pressed once, the food is sprayed in the feeding trough immediately. Timing system: The numbers of the food obtained by one or two of these pigs pressing the panel are functions of the time that the panel is pressed and similarly for the cost paid by laborer (s). Simple system: The numbers of the food obtained by the small pig, the big pig and the two pigs are equal and the cost pain by each of the two pigs is equal as well. Non-simple system: The sequence of numbers of the food obtained by the small pig, the big pig and the two pigs is monotonic non-decreasing and the cost pain by the big pig is not smaller than that done by the small pig. Peace level (0≤L≤1) : A parameter to describe the degree of the two pigs' friendship. Special background: L=0: For a non-patented technology development game, the factory to wait imitation runs orders to promote the sale of his future imitations; L=1: For a non-patented technology development game, the factory to wait imitation will begin imitation after the successful development of another factor. Applicable degree: Degree that a game amount many pigs (can be infinitely many) can be changed into a game between one big pig and one small pig. It is a non-negative real number. In particular, when it is equal to zero, the change method is not feasible. Basic Theory a. Big Pig and Small Pig: Precise definitions of a big pig and a small pig and the relation between degree of size difference of the two pigs and the results of the game are given. b. Inclusion relations of the systems: Simple system ⊂ Instant system ⊂ Timing relation ⊂ L-system. c. Labor enthusiasm theorem: In certain condition, 1) the small "hitchhiking" if and only if the big pig's labor brings profit and the small pig's that does loss, and 2) when the small pig's labor brings profit, his labor enthusiasm is higher than the big pig's that (The theorem is supported by actual cases). d. Mechanism adjustment: Adjust some indices to overcome the small pig "hitchhiking". e. Mechanism control: Set some indicators such that the two pigs' labor enthusiasms to conform intended targets. f. Situation distribution: Order of probabilities of the two pigs' action profits. g. Game amount many pigs: Change a game amount many (can be infinitely many) pigs into a game between one big pig and one small pig. Game between One Big Pig and One Small Pig One big pig and one small pig are put in a special pigsty with one trough under a food dispenser and panels on the two sides of the pigsty. Assume the big pig presses on the panel for A system is said to be 0-peace if it satisfies the 0-peace assumption: The waiting pig immediately eats the food once he sees it. A system is said to be 1-peace if it satisfies the 1-peace assumption: If one of them presses the panel, the other one waits for the laborer to eat the food together with him. Outlooks and Prospects Because the topic of this book comes from applications to economic management problems, it will have broad application prospects, mostly likely in the following three areas. a. Mathematical economics and management science. The phenomenon of the boxed pigs permeates every corner of society, politics, economy, life, and so on. For example, labor enthusiasm theorems are new economic laws. Our principle can be used to control and adjust social members' labor enthusiasm. We anticipate that this book will likely promote research on mathematical economics and management science. b. Animal behavior and experimental game theory. Animal psychologists may promote Baldwin and Meese's experiment (1979) based on our theory. Before performing the experiment, the animal psychologists should know the following. (a) Some indicators such as the quantity of food into the trough (or briefly QFT), the distance between the trough and the panel (briefly, DTP) and costs and so on need be pre-set. (b) The two pigs can eat the food within the trough together, i.e., the big pig cannot bully the small one. (c) The two pigs' running velocities and eating velocities must be measured before the experiment. (d) The two pigs can be replaced by other animals such as two dogs, two cats and so on. Based on many research studies, one can compare the stable situation and theoretical calculation and analyze the reasons for differences, such as intelligence, psychological and genetic factors, and so forth. Therefore, this book puts forward some new research topics to animal psychologists and experiment game theory scholars. c. Rise of applied research of axiomatic theory of boxed pigs. The traditional model of boxed pigs has been applied in various fields, including economic management. However, because there was no strictly scientific theory of boxed pigs, the quality of the research is low. The lack of theoretical results has hindered the progress of application research. Thus, we anticipate that axiomatic theory of boxed pigs will promote the applied study of the boxed pigs and will open up a more profound and scientific research field. Academic Evaluation about Research Signification and Creativity As everyone knows, significance and creativity in one's research result in a life of academic achievements. So far, 30 experts have reviewed this study. The research significance and creativity of the study obtained 29 experts' consistent affirmation. However, in all the inspection reports of the National Natural Science Foundation of China in 2013, the fifth reviewer (in fact, it is very clear that he did not read my application report) wrote that "The applicant reviewed the research significance of his own project, and he thinks that his own project has the original innovation thought and exploratory. However this reviewer does not think so." Thus, the reviewers to negate my research significance and creativity are less than 3.3% of the total number who have examined them. This is no wonder because a new academic thought has to accept a long time contending and needing to overcome all kinds of resistance. The shortage of Research Funding In recent years, the shortage of research funding has forced me into trouble. I do not have money to attend academic conferences and to publish papers to exchange the progress of my axiom theory of the boxed pigs. My application to NNSFC in 2012 was refused based on a reviewer's opinion is that "this project does not belong to the international research hot spot." My application to NNSFC in 2013 was refused again based on the fifth reviewer denying my innovativeness and the significance of my research. Even the lower-grade foundation project, the Natural Science Foundation of Jiangsu Province, refused my applications two times in 2008 and 2012 without any appraisal report. International Support God never shuts one door but he opens another. Mother's cruel weaning can not kill her life hard baby. The poor baby, axiomatic theory of boxed pigs, in the time of crisis was supported by international academic field and international press field. At present, their support includes the following three aspects: (1) Columbia International Publishing of the United States published the monograph "L-system of Boxed Pigs and its Deductive Subsystems", Jiang (2015), and provided the founds for the publication of this book. (2) International Journal of Advanced Computing published freely our team's paper, Jiang et al.(2014). (3) Another international academic publishing company of the United States, Science Publishing Group, invited me again and again to set freely the special issue "Axiomatic Theory of Boxed Pigs" in the international Journal "Economics" and hire me to be its lead guest editor. See: http://www.sciencepublishinggroup.com/specialissue/1770 01. Attention and Support of OR field of China Axiomatic Theory of Boxed Pigs has obtained attention and support of OR field of China, which is embodied in the following two aspects: (1) Sci.and Tech. Institute of China and OR Society of China (2013), a programmatic authority literature of OR society of China, introduced the research progress of axiomatic theory of boxed pigs. (2) A special topic speech with the title "Introduction to Researching Progress of Axiomatic Theory of Boxed Pigs" was invited by OR society of China in 2014 (ORSC2014). Incompleteness of L-System (1) Purpose: Try to find simple, instant and timing K-systems, K=0,1, which are not deduced from L-system. (2) Progress: At present we have found that these non-deductive systems of L-system exist and that there no inclusion relation between the instant system and timing system. However, the simple systems can be deduced from them and L-system (L=0,1), respectively. Negative L-Systems and Super L-Systems In the L-system, L satisfies the condition 0≤L≤1. Next work is extending L such that either L<0 or L>1. Negative L-Systems (L<0) It can be used to describe the game that non-laborers can get in advance profit from laborers. For example, when a small developer in a remote area knows that a big one will develop a large project there, such as building big shopping malls, a large square, residential areas, and so on, the small developer will raise his prices, and his commercial housing becomes hot in sales although those large projects may not be built for many years. Super L-Systems (L>1) It can be used in the field of technical innovation. For example, for non-patented technology, if its technology difficulty is very great, then it needs a long time from imitation to sale. For patent technology, after the developer gains his profit and enjoys the patent protection, the imitators can be allowed to freely copy the productions and sell them. L-Negative Systems In an L-system, the quantity of food into the trough (or briefly QFT) in a simple instant L-system is always assumed to be positive, i.e., q>0. And the laborer needs to pay positive cost c >0, i.e., he can suffer losses. However, there is another case in a social system. When a pig presses the panel, some negative public interest, called also a public nuisance may occur, i.e., q<0 . And he needs to pay the negative cost, c<0, i.e., he gets a positive benefit. L-negative system can be found from the games that some players, called black sheeps, harm public interests for their own benefit.

v3-fos

2019-04-25T13:03:40.282Z

2015-10-23T00:00:00.000Z

55826034

s2

Sprouting Value Index: A Mathematical Approach for Evaluation of Vegetative Propagation in Tree Species Rooting of Saraca asoca stem cuttings using three IBA (Indole 3-butyric acid) concentrations 300 ppm, 500 ppm and 1000 ppm IBA, have been carried out and the data obtained was evaluated with SVI (sprouting value index) method in order to interpret the vigor in rooting/sprouting and the suitable medium, which gives maximum results. Field studies were carried out using three rooting medium at a location in Central Kerala, Peninsular India, for three years at four months regular interval. The control cuttings do not recorded rooting. The results obtained indicate high SVI (sprouting value index), when prop root cuttings planted in root trainers with coir pith compost (RTCP) for all the three concentration of IBA applied. Sprouting percentage was increased and the delay in completion of sprouting/rooting initiation decreased by the use of IBA treatment. Increased CWR and SUP percentages indicate defects in management practices, dormancy and or the genotype of the cultivar. Introduction Plant propagation has a global effect, as it is a fundamental occupation of human kind and its discovery dates back to the origin of civilization and ornamental gardening attained high level during the period between 500 B.C to A.D. 1000 [1], [2]. The vegetative propagation of herbaceous plants can be performed easily as many of them produces bulbs, rhizomes, corms, tubers, offsets, suckers and stolons which are natural vegetative propagating structures. But vegetative propagation in tree species is a difficult process and it is attained through hormone application mostly in stem cuttings, which eventually initiates adventitious root and shoots [3], [4], [5]. Again, there are several mathematical expressions and explanations to measure seed germination and viability in sexually reproducing plants [6], [7], [8], [9], [10] but mathematical expressions for determining efficiency of vegetative planting material or for clonal propagation is scanty. The data obtained through the present investigation are being interpreted using sprouting value index (SVI). Saraca asoca (Roxb.) de Wilde is an enlisted medicinal tree species of the tropics with astringent bark, which is reported to have stimulating effect on the endometrium and ovarian tissue, and is useful for treating menstrual pain and menorrhagia due to uterine fibroids, leucorrhoea, and internal bleeding, haemorrhoids and haemorrhagic dysentery. In Ayurvedic medicine, it is also used for treating dyspepsia, diseases of the blood, biliousness, tumours, abdominal enlargement, colic, piles, ulcers and bone fractures. The flowers pounded in water, are used to treat haemorrhagic dysentery and the dried flowers for diabetes; they are considered to be an excellent tonic, and useful for the treatment of syphilis and biliousness. The edible seeds are diuretic. [11], [12], [13], [14] [15]. The lack of technology for regeneration from cuttings was considered for selecting the plant for the experiment trials. Specimen Collection and Conduct of Field Trials For the present investigation plant samples were collected from T.C Joseph Memorial Botanical Garden, of the Department of Botany, Union Christian College, Aluva, (+10° 7' 30.65", +76° 20' 3.32") Ernakulam district, Kerala State, India. Plant was identified by literature scrutiny, as the plant is not recorded in any local and national herbaria in South India. All field trials was conducted in the plant nursery of T.C Joseph Memorial Botanical Garden, of the Department of Botany, Union Christian College, Aluva, (+10°7'30.65", +76°20'3.32") Ernakulam district, Kerala State, India. For the sake of large scale planting material production in mechanized gardens, trials were conducted in 100cc roottrainer blocks (24 celled) using potting mixture (RTPM), root trainers with vermin compost (RTVC) and root trainers with coir pith RTCP as rooting medium. Experiment Design For all field trials, average of the three replicates during the months of February (P1), and June (P2), October (P3) from Feb 2009 to October 2012 were conducted in three different rooting medium such as root-trainers with potting mixture (RTPM), root trainers with vermi compost mixture (RTVC) and root-trainers with coir pith (RTCP). Stem cuttings with an average size of 7.5 to 10 cm were used. The sample size was kept twenty-four for each trials separately as the 100cc root-trainer block contains 24 cells. A non-auxin control and three Indole 3-butyric acid (IBA) concentrations were designed in this experiment with 300ppm, 500ppm and 1000ppm (parts per million) in order to detect the rooting/sprouting ability by quick dip method. A randomized complete block design was employed. After 45 days, the cuttings were evaluated for rooting/sprouting percentages, mortality percentage and viability percentage. The data obtained were subjected to one factor analysis, employing analysis of variance (ANOVA) and two-way ANOVA. Calculation of SVI In order to develop the new idea of calculating sprouting value index (SVI), data regarding sprouting and successful rooting percentages (SP -sprouting percentages), percentage of planting material with callus production but without rooting (CWR -callus without rooting), percentage of sound unsprouted propagule without callus production (SUP) (was determined by vertical cut test: planting material with more than three nodes living tissues was considered viable) was prepared. Viability percentage can be prepared using the formula (VP = SP + CWR + SUP), peak value (PV = maximum mean sprouting recorded at any time during the test), final mean sprouting (final MDS = cumulative percentage of full sprouting at the end of the test divided by number of days to finish sprouting) were calculated. SVI index method developed through the present study is calculated by the equation, SVI = PV * MDS. Results Sprouting and rooting studies of stem cuttings were carried out in three different rooting medium during three seasons in an year (June, October and February), for three consecutive year and the various attributes obtained are given in table 1 to table 6. Stem cuttings of seven-year-old Saraca asoca plants was used as mother plants in order to obtain stem cuttings at four months interval. Table 1, table 2 and table 3 gives the VP of prop root cuttings in three concentrations used against the control whereas table 4, table 5 and table 6 gives SVI in different concentrations. Callus production as well as sprouting and rooting was very poor in control. High VP was obtained with all the three concentrations of IBA used (ranging between 83.05% to 95.84%) in the rooting/sprouting medium RTCP. With stem cuttings of Saraca asoca, maximum SVI was obtained in RTCP 1000ppm treated cuttings (29.17). The speed of completion of sprouting/rooting in RTVC was found lower than in RTCP. The ANOVA results on callus production show significance at 1% level between concentrations of IBA and significance at 5% level between media of study. The ANOVA results on callus formation and callus with root formation show significance at 1% level between concentrations of IBA and between mediums. The results was non significant at 5% level between years and month of study. The ANOVA results on callus formation and callus with root formation show significance at 1% level between concentrations of IBA and significance 5% level between media of study for callus formation root production show significance at 1% level between medium of study. Discussion The SVI studies worked out in the present study is a mathematical approach to determine the quality of rooting material, suitability of the medium used for rooting and the optimum concentration of IBA to be used. Production of elite genotypes of any plant species can be generated by vegetative method of propagation round the year. Successful propagation using stem cuttings has been reported by several studies in various plants, which uses stem cuttings, rhizomes or other vegetative parts [16], [17], [18], [19], [20] but rooting of stem cuttings of tree species is difficult to the cultivation field. In field trials, all planting materials gave elite performance in RTCP planting medium. In the trials using 1000ppm IBA (Indole 3-butyric acid) treated cuttings also SVI was highest in RTCP (29.17). Significant differences in rooting were found between various rooting media when effect of rooting, in sheanut stem cuttings were performed [21]. Sprouting percentage and speed of completion of sprouting/rooting initiation increased by the use of IBA treatment [14]. However, it is shown by other workers [21] that very high IBA concentration have negative effect in rooting. Comparing the CWR and SUP percentages one can also assess the defects in management practices, dormancy and the genotype of the cultivar. Sprouting index value (SVI) proposed through the present study is a modified form of Germination value (GV), proposed by Czabator [6] for seed germination studies. It is also suitable in field and nursery trials for vegetative propagation and IBA treated rooting of cuttings. The incorporation of CWR (callus production without rooting) along with SUP (sound unsprouted propagule) in calculating VP (Viability percentage) is effective in finding the field oriented defects. SVI and VP is an integrated measure of planting material quality. The speed of sprouting/rooting ability along with the completeness of sprouting can also be determined vegetative planting materials. Suitable rooting media hold considerably high rooting ability [14] [21]. Even though SVI for control were found zero, the VP for all the IBA treated trials are > 90% (table 1, table 2 and table 3) which indicates that by using alternate methods like pretreatments and management practices, SVI can be increased. The internal physiology of the planting material may be the reason for the same. The change in planting material and IBA treatment used affects the rooting process as in the present study; SP was increased in higher concentration (1000ppm IBA). Methods for reducing CWR and SUP values can increase SP and the sample in rooting medium with least difference in SP and VP value will give maximum performance. Results of the rooting experiments showed that with very high IBA concentration in sheanut tree cuttings, rooting ability decreased [21] and hence three concentrations of IBA were used in the present experiment. Conclusions The present study focuses on regeneration of plantlets from stem cuttings of Saraca asoca and the data obtained were interpreted using sprouting value index (SVI) to establish suitable vegetative planting material and suitable rooting medium through field trials. Trials conducted in different rooting medium reveal the sprouting efficiency and vigor in different medium. SVI is a statistically treated data, which is obviously a modification of germination value for seed germination proposed by Czabator [6] the incorporation of CWR (callus production without rooting) along with SUP (sound unsprouted propagule) in calculating VP (Viability percentage) makes it suitable for vegetative cultivation practices and it interprets the quality of planting material, failure due to management practices and the selection of suitable rooting medium. This method of planting stock preparation is valuable in large-scale cultivation and much promising in producing quality clonal planting material production in economically important plants in future.

v3-fos

2019-03-31T13:46:25.093Z

2015-01-01T00:00:00.000Z

26647244

s2

PRODUCTION OF EXTRACELLULAR PIGMENT FROM MICROBES AND ITS APPLICATION Vegetable sample procured from local markets of Tamil Nadu were used in this study to isolate pigments producing bacteria. Red pigment bacteria were isolated and developed into nutrient media. Morphological observations revealed that the isolated bacteria are Gram Negative, cocci and biochemical characterization was carried out and isolated are identified as Rhodotorula sp. Maximum production of pigments was observed at 35°C and pH 7. Pigment was extracted from the isolated microorganism and it was dried and powdered. Extracted pigment was check for the microbial action and it was used to dyeing the cloth. INTRODUCTION Natural colours are generally extracted from fruits, vegetables, roots and microorganisms and are often called "biocolours" because of their biological origin ( Pattnaik et al.., 1997). Heath environmental concerns due to unmonitored utilization of synthetic colorants revived interest in natural dyes as they are safer, healthier, biodegradable, and exhibit higher compatibility with the environment ( Fatima shatila et al.., 2013 ) Many artificial synthetic colorants are widely used in food stuff, dye, cosmetic and pharmaceutical manufacturing processes. The synthetic pigments which are used produce harmful effect to human and pollute water and soil. In textile industry during manufacturing and usage approximately 10-15% of the dye is lost directly to wastewater and pollute the environment ( Palanivel velmurugan et al.., 2009 ). Natural pigments can be obtained from two major sources namely plants and microorganisms. The accessible authorized natural pigments from plants have numerous drawbacks such as instability against light, heat, or adverse pH and low water solubility. The advantage of pigment production from microorganisms include easy and fast growth in the cheap culture media. Microoganisms produce various pigment like carotenoids, melanins, flavones, quinines. The various types of microorganisms like bacteria, fungi, algae, yeast are present in different colours. The coloured microorganisms are isolated from various samples and pigment was extracted from the microorganisms and it used for industry ( Sahara sayeed khan et al.., 2013 ). Different pigmented microorganisms were isolated from the vegetable effects on temperature and pH for pigment production was identified. The particular strain was identified by using different biochemical test. Hence microbial pigment production is one of the emerging fields of research to demonstrate its potential for various industrial application. There is an increasing demand for natural colour in the food, pharmaceutical, cosmetics, textile, printing and dye industry. Collection of Samples: Vegetable (beetroot) sample was collected from local market in Erode, Tamil Nadu, India. Sample preparation: 1gm of vegetable (beetroot) was mashed with 10 ml of distilled water and it was used. Isolation and screening of pigmented microorganisms: The sample was serially diluted up to 10 -3 and 0.1 ml of the diluted sample was spread on nutrient agar plate. Incubated for 24hrs at 30 0 C and it was checked for the pigmented production. The pigment produced colonies were used for the future studies. Purification of cultures Pigmented Bacterial isolates was purified by streaking onto nutrient agar plate and it was incubated for 24hrs at 30 0 C. Maintenance of culture Pigmented Bacterial cultures were grown on nutrient agar and it was maintained at 2-4 0 C temperature in refrigerator and sub cultured into respective medium. Characterization of isolated pigmented microorganisms: Gram stain: The stain makes use of the differing membrane structures between Gram positive (single cell membrane with a tough outer cell wall of peptidoglycan), and Gram negative organisms (have two layers of membranes, with a thin layer of peptidoglycan sandwiched between them). Prepare a bacterial smear and heat fixed on a slide, pour a few drops of crystal violet on a smear waite for 1 minute and wash with water. Now fixed the smear with Gram's iodine for 1 minute and wash again with water and decolourize the stain with 95% ethyl alcohol dropwise, wash with water and conter stain with safranine ( 45 sec ) and again wash with water. After drying examine under oil immersion. Indole test: Used to determine the ability of an organism to split indole from the amino acid tryptophan using the enzyme tryptophanase. Red layer formed on surface of the media showed Positive result and yellow layer showed negative result. Methyl Red Test It is used to determine the ability of an organism to produce mixed acid end products from glucose fermentations. Some organisms produce large amounts of various acids (lactic, acetic, succinic, formic) plus H2 and CO2. The large amounts of acids lower the pH to lower than 5.0. These organisms also produce great amounts of gas due to the presence of the enzyme formic hydrogenlyase. Red color developed indicates Positive result and yellow color developed indicates Negative result. Voges-Proskauer Test It is used to determine the ability of an organism to produce acetoin; 2,3 butanediol; and ethanol which causes lowering of the pH than the methyl red positive organisms. VP test detects the presence of acetoin, which is a precursor to 2,3 butanediol. Citrate Utilization It is used to determine if an organism is capable of using citrate as the sole source of carbon with production of the enzyme citratase. The media contains sodium citrate as the carbon source, and ammonium salts as the nitrogen source, with bromothymol blue as the pH indicator. An organism that uses citrate breaks down the ammonium salts to ammonia, which creates an alkaline pH. Alkaline pH causes media to change from green to Prussian blue shows positive result and no color change in the medium denotes negative result. Catalase Test It is used to test for the presence of enzyme catalase. Hydrogen peroxide (H2O2) is formed as an end product of the aerobic breakdown of sugars. When H2O2 accumulates, it becomes toxic to the organism. Catalase decomposes H2O2 and enables the organism to survive. Only obligate anaerobes lack this enzyme. Bubbling (O2 gas is liberated from the H2O2) shows positive result and no bubbling denotes negative result. Nitrate Reductase Test It is used to determine the ability of an organism to reduce nitrate (NO3) to nitrite (NO2) or nitrogen gas (N2) by the production of the enzyme nitratase. The reduction of nitrate to nitrite or nitrogen gas takes place under anaerobic conditions in which an organism derives its oxygen from nitrate. Appearance of red color denotes that nitrate is reduced to nitrite and it is a positive result. If there is no color change the confirmation test was done by adding a small pinch of zinc powder. Urease Test Used to determine the ability of an organism to split urea to form ammonia (an alkaline end product) by the action of the enzyme urease. Media also contains the pH indicator phenol red, which turns an intense pink at alkaline pH. Intense pink/red color indicates positive result and no color change denotes negative result. Triple sugar iron agar test (TSI) This test is specifically used for the identification of enteric bacteria, but can also be used for other organisms. This agar contained 0.1 % glucose, 1.0% lactose, and 1.0% sucrose in one tube. Along with the three sugars, phenol red was also present to verify if fermentation occurred. Lipid hydrolysis It is used to determine the ability of an organism to produce the enzyme lipase which hydrolyzes fat. Lipase splits fats into glycerol and fatty acids that can be used for anabolism or energy production. Extraction of pigments Different solvents like ethyl acetate, methanol, acetone, hexane was used to check for the maximum solubility of pigments. And the solvent was selected by checking the maximum solubility of pigment in it. After incubation the bacterial cells were washed with methanol and it was transferred to centrifuge tube. The tube was centrifuged at 5000 rpm for 15 minutes. The colored supernatant and pellet was separated. The colored supernatant was used directly and pellet was transferred to evaporating dish for one day and kept at room temperature. From the vegetable (beetroot) extract, one pigmented microorganism was isolated. Effect of different pH & Temperature on the pigment color Selection of pH & temperature for pigment producing microorganisms. When the Red pigment producing colonies are treated with different pH, different coloured pigments are produced. Table 1 shows the various colours produced when pH & Temperature is changed ( Ahmad et al., 2012 ). When the temperature is changed from 25 0 C to 60 0 C there was no change but above 60 0 C the Red colour colonies were changed to pink colony. Biochemical test results The biochemical results for the Red pigmented microorganism was shown in the following Table 2. The table shows that the Red pigment producing microorganism can split indole & can produce mixed acid end products from glucose fermentations with production of citrate, catalase, nitrate reductase & urease enzymes, TSI test shows that this organism can ferment glucose only. These organism can also hydrolyse lipids Extraction and drying of pigment Pigments were extracted from the isolated microorganisms. Methanol was used for the pigment extraction and the extracted pigment was dried and it was powdered. Pigments that are isolated from the microorganisms was checked for the microbial action. Extracted pigment was streaked on to the nutrient agar plate and it was incubated at 30 0 C for 24 hrs. After incubation there was no microbial growth. Extracted extracellular pigment was used for dyeing the cloth. These microbial pigments can also used for coloring candles, paper, soap, pencil, and high lighter pen. Fig8.Identification of microorganisms Microorganisms were identified using known microorganisms. CONCLUSION Colorants are used in a vast majority industries from clothing and textiles. Most of the synthetic colorants are harmful to the environment and are difficult to biodegrade. There is an increasing interest involving microorganisms as an alternate source of synthetic colorants. In this consideration the present study was carried out to reduce the effects of non biodegradable pollutants. In the above study soil was used for isolating microorganisms. The pigment ( Red ) producing microorganisms are isolated from the source and their pigment production was increased in the nutrient medium at pH 7 at 30 0 C. This indicates that pigment production in influenced by physical factors like temperature & pH of the culture medium.The biochemical characterization for the Red pigment producing microorganism showed positive result and strain characterization revealed that it is small, circular and gram negative bacteria and this was compared with the known species Rhodotorula which resembled similar. The pigments produced were extracted with methanol and were tested for the presence of microbes in the pigments. The extracted pigment was applied for dyeing fabric. The results showed that the fabric can uptake the dye after 24 hrs of soaking. In future the present study can be implemented to replace the synthetic colorants used in textile and dying industries. This aims to reduce the toxic effects of synthetic colorants in aquatic ecosystems.

v3-fos

2019-03-30T13:08:17.712Z

2015-01-01T00:00:00.000Z

21152764

s2

Molecular Genetic Variations among Some Punjab Sheep Breeds Using RAPD Analysis The present study was carried out on seven sheep breeds (Thalli, Lohi, Salt Range, Kajli, Sipli, Buchi and Hissradale) by Random Amplified Polymorphic DNA (RAPD) analysis using 21 random decamer primers. Blood samples were collected from different numbers of animals per breed, of both sexes. After DNA extraction PCR was carried out by using 25 μL reaction mixture containing 3 mM MgCl2, 100 μM each of dATP, dCTP, dGTP, dTTP, 0.2 μM primer, 15 ng of genomic DNA, and 5 unit of Taq polymerase, through programmable thermal cycler (Crea Con, TCY, USA).Out of 21 decamer primers 16 primers yielded easily scorable bright DNA bands while other five yielded smeared and nonspecific fragments. The genetic similarities of seven sheep breeds were high, ranging from 74.42% to 94.29 %. Thalli and Latti had greatest similarity (94.29%). The lowest genetic similarity was between Lohi and Hissardale (74.42%). The unweighted pair group method of arithmetic means (UPGMA) dendrogram obtained from the cluster analysis revealed two clusters. The study confirms that the molecular genetic techniques such as RAPD-PCR can economically and efficiently be used to establish genetic distances and similarities among and within breeds as well as to find out breed specific genetic markers. The seven sheep breeds in the study could be identified by using one breed specific RAPD marker or a combination of two or more markers. Primer GLB-08 can identify Lohi breed and primer GLA-14 can identify Hissardale. Primer GLA-19 can identify both Kajli and Sipli breeds simultaneously. Introduction Sheep and goat were perhaps the first ruminants to be domesticated around 10,000 B.C. Sheep were first domesticated probably in Iran and Baluchistan and what is now the Near East. Domesticated sheep existed in Harappa and Mohenjo-Daro. Most of the present breeds of sheep have traces of Mediterranean as well as Asian wild sheep [12]. The Pakistani sheep breeds most probably descended from urial (Ovis vignei), the wild sheep of Baluchistan, Afghanistan, and the central Asian area, as well as from the argali (Ovis ammon), and the Marco Polo sheep of China. At present there are 28 sheep breeds in Pakistan [21]. There are 25.5 million heads of sheep in Pakistan producing 40.7 metric tons of wool and contributing a reasonable amount of mutton (782.1 metric tons) together with goat population [3]. Livestock diversity is shrinking rapidly. Among the domesticated animal populations, it is estimated that 1 to 2 breeds are lost every week. So there is an urgent need to define strategies to prioritize breed conservation [10].The need for conservation comes from the potential rate of decrease of genetic variation. The loss of genetic variation within and between breeds is detrimental. Once animal genetic diversity has been lost, it can not be replaced. Indigenous and locally developed sheep breeds are an important asset for many reasons, particularly because, over time, they have developed unique combinations of adaptive traits to best respond to pressures of local environment [4]. Conservation of genetic resources requires the characterization of the available stock for preservation and management as well as evaluation of the phylogenetic origins of the genetic groups available [18]. In the recent years, several new and sophisticated methods have been developed to screen the polymorphism at the DNA level. A recent technique capable of detecting DNA based polymorphism is random amplified polymorphic DNA (RAPD) analysis. It is based on random amplification using polymerase chain reaction (PCR) with oligonucleotide primers. Single primer can be used to amplify genomic DNA and that polymorphism can be detected between the amplification products of different individuals. Polymorphism between individuals can arise through (1) nucleotide changes that prevent amplification by introducing a mismatch at one priming site (2) deletion of a priming site (3) insertion that render priming sites to distant to support amplification (4) insertions or deletions that change the size of the amplified product [26]. Efficient use of RAPD markers requires DNA isolation, optimum amplification conditions and appropriate data analysis. Thus, the present study was planned to determine molecular genetic variation among sheep breeds (Buchi, Hissardale, Kajli, Lohi, Sipli, Thalli and Salt Range) of Punjab province, Pakistan by using RAPD analysis with the following objectives:  To optimize conditions for RAPD-PCR amplification for sheep genomic DNA.  To construct a dendrogram among seven sheep breeds of the Punjab province in Pakistan on the basis of genetic variation. Materials and Methods The animals from sheep breeds (Buchi, Hissardale, Kajli, Lohi, Sipli, Thalli and Salt Range) of Punjab province were sampled according to their spatial distribution keeping in view the breed characteristics and where possible, pedigrees were consulted to ensure that animals were unrelated. Blood samples were collected from animals kept at different livestock farms, under the supervision of Livestock and Dairy Development Department (L&DD) Punjab. Blood samples were taken from various numbers of animals per breed, aseptically from jugular vein in properly labeled 5ml potassium Ethylene Diamine Tetra acetic Acid (EDTA) vacutainers. Blood samples were immediately transferred to ice and later stored at -20 o C till further processing. High molecular weight genomic DNA were extracted from the frozen peripheral whole blood according to the FBI protocol reported by Signer et al. (1988) and Grimberg et al. (1989) with some slight modifications [9,22]. The concentration of total genomic DNA was calculated by measuring Optical Density (OD) at 260 nm (1 OD 260 = 50 µg of double stranded DNA/ml) on UV visible spectrophotometer. The DNA bulks of each breed were prepared by pooling equal quantities of DNA from available number of individuals. PCR was carried out in 25µL reaction mixture containing 3 mM MgCl 2 , 100 µM each of dATP, dCTP, dGTP, dTTP, 0.2 µM primer, 15 ng of genomic DNA, and 5 unit of Taq polymerase, through programmable thermal cycler (Crea Con, TCY, USA). A total of 21 primers were used for the analysis. The thermal cycler was programmed for 5 minutes initial denaturation at 94 o C for first cycle, followed by 1 minute denaturation at 94 o C, 1 minute primer annealing at 36 o C and 2 minutes extension at 72 o C for next 40 cycles and then final extension at 72 o C for 10 minutes. PCR reaction components are given under in table 1. The PCR products were electrophoresed at 50 V in 1.2 % agarose gel for approximately 2 hours using 0.5 X Tris Boric Acid EDTA (TBE) buffer containing ethidium bromide (10 ng/100 mL agarose solution in TBE). The fingerprints were examined under ultra violet illuminator and photographed using the Uvitec Gel Documentation Still Video System. Amplification profiles of seven sheep breeds were compared with each other and bands of DNA fragments were scored as present (1) or absent (0). The data of the primers were used to estimate genetic similarity (GS) on the basis of number of shared amplification products [16]. The coefficients were calculated by the following statistical equation; GS is the genetic similarity coefficient and D is the genetic distance in which N a and N b are the numbers of fragments in breed a and b, respectively, whereas Nab is the fragment shared by the two populations. Similarity coefficients were utilized to generate a phylogenetic tree (dendrogram) by using unweighted pair group method of arithmetic means (UPGMA) [23]. Results and Discussion Pooled DNA of seven sheep breeds were amplified with 21 different random primers. Five primers amplified no fragments or yielded smeared bands that could not be clearly identified. A total of 53 DNA fragments were generated by the remaining 16 primers with an average of about 3.3 bands per primer. The number of DNA fragments amplified with each primer was in a range reported in different studies using the random primers [5,19]. Bands that a primer yielded in this study ranged from one to six. Generally, the size and number of the fragments produced strictly depended upon the nucleotide sequence of the primer used and upon the source of the template DNA. To check the consistency of the amplified products, reactions were duplicated from time to time. Only easily resolved bright DNA bands were considered and scored. All the breeds showed variation with each other on their amplification profile bases. Of the total 53 DNA bands amplified by 16 primers, 16 fragments showed polymorphism among seven sheep breeds, which is an approximately 30.19% polymorphism. The rest of the 37 bands (69.81%) were monomorphic in all seven sheep breeds. These results indicated that the level of DNA variation was low among the seven sheep breeds. These seven sheep breeds could be identified by using one breed specific RAPD marker or a combination of two or more markers. Primer GLB-08 can identify Lohi breed and primer GLA-14 can identify Hissardale. Primer GLA-19 can identify both Kajli and Sipli breeds simultaneously. Therefore RAPD markers can be used for identification of sheep breeds. Out of seven breeds studied, Kajli sheep produced the maximum number of DNA fragments (46) while minimum numbers of fragments (40) were produced by Hissardale. Genetic Similarity among sheep breeds The genetic similarity matrix of RAPD data for the seven sheep breeds was constructed based on Nei and Li's (1979) coefficient of similarity and is shown in table. The genetic similarity of seven sheep breeds were high, ranging from 0.9429 to 0.7442. Genetic Similarity among sheep breeds Nei's standard genetic distance (D) was calculated from RAPD-PCR data to estimate the molecular genetic variation among seven sheep breeds. Genetic distance among these breeds was very low in the range of 0.0588 to 0.2954. the maximum genetic distance was found between Lohi and Hissardale, while, minimum genetic distance was found between Thalli and Salt Range (Latti) (0.0588). Template DNA concentration of 15 ng/uL was found to be optimum. The optimum concentration of MgCl 2 was found to be 3 mM. MgCl 2 concentration has a profound effect on DNA amplification. Five unit/25uL reaction mixtures were found optimum. The optimum annealing temperature found in this study was 36 o C. Forty cycles per reaction was found optimum. Random amplified polymorphic DNA (RAPD) produced consistent results with optimized conditions and have potential to be employed for molecular genetic variations and taxonomy classification. The observed polymorphism in the present study was 30.19%, while 69.81% fragments were monomorphic. The probability of finding these short decamer primer cognate sequences in the target DNA varies and may be numerous, such that a single primer generates multiple amplification fragments. Forty two animals were analyzed out of the sixty nine selected for blood collection. Genomic DNA of six animals from each breed was extracted and pooled to make bulks by mixing equal quantity of DNA from each individual. Nei's standard genetic distance among seven breeds was very low in the range of 0.2954 to 0.0588. In the present study the maximum genetic distance was found between Lohi and Hissardale (0.2954), while, minimum genetic distance was found between Thalli and Salt Range (Latti) (0.0588). On the other hand the genetic similarities of seven sheep breeds were high, ranging from 94.29 % to 74.42 %. Thalli and Latti had greatest similarity (94.29 %). The unweighted pair group method of arithmetic means (UPGMA) dendrogram obtained from the cluster analysis revealed two clusters. Conclusions Genetic distance among these breeds was low in the range of 0.0588 to 0.2954. The low levels of genetic variation among these breeds indicate the high degree of genetic relationships. This increase in the relatedness among these indigenous breeds is the result of haphazard and indiscriminate crossbreeding in the home tracts of the respective breeds, and lack of the implementation of the provincial and national breeding policies in the country. For molecular genetic studies, sophisticated inputs such as; authenticity of pedigree, appropriate selection of molecular technique, availability of a prior knowledge about the genome of respective species, stipulation of good working conditions, latest equipments and laboratories are of supreme importance. A large number of primers from different companies, more number of animals from each breed with authentic pedigree recording and sampling from breed home tracts is also suggested along with more advanced molecular genetics techniques using microsatellites such as SSR, in order to refine the relationship.

v3-fos

2019-08-18T21:53:17.714Z

2015-01-01T00:00:00.000Z

251346234

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‘Polares’ Blackcurrant Polares is a new blackcurrant (Ribes nigrum L.) cultivar released from the Ribes breeding program conducted for many years at the Research Institute of Pomology and Floriculture (now the Research Institute of Horticulture) in Skierniewice, central Poland (Pluta et al., 2000; Pluta and _ Zurawicz, 1993, 2009). Bushes of ‘Polares’ have a low-plant vigor, upright plant habit, and are moderately productive. They have low susceptibility to economically important leaf diseases and are resistant to the blackcurrant gall mite (Cecidophyopsis ribis Westw.). Gall mite (not found yet in the United States) is the most serious pest of blackcurrant, especially in Europe and New Zealand, devastating its flower and leaf buds and spreading the Blackcurrant reversion virus (Brennan et al., 2008). ‘Polares’ is resistant to the powdery mildew (American gooseberry mildew) (Sphaerotheca mors-uvae Schwein./Berk. et Curt.), moderately susceptible to anthracnose (Drepanopeziza ribis Kleb.) and slightly susceptible to white pine blister rust (Cronartium ribicola Fish.). Fruit of ‘Polares’ are rather small, with a medium level of soluble solids and acidity, and are very rich in anthocyanins and ascorbic acid. ‘Polares’ is a late-season cultivar, which requires fertile soils for successful fruit production. Fruit of ‘Polares’ can be easily collected by different types of harvesters. Polares is a new blackcurrant (Ribes nigrum L.) cultivar released from the Ribes breeding program conducted for many years at the Research Institute of Pomology and Floriculture (now the Research Institute of Horticulture) in Skierniewice, central Poland (Pluta et al., 2000;_ Zurawicz, 1993, 2009). Bushes of 'Polares' have a low-plant vigor, upright plant habit, and are moderately productive. They have low susceptibility to economically important leaf diseases and are resistant to the blackcurrant gall mite (Cecidophyopsis ribis Westw.). Gall mite (not found yet in the United States) is the most serious pest of blackcurrant, especially in Europe and New Zealand, devastating its flower and leaf buds and spreading the Blackcurrant reversion virus (Brennan et al., 2008). 'Polares' is resistant to the powdery mildew (American gooseberry mildew) (Sphaerotheca mors-uvae Schwein./Berk. et Curt.), moderately susceptible to anthracnose (Drepanopeziza ribis Kleb.) and slightly susceptible to white pine blister rust (Cronartium ribicola Fish.). Fruit of 'Polares' are rather small, with a medium level of soluble solids and acidity, and are very rich in anthocyanins and ascorbic acid. 'Polares' is a late-season cultivar, which requires fertile soils for successful fruit production. Fruit of 'Polares' can be easily collected by different types of harvesters. Origin 'Polares', tested as PC-7/13, is a seedling from a S12/3/83 3 EMR 1834/113 cross made at the Fruit Breeding Department of the Research Institute of Pomology and Floriculture, Skierniewice, Poland. Crossing of parental forms was made in Spring 1994, by S. Pluta, and the seedling, from which the cultivar was developed, was selected in 1999. The female parent, S12/3/83, originated from the blackcurrant breeding program conducted by R. Brennan Knight et al. (1974). EMB 1834/113 inherited a dominant Ce gene, controlling resistance to gall mite. This gene was introduced to blackcurrant from gooseberry (Ribes grossularia L.). Large-fruited, selffertile blackcurrants of commercial potential were obtained in the third backcross (Keep et al., 1982;Knight et al., 1974). In the agroclimatic conditions of Poland both parental cultivars differ in many traits determining their productive value. The cross pollination was intended to produce late-season genotypes combining high productivity and good fruit quality as well as low susceptibility to leaf diseases and high resistance to gall mite. Seedling plants of the cross were planted in the selection field of the Experimental Orchard of the Research Institute of Pomology and Floriculture at Dąbrowice (central Poland) in Spring 2000. 'Polares' was selected for its good fruit productivity, upright plant habit, good fruit quality, and high resistance to fungal diseases in July 2006 by S. Pluta. The name of 'Polares' derives from initial letters of words-Poland and resistance. Description and Performance 'Polares' has been tested in a field trial at the Experimental Orchard in Dąbrowice, Poland. The cultivar trial was established in Autumn 2002 and was conducted until 2010. In the experiment beside 'Polares' nine other cultivars were planted. These cultivars were or are commonly grown in Poland, including ' € Ojebyn' and 'Ben Lomond', which served as the standards. Cultivars grown in the experiment are shown in the tables (Tables 1-3), and they are arranged according to their fruit ripening time. The experiment was conducted on a soil of medium fertility, forecrop was a mustard-green manure. It was established in a random block design, in three replications. Bushes of all cultivars were planted in a density of 3.0 3 0.75 m, with five bushes on a plot. All management practices were applied as recommended for commercial blackcurrant plantations in Poland. No plant protection against main diseases was conducted and only very limited sprayings against aphids and red spider mite were applied. Observations and measurements included plant growth, flowering and harvesting time, fruit yield, fruit size, chemical composition of fruit, and susceptibility of plants to leaf diseases. Results as the averaged values for 5 years of full cropping (2006-10) are presented in tables (Tables 1-3). Bush growth and flowering. 'Polares' is characterized by a low-plant vigor shown by the bush size index (width 3 height of the bush) and for this cultivar is only 1.66 m 2 ( Table 1). The trial shows that 'Polares' is significantly less vigorous than the standard cultivar € Ojebyn (2.21 m 2 ), and also has smaller bush size index than most of the cultivars evaluated in the trial; however, its vigor is similar to the second standard cultivar Ben Lomond (1.87 m 2 ). This indicates that 'Polares' requires fertile soils and provision of good cultivation measures on the plantations. 'Polares' also has a high plant habit index (0.75), which is significantly higher than that of both ' € Ojebyn' and 'Ben Lomond' (0.63 and 0.61, respectively). It shows that 'Polares' has a compact and upright plant habit (Fig. 1). Leaves of 'Polares' are of medium size, five-lobed, and light green, with an extended center lobe (Fig. 2). The inflorescences are single and/or double, of mediumlength. In central Poland, the bloom period of 'Polares' is medium-late (end of April or beginning of May depending on the year). In that regard 'Polares' is similar to 'Ben Lomond' and 4-5 d later than ' € Ojebyn'. Harvest date, yield, and fruit size. 'Polares' is a late-ripening cultivar. Under the agroclimatic conditions of central Poland its fruit ripen in the second half of July, on average 8 d later than the fruit of ' € Ojebyn' and 2 d later than the fruit of 'Ben Lomond' (Table 1). 'Polares' is highly self-fertile, similar to the standard cultivars € Ojebyn and Ben Lomond and it produces similar yields as both standard cultivars. However, 'Polares' is significantly less productive than the Polish blackcurrant cultivars tested in this experiment, such as Tisel, Tiben, and Ruben, which are also characterized by much greater plant growth vigor _ Zurawicz, 2002, 2004). In the experiment, fruit were picked by hand and the average fruit yield of 'Polares' was 1.45 kg/bush (7.2 t · ha -1 ). It was similar to the fruit yield harvested from plants of ' € Ojebyn' (1.75 kg/bush) and 'Ben Lomond' (1.77 kg/bush), but almost 50% less than for three other Polish cultivars-Tisel, Tiben, and Ruben. The mature bush requires regular pruning, which stimulates an intensive growth of new shoots. Fruit of 'Polares' ripen uniformly within the strig and the bush, so machine harvest is easy. For evaluation of fruit size, samples of about 1.5 kg of berries were collected from Received for publication 7 July 2015. Accepted for publication 28 Aug. 2015. This study was co-financed by the European Union from the European Regional Development Fund under the Innovative Economy Operational Programme, contract no. UDA-POIG.01.03.02-00-033/12-00. We thank J. Markowski and his team for performing chemical analyses of the fruit. 1 Corresponding author. E-mail: Stanislaw.Pluta@ inhort.pl. each plot, from which three samples of 100 fruits were randomly chosen, and then the average fruit weight (in g) was calculated. As shown in Table 2 and Fig. 2, 'Polares', similar to the British cultivar Foxendown, produces small fruit, significantly smaller than the fruit of all other cultivars in the trial. In another trial conducted at the Experimental Orchard at Dąbrowice near Skierniewice, Poland, the suitability of 'Polares' for machine fruit harvesting was studied from 2009 to 2012. The research was carried out on a 3.0-ha trial plantation including several Polish cultivars (Tisel, Tiben, Ores, Ruben, Tines, Gofert, and Polares), as well as two standard cultivars: € Ojebyn and Titania. The plantation was set up in Autumn 2005 and bushes were planted at a spacing of 3.8 · 0.5 m, separately in adjacent rows, each 250 m long and containing 500 bushes. The experiment had a randomized block layout with four plots (replicates) of 50 plants each. Fruit harvesting was carried out with the use of the self-propelled Polish made harvester (type KPS-4B, Research Institute of Pomology and Floriculture, Skierniewice, Poland). An average fruit yield for 'Polares' of 5.3 t · ha -1 was achieved, very similar to ' € Ojebyn' (4.9 t · ha -1 ) and 'Titania' (5.7 t · ha -1 ). The combined fruit harvesting efficiency of 'Polares' was above 96%. The shoots of 'Polares' were sufficiently flexible, which allowed the fruit to be shaken from the bush. No visible symptoms of shoot damage caused by the harvester have been observed (data not shown). Chemical composition of fruit. Content of chemical compounds comprised soluble solids (%), acidity (%), anthocyanins (mg/100 g of fresh fruit) and ascorbic acid (mg/100 g of fresh fruit) ( Table 2). For chemical analysis, samples containing about 1.5 kg of fully matured fruit were collected randomly at harvest from each plot. Collected fruit were washed, sealed inside plastic bags, and frozen at -25°C. Before analysis, the samples were disintegrated in a frozen state and mixed thoroughly to obtain uniform material. In the case of organic and ascorbic acid determination, the frozen material was homogenized in 6% HPO 3 solution and, after filtration, analyzed by high-performance liquid chromatography using a Hewlett-Packard 1100 chromatograph equipped with two Supelco LC-18 25 columns in sequence. For the determination of anthocyanins, calculated as cyanidin-3-O-rutinoside/100 g of raw material, the extinction coefficient for the calculation was 28,800 and the molecular weight 595 (Kapasakalidis et al., 2006;Wrolstad, 1976). Content of soluble solids, anthocyanins, titratable acidity, and ascorbic acid were analyzed in five consecutive seasons (2006-10) and average results are presented in . The berries contain significantly less of these compounds than two standard cultivars € Ojebyn (16.9%) and Ben Lomond (16.7%) and much less than, for example, Tiben (17.7%) or Tisel (19.3%). Fruit of 'Polares' are also low in acidity (2.58%), significantly lower than the standard cultivars € Ojebyn (2.88%) and Ben Lomond (3.47%) and most of the other cultivars tested in this experiment. However, fruit of 'Polares' are very rich in anthocyanins and ascorbic acid. In terms of the content of anthocyanins, 'Polares' (406.4 mg/100 g of fruit) significantly exceeds the two standard cultivars. In regard of ascorbic acid, 'Polares' (283.0 mg/100 g of fruit) is also significantly better than other cultivars in this experiment with the exception of Tisel, well known for its high content of ascorbic acid in fruit (Pluta and _ Zurawicz, 2002). Frost, pest, and disease susceptibility. The bushes of 'Polares' are winter hardy. Until now in Poland, no frost damage on shoots has been observed, even if winter temperatures were very low (-30°C) and snow cover was not always present. However, flower buds, flowers, and fruit sets of 'Polares' suffer from late-spring frosts. Susceptibility to fungal diseases included powdery mildew, anthracnose,, and white pine blister rust. For evaluation of these diseases, a ranking scale (1-5) was used, where 1 = no visible symptoms of infection, 3 = moderate symptoms of infection, and 5 = highest symptoms of infection, and the results are presented in Table 3. Based on the average results (2007-10), 'Polares' is resistant to the powdery mildew, similar to the other cultivars evaluated in the trial except Ben Lomond, which was moderately affected by the pathogen. None of the tested cultivars was resistant to anthracnose; however, significant differences were evident. In that respect, 'Polares' was classified as moderately affected by the disease (3.0 points on the 1-5 ranking scale), quite similar to both standard cultivars. More pronounced differences between the evaluated cultivars were observed in the susceptibility to white pine blister rust. 'Polares' was classified as slightly susceptible (1.58 points on the 1-5 ranking scale) and was significantly less susceptible than both standard cultivars-€ Ojebyn' (2.76) and Ben Lomond (2.20). Of the tested cultivars, only Tisel, Ores, and Ruben were resistant to this pathogen. In the trial, 'Polares' was only slightly affected by red spider mite (Tetranychus urticae Koch.); however, in some years 'Polares' showed medium susceptibility to blackcurrant aphid (Cryptomyzus ribis L.) and blackcurrant stem midge (Resseliella ribis Marik.) (data not included). In another field trial, conducted in three consecutive years from 2008 to 2010 at the Experimental Orchard in Dąbrowice, Poland, 'Polares' was grown among blackcurrant bushes heavily affected by gall mite. No symptoms of gall mite infestation were observed on 'Polares' bushes (Łabanowska and Pluta, 2010). Molecular analyses performed at the James Hutton Institute showed that 'Polares' contains the major Ce gene, which confers the full resistance against this pest (R. Brennan, personal communication being already propagated by a licensed nursery in the United States, the names of which will be supplied on request. Growers and nurseries interested in cultivating or propagating this cultivar in the United States and Canada may contact Greg Quinn, Walnut Grove Farm, LLC, 59 Walnut Lane, Staatsburg, NY 12580; E-mail: [email protected].

v3-fos

2018-04-03T06:04:16.659Z

2015-01-13T00:00:00.000Z

18415884

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Managing and eradicating wildlife tuberculosis in New Zealand Abstract Tuberculosis (TB) due to Mycobacterium bovis infection was first identified in brushtail possums (Trichosurus vulpecula) in New Zealand in the late 1960s. Since the early 1970s, possums in New Zealand have been controlled as part of an ongoing strategy to manage the disease in livestock. The TB management authority (TBfree New Zealand) currently implements three strategic choices for disease-related possum control: firstly TB eradication in areas selected for eradication of the disease from livestock and wildlife, secondly Free Area Protection in areas in which possums are maintained at low densities, normally along a Vector Risk Area (VRA) boundary, and thirdly Infected Herd Suppression, which includes the remaining parts of VRA where possums are targeted to minimise the infection risk to livestock. Management is primarily through a range of lethal control options. The frequency and intensity of control is driven by a requirement to reduce populations to very low levels (usually to a trap-catch index below 2%), then to hold them at or below this level for 5–10 years to ensure disease eradication.Lethal possum control is implemented using aerial- and ground-based applications, under various regulatory and operational constraints. Extensive research has been undertaken aimed at improving the efficacy and efficiency of control. Aerial applications use sodium fluoroacetate (1080) bait for controlling possums over extensive and rugged areas of forest that are difficult to access by foot. Ground-based control uses a range of toxins (primarily, a potassium cyanide-based product) and traps. In the last 5 years there has been a shift from simple possum population control to the collection of spatial data on possum presence/absence and relative density, using simple possum detection devices using global positioning system-supported data collection tools, with recovery of possum carcasses for diagnostic necropsy. Such data provide information subsequently used in predictive epidemiological models to generate a probability of TB freedom.The strategies for managing TB in New Zealand wildlife now operate on four major principles: firstly a target threshold for possum population reduction is defined and set, secondly an objective methodology is applied for assessing whether target reductions have been achieved, thirdly effective control tools for achieving possum population reductions are used, and fourthly the necessary legislative support is in place to ensure compliance. TBfree New Zealand's possum control programme meets these requirements, providing an excellent example of an effective pest and disease control programme. Tuberculosis (TB) due to Mycobacterium bovis infection was first identified in brushtail possums (Trichosurus vulpecula) in New Zealand in the late 1960s. Since the early 1970s, possums in New Zealand have been controlled as part of an ongoing strategy to manage the disease in livestock. The TB management authority (TBfree New Zealand) currently implements three strategic choices for disease-related possum control: firstly TB eradication in areas selected for eradication of the disease from livestock and wildlife, secondly Free Area Protection in areas in which possums are maintained at low densities, normally along a Vector Risk Area (VRA) boundary, and thirdly Infected Herd Suppression, which includes the remaining parts of VRA where possums are targeted to minimise the infection risk to livestock. Management is primarily through a range of lethal control options. The frequency and intensity of control is driven by a requirement to reduce populations to very low levels (usually to a trap-catch index below 2%), then to hold them at or below this level for 5-10 years to ensure disease eradication. Lethal possum control is implemented using aerial-and groundbased applications, under various regulatory and operational constraints. Extensive research has been undertaken aimed at improving the efficacy and efficiency of control. Aerial applications use sodium fluoroacetate (1080) bait for controlling possums over extensive and rugged areas of forest that are difficult to access by foot. Ground-based control uses a range of toxins (primarily, a potassium cyanide-based product) and traps. In the last 5 years there has been a shift from simple possum population control to the collection of spatial data on possum presence/ absence and relative density, using simple possum detection devices using global positioning system-supported data collection tools, with recovery of possum carcasses for diagnostic necropsy. Such data provide information subsequently used in predictive epidemiological models to generate a probability of TB freedom. Introduction In New Zealand, and a number of other countries, tuberculosis (TB) due to Mycobacterium bovis infection has become established in one or more wildlife hosts capable of independently maintaining the disease. The host or suite of hosts differs between countries and in New Zealand is uniquely centred on an introduced marsupial, the brushtail possum (Trichosurus vulpecula). Possums were introduced to New Zealand from Australia in 1858 to establish a fur trade (Clout and Ericksen 2000). With natural spread and subsequent human relocations, possums now occupy virtually all vegetated habitats in the North and South Islands of New Zealand from the coast to high mountains, including extensive rugged forest lands, farmland and semi-urban habitats (Clout and Ericksen 2000). Before possums were acknowledged as a wildlife vector of TB in 1971 (Davidson 1976) they had already been recognised as a significant conservation pest, causing major vegetative damage and even canopy collapse in some indigenous forest types, and selectively removing palatable forest understorey species such as fuchsia and mistletoe (Payton 2000). Organised possum population control began in the 1950s and continues to the present day as a means of curtailing possum impacts on indigenous forests and biodiversity values in priority conservation areas. Following their confirmation as TB hosts, possums were also controlled for TB management purposes to prevent, or at least reduce, the potential transmission of disease to farmed livestock, predominantly cattle (beef and dairy) and red deer (Davidson 1976). TB was first identified in wild possums in 1967 (Ekdahl et al. 1970), and subsequently identified in possum populations in 32 geographically discrete areas of New Zealandreferred to as Vector Risk Areas (VRA) which in total amounted to 10.6 million hectares (∼38% of New Zealand's land area) in 2012/13. A VRA is a defined geographical area where TB is established in a wildlife maintenance host (Livingstone et al. 2015). The importance of possums as a cause of TB in cattle, and the effectiveness of intensive lethal control as a way of managing this problem, were both demonstrated soon after TB was first identified in possums. As early as 1972, in the Buller South district of the West Coast region of the South Island, control of possum populations sympatric with M. bovis-infected beef and dairy herds resulted in an immediate and significant reduction in herd infection (Livingstone et al. 2015). This identified a strong causal link between TB in possums and livestock infection, and demonstrated that killing possums was highly effective in breaking that link. Subsequent findings of TB infection in cattle herds related to possums in a number of discrete areas (Adlam 1977) further confirmed possums as the main wildlife vector of the disease. As a consequence, a programme of possum population control was instigated in and around areas where TB in possums was found. Subsequent epidemiological modelling and empirical data since the 1970s have indicated that TB can be eradicated from possum populations by reducing and maintaining their numbers at low densities for approximately 5 years (Barlow 1991a;Caley et al. 1999;Ramsey and Efford 2005), which is usually achieved by large-scale lethal control (as discussed later). Together with testing and movement control policies for livestock (Buddle et al. 2015), possum control has been the critical element that has led to a 95% reduction in the number of infected cattle and deer herds, from a peak of 1694 in June 1994 to 66 in June 2012 (Livingstone et al. 2015). Because tuberculous possums and possum control are problems exclusive to New Zealand, unique solutions had to be developed that took into account this species' biology in a non-native country, including TB epidemiology, available control technology and its effects on indigenous non-target species, and the regulatory requirements and public expectations regarding large-scale pest control. Moreover, special consideration was required with regards to the practical aspects of pest control over the vast areas of New Zealand's steep forested landscapes, throughout which possum population control was required. Since 1991 the Animal Health Board (AHB; now TBfree New Zealand) funded research to improve the management of TB in New Zealand and to help achieve the goals of the National Pest Management Plan for Bovine Tuberculosis (Livingstone et al. 2015). The practices of possum control have been underpinned by both operational trials and specific research projects aimed at improving the strategic application and effectiveness of control programmes (i.e. intensity, frequency and spatial scale of control), and the more specific tactical application of operational practices (i.e. bait sowing rates, trapping methodology, outcome monitoring, and target specificity). This paper reviews the main strategic, tactical and operational practices that have developed over the past three decades for reducing or completely eliminating TB in possums in particular, but also for disease surveillance in other wildlife species, e.g. ferrets (Mustela furo) and feral pigs (Sus scrofa), that are involved in the epidemiology of TB in New Zealand. Strategic options In the broadest terms, the strategic options for pest and disease management are to do nothing, to reduce pest density or disease prevalence and maintain it at some level that is acceptable, or to eliminate the pest or disease completely from an area and prevent its reinvasion. All three options are applied in New Zealand to wildlife TB management. Historically, because of limited funds, possum control was mainly applied on or near farmed areas that had the worst or most intractable livestock TB problems. Successive amendments to the National Pest Management Plan, along with associated increases in funding for possum control, provided the ability to firstly define and contain the spread of infected possums, secondly develop extensive conjoined areas of possum control within VRA where possums could be maintained at low densities, thus reducing the risk of TB in possums migrating within the area under control as well as minimising their contact with cattle and deer, and thirdly eradicate TB from the possum population in a few small VRA by maintaining possum densities at low levels. In July 2011, the latest amendment to the National Pest Management Plan was implemented, with an objective to eradicate TB from a minimum of 2.5 million hectares of VRA by 2026 (Anonymous 2012a), based on annual funding capped at the same level as for 2002/03. To achieve this objective, approaches to possum control had to be refined, with funding prioritised on the basis of strategic choices for particular VRA or parts of VRA. The ability to prioritise areas has evolved over time as technical and control capabilities have improved, in line with improved understanding of possum ecology and TB epidemiology. Three strategic choices are currently used to guide disease-based possum control: firstly TB eradication in those parts of selected VRA that together make up a minimum of 2.5 million hectares, TB is required to be eradicated from the possum population, with a probability of freedom of ≥0.95; Secondly Free Area Protection in those areas where possums are maintained at low densities, normally along a VRA boundary (buffer) to prevent TB in possums migrating and establishing infection in the adjacent Vector Free Area. Buffers vary in width from 5-15 km depending upon the extent and type of possum habitat within the buffer and adjacent non-controlled areas; thirdly Infected Herd Suppression, including the remaining parts of VRA where possums are targeted to minimise the probability of TB in possums infecting cattle and deer. The level of control is designed to ensure that the national annual M. bovis infected herd period prevalence remains <0.04%. The Infected Herd Suppression areas include farms adjacent to some relatively large areas of forest where possums are unmanaged under current resource availability. For TB management, and disease management generally, the strategic options of suppression in perpetuity or eradication tend to overlap in practice, because both aim to reduce the reproductive rate of the disease to below 1.0 and hold it there (Anderson and May 1979). The principle difference is that where local eradication is the goal, a large surrounding area must also be free of disease to prevent reinvasion, whereas with suppression such separation is not required. Tactical options There have been three possible tactical options for managing TB in New Zealand wildlife that have received research consideration: wildlife population reduction through lethal control, including harvesting (Coleman and Livingstone 2000), wildlife reproductive control (Cowan 2000) and vaccination of wildlife against TB (Barlow 1991a). During the 1990s, these options were examined on a theoretical basis using population-and individual-based simulation models (Barlow 1991b(Barlow , 1993(Barlow , 2000aRamsey et al. 2002) to determine their relative effectiveness in possums, when used either singly or in combination. Barlow (2000b) summarised the findings and predictions of these models for each of the options, while Ramsey and Efford (2010) published the predictions of a more sophisticated individual-based spatial model to evaluate the same approaches, both individually and in combination. Although the models predicted that integrating culling with either fertility control and/or vaccination of possums could provide cost-effective alternatives to sole reliance on lethal control, neither fertility control nor vaccination has developed to the stage that they are available for operational application despite considerable research effort and expenditure. For detailed coverage of the New Zealand research underpinning development of possum vaccines and fertility control tools, the reader is referred to the relevant sections of recent review articles by Cross et al. (2011), Waters et al. (2012 and Buddle et al. (2013). Overall, lethal control remains the only viable option available for managing M. bovis infection in New Zealand wildlife. The initial modelling of a lethal control strategy predicted that if possum populations were first reduced by 75% (knockdown) and then maintained below 40% of the population carrying capacity (maintenance), TB could be eliminated within 10 years, provided there was no immigration of M. bovis-infected possums during this time (Barlow 1991a). These predictions have generally been supported by field studies where both TB prevalence and relative possum abundance have been monitored (Warburton 1996;Caley et al. 1999), although in practice possum densities may have been held at lower densities than required by model predictions. Further modelling (Ramsey and Efford 2005) suggested that if possums were reduced to and maintained below a trapcatch index (TCI) of fewer than two possums captured per 100 operational trap-nights (2%), and provided there was no immigration of tuberculous possums, TB could be eliminated within 5 years. A comparison of different TCI targets also suggested that 2% would be the most cost-effective (Ramsey et al. 2008). That is, maintaining possums merely below a TCI of 5% would eventually eliminate the disease, but it would take longer, and the extra costs of extended control would exceed the costs of maintaining possums below 2% for a shorter period of time. The concept of an initial knockdown operation to reduce the possum population from its uncontrolled state to a TCI target of 2% or below, followed by annual maintenance control to hold the possum population at or below a required TCI is followed in practice for TB control. The precise intensity and frequency of the lethal control applied varies depending on the strategic aims of the operation, i.e. TB Eradication, Free Area Protection or Infected Herd Suppression. The criteria differentiating these three classifications, and the different approaches applied for wildlife management to each, are described in detail in Supplementary Information 1. 1 Aerial operations Aerial poisoning is restricted to the use of the metabolic toxin sodium fluoroacetate (1080), incorporated into bait at 0.15% w/w (or occasionally at 0.08%), but never >0.2% w/w as concentrations above this can result in detection and consequent avoidance by possums . Aerial operations can cover large areas of forest, e.g. up to 85,000 hectares have been treated in one operation (Coleman et al. 2006), and with several aircraft sowing bait simultaneously such operations can be completed in one or two days. Given high efficacy, aerial baiting is generally the most cost-effective option for controlling possums over extensive and rugged areas of forest that are difficult to traverse efficiently on foot, and in some situations it is the only practicable option. Aerial control has been underpinned by extensive research aimed at increasing percentage kills of such operations, and reducing per hectare costs, as well as addressing non-target and welfare impacts, user safety and ensuring operational reliability. Other research has aimed to gain a better understanding of, and address public concerns related to, the impacts of 1080 on the environment and possible residues in water (Eason et al. 2011;Northcott et al. 2014). When aerial 1080 operations were first used in the 1960s and early 1970s, they produced variable results, with the estimated percentage of possums killed from 18 operations and 15 trials averaging only 69% (Batcheler 1978). Subsequent research examining bait quality (Batcheler 1982;Frampton et al. 1999), bait acceptance (Morgan 1982) and bait distribution (Morgan 1994) changed operational best practice, and kills exceeding 85% were then consistently achieved (Coleman et al. 2006). This and other research led to the development of bait-quality standards that addressed issues such as toxin concentration, bait palatability, moisture content, bait hardness, and bait storage . As part of current best practice, all aircraft sowing 1080 baits must use global positioning systems (GPS) to enable the pilot to disperse bait along lines at the required spacing, and to enable operational staff to audit the flight paths to ensure all of the operational area is treated and that bait has not been sown outside the designated control boundaries. In addition, all aerial 1080 operations undertaken for TBfree New Zealand must meet its standard operational procedures (Anonymous 2012b). Most aerial 1080 operations currently utilise helicopters to deploy bait. To reduce aerial control costs, the use of fixed-wing aircraft as an alternative to (or in tandem with) helicopter application is being assessed. Available aerial top-dressing aircraft can carry one tonne of bait, have an hourly operational cost similar to helicopters, but sow at twice the speed. Initial trials using 1 kg/ha of non-toxic bait and 0.5 kg/ha of 1080 baits, applied in strips by fixed-wing aircraft, obtained a similar reduction in possum density as standard helicopter broadcast baiting (Nugent et al. 2012). However, fixed-wing aircraft require an airstrip for loading bait, and if this is distant from the application area, then cost savings relative to helicopter use may be lost. Improving operational efficacy by the use of pre-feeding To ensure possum populations are reduced consistently to very low levels, aerial baiting operations now routinely include a single application of non-toxic pre-feed baits (i.e., the same bait type as for the toxic application, but without 1080) to increase subsequent toxic bait consumption and overcome any bait aversion . If possums are pre-fed before aerial application of 1080 baits, on average more possums are killed than if there is no pre-feeding (Coleman et al. 2006). The value of pre-feeding is also supported by a range of pen and field-based research trials Nugent et al. 2011a). While possum control is the primary focus of TB-related animal pest control operations, aerial baiting with 1080 can have significant impacts on populations of other invasive predatory species such as ship rats (Rattus rattus) and, secondarily, stoats (Mustela erminea) (Murphy et al. 1999). These impacts in turn can generate either beneficial or adverse outcomes for conservation of indigenous fauna, especially birds and some large invertebrates, of which rats and stoats are significant predators. In some forest types, ship rats have been observed to increase in number to higher levels than they were before control, potentially posing increased predation pressure on native fauna (Ruscoe et al. 2011). To offset this risk it is desirable to achieve high rat mortality as part of the control operations to ensure any benefits are maximised. High rat kills can be achieved by non-toxic prefeeding prior to poison deployment (Nugent et al. 2011a), which is an additional justification for this practice. Rat (and consequently stoat) numbers can also increase significantly during years when native beech forest seeding is synchronised (masting events), so control operations for TB in possums are aligned with conservation management needs to generate benefits for indigenous species which are at high risk from rat and stoat predation, especially in these mast years. Such applications of 1080 can result in increased abundance of native birds (Powlesland et al. 1999;O'Donnell and Hoare 2012). Providing sufficient 1080 bait to kill all possums Current best practice aerial baiting strategies are designed to ensure possums will encounter at least a single toxic bait carrying a lethal dose. To this end, 12 g baits containing 0.15% w/w 1080 are distributed as uniformly as possible across the target area generally at an application rate of 2 kg bait/hectare, equivalent to one bait per 60 m 2 . Baits are applied using helicopters with underslung sowing buckets which can broadcast bait across swaths up to 220 m wide, or along much narrower "trickle-fed" paths where accuracy is crucial. The aim when broadcasting baits is to achieve a uniform distribution of baits across the control area with no gaps in bait distribution large enough to prevent any possums from encountering bait. In practice, baits often fragment at various stages of their operational-cycle (e.g. bagging, transport, storage, handling, loading, sowing, and partially eaten baits) and because fragmented baits are not of uniform size and weight, they tend to be unevenly distributed, with heavier baits distributed more towards the outside and lighter baits towards the middle of the application swath (Nugent et al. 2011a). Therefore bait density needs to be sufficiently high so that possums which do not find a whole bait will still find and eat a lethal dose from several bait fragments before the onset of toxicosis (within about 30-40 minutes) at which point further bait intake ceases; sublethally poisoned possums will be likely to avoid bait in the future. While an overall bait density of 2 kg of bait/hectare largely meets this need, control efficacy could be increased (with possibly lower sowing rates) through development of bait types less prone to fragmentation, but still palatable to possums. Research and development work is in progress towards such bait improvements. Investigation into the extent of bait fragmentation and its effect on bait quality (Nugent et al. 2011b) together with bait density and the influence this has on probabilities of possums encountering lethal quantities of bait, has led to the testing of alternative bait distribution patterns including strip and cluster sowing Morriss 2010, 2011;Nugent et al. 2012). Sowing baits within a relatively narrow strip or in clusters ensures baits are at high density where they occur which in turn ensures that sufficient quantities of toxic baits are readily available for possums to encounter and eat a lethal dose before toxicosis sets in, irrespective of bait fragmentation, and at possibly lower overall application rates. In strip and cluster sowing, the control area is first strip-sown with non-toxic pre-feed before the application of toxic bait in strips or clusters, along the same GPS guided lines recorded from pre-feeding. Possums exposed to pre-feed increase their searching behaviour for a period of several weeks; thus, provided aerial cluster or strip sowing of toxic bait occurs within this time period, there is a high probability that possums will find a strip or cluster of baits in sufficient quantity to avoid problems of only encountering a bait fragmentation and therefore a sublethal dose ). Recent research on strip and cluster sowing methodology suggests that relative to broadcasting, bait sowing rates can be significantly reduced without loss of effectiveness, especially where possum density is relatively low Morriss 2010, 2011). Use of these new sowing options could significantly reduce the amount of bait needed to be sown, and the amount of 1080 being applied to the environment. However, further work is still required to improve consistency of possum kill (for all habitat types currently subject to broadcast aerial sowing) while reducing bait application rates and costs. Minimising the impacts of 1080 baits on non-target species Because 1080 is a relatively broad-spectrum poison, care must be taken to avoid adverse effect on valued non-target species. There is some history of such adverse effects, and mitigation of them over time. From the 1960s to 1980s, 1080 was often applied to chopped carrot bait for aerial application. As the chopped carrot bait was not routinely screened to remove chaff (small fragments of carrot containing 1080), bird deaths, especially New Zealand tomtit (Petroica macrocephala) and robin (Petroica longipes) were commonly recorded (Harrison 1978;Spurr and Powlesland 1997). Subsequent refinement of 1080 best practice (Morgan 2004), with the now much greater use of cereal pellets, has resulted in operations that pose little risk to non-target native bird species (Spurr and Powlesland 1997;Powlesland et al. 1999Powlesland et al. , 2003. More recent surveys of bird populations, especially of threatened species such as kiwi (Apteryx spp.) and kaka (Nestor meridionalis), have used radio-tags to track individual birds and monitor mortality, and this has provided more robust information on the risks that 1080 operations pose to birds (Veltman and Westbrooke 2011) with general reassurance that such risks are low. A recent exception was the death of seven of 17 radio-tagged kea (Nestor notabilis) killed during a 1080 operation in 2008, although it was believed these birds may have been habituated to scavenging refuse and therefore may have been more willing to seek and consume cereal pellets (J Kemp, 2 pers. comm.). In response to these deaths, the Department of Conservation (DOC) has monitored 89 radio tagged kea through seven subsequent aerial 1080 operations with no further deaths recorded. Following the original kea deaths, DOC (and latterly TBfree New Zealand) carried out limited trials on captive kea comparing the birds' relative acceptance of two different types of cereal bait, namely RS5 and No. 7 pellets (Animal Control Products Ltd, Whanganui, NZ) (Blyth 2011). As a result of these trials, DOC now requires all 1080 operations within kea range to use RS5 baits which were less preferred by kea and which are more readily detoxified by rainfall. Additionally, trials testing anthraquinone and d-pulegone as added repellents in cereal pellets resulted in an 88.7% reduction in bait consumption by kea (Orr-Walker et al. 2012). Although these repellents appear to be effective at repelling kea, research is now underway to assess their effects on the acceptability of baits to possums and ship rats (Cowan et al. 2013), as well as understanding the volatility of d-pulegone and its impact on repellency effectiveness (M Crowell 3 , pers. comm.). Aerial 1080 baiting operations can also cause mortality in wild deer, although at apparently highly variable rates. To mitigate the impacts of this on commercial or recreational deer hunting recent research has led to development of a deer repellent (Epro Deer Repellent; Epro Ltd, Taupo, NZ) aimed at reducing deer mortality while not reducing the percentage kill of possums or ship rats (Nugent et al. 2004;Morriss et al. 2006;Morriss and Nugent 2008). Results indicated that deer deaths can be reduced by about 90% when using the repellent, with no measurable reduction in either possum or ship rat kills (Morriss et al. 2003;Speedy 2005;Morriss and Nugent 2008). However, inclusion of the repellent in 1080 cereal bait doubles the bait price, which effectively reduces the area over which possum control can be applied under a fixed budget (Morriss and Nugent 2009). Nevertheless, in 2011/12, it was used in about half of the aerial 1080 operations carried out by TBfree New Zealand. Conversely, in situations where TB is present in both the possum and wild deer populations, it may be beneficial to allow or even maximise by-kill of deer from a 1080 operation targeted at possums, because this will reduce the time-frame to eradicate TB from the wild animal population (Barron and Nugent 2011). Risks to dogs and livestock are managed through public notification, poison warning signs and communication with land users in the vicinity of the operations. Dogs are particularly susceptible to 1080 poisoning, with a stated lethal dose of <0.1 mg/kg bodyweight (Goh et al. 2005). In New Zealand this risk is mostly through the consumption of 1080-poisoned possum carcasses. Although research has attempted to identify a potential antidote for dogs, to date this has been unsuccessful (Goh et al. 2005). For livestock, trials assessing the effectiveness of deer repellent on bait to minimise the risk to sheep and cattle have indicated that the compound works effectively for sheep but not for cattle (Morriss and Nugent 2009). Animal welfare impacts of 1080 The use of 1080 also raises animal welfare concerns, with arguments that possums and other target and non-target animals suffer unacceptable levels of pain and distress following poisoning with 1080 (Sherley 2007). Others have argued that, on the basis of possums' physiological and behavioural responses and the duration of effects, 1080 poisoning is not unduly inhumane (Twigg and Parker 2010). The behaviour of animals after 1080 intoxication has been investigated to assess its relative humaneness. To determine the welfare impacts of 1080, the behaviour of eight experimentally poisoned possums was assessed in a controlled study (Littin et al. 2009). Half of the animals displayed abnormal appearances and postures 1 hour 50 minutes after consuming baits, seven of the eight animals exhibited retching, and three vomited, over a 27-minute period (these symptoms starting 2 hours 53 minutes after dosing). Lack of coordination began 3 hours 37 minutes after dosing, with possums then spending most of the time until death prostrate and showing spasms and tremors; mean time to death was 11 hours 26 minutes (Littin et al. 2009). In comparison to other available vertebrate toxic agents (none of which are registered for aerial application), the welfare impacts of 1080 are ranked as moderate, intermediate between cyanide (mild) and anticoagulants (severe) (Fisher et al. 2010). Although 1080 use does have a welfare cost, it is the only vertebrate toxin currently registered for aerial application on the main islands of New Zealand and therefore its use continues to be necessary if possums and other mammalian pests are to be managed cost-effectively over large areas. It is important that any pest control that involves killing sentient animals is carried out within a strategy that has wide social acceptance, along with well-based confidence that its objectives and benefits are necessary, measurable and achievable (Warburton and Norton 2009). Additionally, the most humane control method available that is also suitable for purpose should also be selected. The strategy and operational procedures of TBfree New Zealand are considered to meet all the necessary requirements for a robust and defendable pest management programme (Warburton and Norton 2009). Effects of 1080 baits on water quality Recent reviews have highlighted the issues regarding the potential toxicity of 1080 in the environment (Eason et al. 2011;Wright 2011), and public concerns of 1080 and water generate considerable scrutiny of aerial 1080 operations. Aerial 1080 baiting operations require regulatory approval, and water monitoring may be a condition of the approving agency e.g. post-application testing of water flowing from the application area. As a result of these requirements, our own toxicology laboratory (Landcare Research, Lincoln, New Zealand) tested 2,639 water samples between 1990 and August 2012, collected from areas after aerial applications of 1080. There was no detectable 1080 in 96.7% of these samples using gas chromatography with a lower detection limit of 0.1 ppb. Concentrations of 1080 in the remaining 3.3% (n=88) samples ranged from 0.1-9 ppb. Of the total samples taken, 887 (34%) were from water used for human or stock drinking supplies, and four of these contained detectable 1080 residues at 0.1 ppb (one sample) or 0.2 ppb (three samples). None of these, however, exceeded the New Zealand Ministry of Health's provisional maximum acceptable level for drinking water of 3.5 ppb (Anonymous 2008). Since 2008, the Environmental Protection Authority (formerly the Environmental Risk Management Authority) has released annual reports on the aerial use of 1080, which contain specific operational details and water monitoring results. The data cited above are incorporated into the overall figures reported by the Environmental Protection Authority. Review of use of aerial 1080 control In response to public concerns about, and opposition to, 1080 use for vertebrate pest control, the Environmental Risk Management Authority carried out an extensive reassessment of 1080 in 2007, and concluded that the benefits of using 1080 outweighed the adverse effects, and that any adverse effects of using 1080 could be managed adequately, provided it was applied following standardised best practice. Some new controls were imposed on aerial application of 1080 to make it safer, including mandatory use of GPS guidance of aircraft, post-operational reporting, and several requirements related to signage and notification (Anonymous 2007). The review assessed the technical risks but Green and Rohan (2012) suggested that to address community concerns improvements in engagement with relevant parties were needed, and building dialogue and collaboration with key communities was required. The reassessment by the Environmental Risk Management Authority provided support for ongoing use of 1080, but it did not quell continued opposition, and some members of parliament called for a moratorium on its use. As a consequence, in 2011, the Parliamentary Commissioner for the Environment carried out an extensive review of the use of 1080 in New Zealand including alternatives, and concluded "It is my view, based on careful analysis of the evidence, that not only should the use of 1080 continue (including in aerial operations) to protect our forests, but that we should use more of it" (Wright 2011). Other research has investigated the fate of 1080 in the environment once it has been deployed in bait form, assuming it has not been consumed by a pest, and shown that 1080 can leach from baits (Suren 2006), but that it is readily degraded in soils at ground temperatures over 5°C (Northcott et al. 2014). Further, of the 1080 that typically leaches from baits following rain, less than 1% is subsequently found in surface water where it could feasibly pose a risk for terrestrial animals (Srinivasan et al. 2012). Ground-based operations Because of the risk posed to livestock, and especially to farm dogs, from uneaten 1080 baits and carcasses poisoned with 1080, aerial application of 1080 baits cannot be used for controlling possums over farmland, unless dogs and livestock can be removed until baits and/or possum carcasses are detoxified, with the latter taking some months. Nor can aerial application of 1080 be carried out near dwellings, residential areas or other areas with high unrestricted public access and use. Consequently, possum control in such areas mostly relies on ground-based methods including traps and poisons. Most of these control operations target possum habitat on farms ranging from isolated trees and shelter-belts, to patches of scrub, forested gully systems, plantation forests, and areas of remnant indigenous forest that might be several hundred hectares in area. In order to reduce the prevalence of TB in livestock, ground-based methods must effectively reduce the local relative possum abundance to below 2% TCI Under a performance-based contract system for delivering possum control (see below), contractors for ground-control work select tools, and develop strategies for applying them, in order to maximise the probability they will meet TCI targets. In the three years from 2011-2013, the mean TCI achieved from all recorded performance contracts were 0.65, 0.46, and 0.4% respectively, considerably better than the contracted TCI targets of 2%. Additionally, 93% of about 200 performancebased operations contracted each year achieved their targeted TCI on their first post-control monitor, with the small percentage of failed contracts requiring extra work and re-monitoring to determine if the target had been achieved (data sourced from unpublished metadata in the VectorNet database, TBfree New Zealand Ltd, Wellington, NZ). These results indicate that ground-based contractors are routinely achieving the reductions required to eliminate TB from wildlife and protect livestock. In practice, as for the stages already described for aerial operations, there are three main stages of ground-control operations, but in this case each is linked to contract-specifications (these are described in detail in Supplementary Information 2 4 ). The operational specifications for ground-based control contracts vary depending on the stage of control, and costs vary considerably. Additionally, because ground-based contracts are typically carried out on farmland, the actual area of possum habitat treated is often much less than the total area contracted. On some highly developed farmland (e.g. dairy farms) <10% of the total area might be actual possum habitat that receives control. Consequently, the cost of ground control when expressed as $/total hectare can be misleading especially when compared to the cost of aerial control, where the total area and treated habitat area are the same. The costs for the various contract options for 2011-2013 are shown in Table 1. These show that the more intensive coverage required by detection surveys during the eradication phase increases the cost compared with straightforward input contracts or output control contracts, which have similar costs, but all costs can vary from year to year. However comparing costs alone assumes equal effectiveness, while operational effectiveness (i.e. percentage kill) is not measured directly, so any cost comparisons must be done cautiously. In comparing costs between ground and aerial control it must be noted that although one-off costs might be similar, ground control is typically repeated at least every second year (and often annually), whereas aerial control is generally not repeated for at least 3-5 years. Consequently, the real annual cost of ground-based control is generally greater than for aerial control. Initial control methods Ground-based contractors carrying out initial control generally use a potassium cyanide based product (Feratox, Connovation Ltd, Auckland, New Zealand). This encapsulated form of potassium cyanide was developed in the 1990s (Warburton and Eason 1992; to address increasing possum shyness to the then commonly used cyanide paste (mostly sodium cyanide). The pellets are small pea-sized balls with a core of dry cyanide powder encased in a hardened resin that prevents emission of hydrogen cyanide. Each pellet contains a lethal dose of cyanide for a possum and they are usually presented mixed in a peanut butter ball. These are placed into a small biodegradable labelled bag that is stapled to a tree or fence post to be freely accessible to possums that tear open the bag, eat the peanut butter ball and when chewing on the potassium cyanide pellet, crack the casing and release the cyanide. Some contractors vary their use of potassium cyanide by placing the ball baits in fixed bait stations. Most possums lose consciousness within 7 minutes of ingestion of a potassium cyanide pellet (Gregory et al. 1998) and are found dead within 1-5 m of the site. Potassium cyanide is also presented in other forms such as potato starch cubes and in cereal pre-feed mixes. Use of Feratox enables contractors to achieve high kills over relatively large areas cost-effectively. If the control target is less than 5% TCI and the areas have had several years of Feratox use, most contractors also use trapping as a backup to ensure any possums that are shy of the poison bags are subsequently removed by trapping. A variety of leg-hold traps are used, with Victor No. 1 (Pest Management Services, Christchurch, New Zealand) traps being the most commonly used type. Traps are generally set using a mixture of flour and icing sugar as a visual lure and bait. When controlling possums in larger areas of forest, and depending on the terrain, some contractors use 1080 cereal pellets, either spread onto the ground by hand or in bait stations. Where access is difficult (e.g. steep gullies and dense patches of gorse), 1080 pellets are sometimes broadcast by hand to provide a wider bait distribution. In some accessible forest areas with easy terrain, helicopters are sometimes used to sow non-toxic pre-feed pellets in GPS located strips that are then ground poisoned using 1080 pellets, Feratox or cyanide paste distributed by hand. Maintenance control During the initial stages of maintenance control, contractors may continue to use potassium cyanide or use more traps. Some contractors use alternative poisons, such as cholecalciferol or brodifacoum, in various types of bait stations in areas that have had several prior years of exposure to Feratox pellets and leg-hold traps. For areas in the pre-eradication phase, all habitats must receive control to ensure there are no patches or clusters of possums left that could enable TB to persist. To ensure total coverage and sufficient intensity of control at each site, control contracts are generally changed from performance-(output) based control to input-based control, with control coverage and intensity of control being specified in the contract. New Zealand contracting system and monitoring The contracting system Since about 1996 vertebrate pest control operations, especially those carried out as part of TB management, have been implemented through a competitive contract system (Warburton and Cowan 2008). TBfree New Zealand (the predominant contracting agent) typically publicises a range of control operations for competitive tender. Although price is an important selection criterion, other criteria are used, such as competence in health and safety management, relevant experience and track record, technical skills, equipment, management skills, and proposed methodology. Performance-based contracts generally have a residual TCI target to be achieved before payment is made, such as to reduce the mean relative abundance of possums to below a TCI of 2% with no individual trap-line catching more than two possums. Most performance-based contracts have few restrictions on what control methods can be used, and it is up to the contractor to select the most cost effective methods to achieve the contracted target density. In areas that have already had several years of control resulting in low possum numbers, contracts are generally input-based, partly to eliminate the need for each contract to be independently monitored. Input contracts are essentially method-driven, with contractors being required to apply a particular control method at a prescribed intensity, such as a stipulated trap-spacing and number of trap nights. Input contracts are often used to ensure control is applied to all habitat (to decrease the probability that clusters of possums are missed), and recently have included the use of detection devices to better inform where trapping effort should be applied. For data recording and auditing purposes, all contractors must use GPS capable personal digital assistants to record the location of all detection devices and traps. The GPS data can then be uploaded and checked against habitat maps. Periodic audits are carried out to determine if contractors comply with contract requirements. TBfree New Zealand has developed bespoke databases (i.e. VectorNet and VectorTrax) to manage the large amounts of spatial and activity data collected by contractors, and such data are used to support TB management decisions based on probability predictions from the proof of freedom utility (see Anderson et al. 2015) in cases where the objective is TB eradication. Monitoring post-operation residual possum levels Performance contracts require the use of TCI as a standardised and reproducible method for assessing whether their control operations have successfully reduced the possum population to the contracted target density. The standardised trap-catch methodology for TCI calculations was developed in the 1990s (Warburton 1996), based on setting a defined number of randomly allocated trap lines, each containing a series of 10 consecutive leghold traps, spaced evenly throughout the habitat for three contiguous nights of fine weather. From this, for example, two possums caught over 100 trap nights would indicate a TCI of 2%. To ensure this methodology is applied in a standardised way, a national protocol (Anonymous 2011) and training courses have been developed by the National Pest Control Agencies. The trap-catch protocol ensures the method is applied in a standardised way, but the index itself is sensitive to seasonal, habitat or density variations in possum capture (Forsyth et al. 2005). The post-operation possum populations being monitored are always low (i.e., TCI usually <2%), but clustering of residual possums is a concern. To assess whether the trap-catch method is effective for detecting residual possum clusters, a modelling approach was developed in the 2000s to assess the statistical limits of the protocol. Ramsey and Ball (2004) recommended that the number of trap-nights be extended from three to six and that maximum catches per trap line should be set. Setting line maxima was adopted as routine practice, but increasing trap nights from three to six has not been incorporated into New Zealand best practice methods because of cost. Detecting possums at very low densities Where the aim of TB control is eradication, possum densities are very low in the latter stages of pre-eradication and in the eradication phase itself (i.e. often less than 1% TCI). Consequently most traps do not catch any possums and setting them is essentially a wasted effort. To address this problem, detection devices were developed as a low-cost/low-effort way to identify any continued presence of possums and the locations of such animals, so that subsequent trapping effort could be targeted to those areas where a device had returned a positive result. This type of informed trapping was first attempted by using clumps of flour and icing sugar (Thomson et al. 2002), but more recently specific devices such as WaxTags (Pest Control Research, Christchurch, New Zealand) and chewcards (Connovation Ltd, Auckland, New Zealand) have been developed and used in control programmes (Thomas et al. 2007;Nugent 2008, 2011). Where detection devices show a positive detection, from tooth mark impressions left on the device, three or four followup traps are set around the device for three or four nights and all captured possums are recovered for necropsy. All detection devices and trap locations have their GPS locations recorded on personal digital assistants along with the details of activities e.g., initial trap setting, trap checking. The data recorded become part of the dataset used for generating a probability of TB freedom (Anderson et al. 2013). Surveillance techniques for different wildlife species Determining the presence or absence of TB in possum populations is crucial to measuring progress towards, and achievement of, TB management plan objectives. Detecting the presence of TB in possum populations when the prevalence is very low is expensive, and can be impracticable in areas of extensive forest. Nevertheless, surveillance for the continuing presence or absence of TB from wildlife forms the current basis of next-step TB management decisions in New Zealand, by way of predictions of likelihood of disease absence derived from the probability of freedom utility (Anderson et al. 2015). To support this, possum carcasses infected with TB, as well as carcasses or offal from other infected wildlife, provide sources of material from which to detect potential M. bovis infection; this applies for a range of mammalian wildlife scavengers which are recognised as spillover hosts of TB in New Zealand and is outlined below for each species. Possums Where it is economical and practical, all carcasses of possums killed during the eradication phase or during surveys undertaken in Free Area Protection zones are subject to necropsy and submission of selected lymph nodes for M. bovis culture (Nugent et al. 2015a). In farmed areas where it is relatively easy to transport carcasses, possum surveys provide an economical means of direct TB surveillance, provided the density of possums is very low (relative abundance <2% TCI). However, when possum density is high, or where access is difficult, direct surveillance of possum carcasses becomes expensive, time-consuming and logistically impractical. Currently research is being undertaken to use information from possum surveys as a means of estimating the size of the possum population in a defined area. Knowing what proportion the possum sample is of the total population enables the sensitivity of detecting TB in the possum population to be estimated, given a defined design prevalence (Martin et al. 2007). Prototype methodologies have been described for identifying individual possums during population estimation, some based on DNA identification . One prototype investigation method involves using a remote system to collect individual samples of possum DNA as the capture stage, and to then cross-reference DNA extracted from samples taken from each possum killed during the survey as the recapture stage. Another prototype investigation relies on placing GPS collars on random possums caught prior to surveying to form the capture sample, and use the number of collared possums caught during the surveillance stage as the recapture sample. Wild pigs Where present, wild pigs provide the most sensitive and economical means of detecting TB in the possum population in extensive, rugged and forested areas (Anderson et al. 2015;Nugent et al. 2015b). Surveillance of TB using wild pigs is usually passive through hunters supplying pig heads with predilection-site submandibular lymph nodes intact, together with the GPS location of the kill sites, for subsequent necropsy. For surveys in areas where wild pigs are uncommon, captured disease-free wild pigs can be fitted with radio and GPS collars, and then released into the area of interest as bespoke disease sentinels. All data from pig surveys are used to help provide a statistical probability as to whether TB has been successfully eradicated from the possum population (Anderson et al. 2015). Ferrets Although ferrets play a role in TB dynamics in New Zealand, they are generally not sufficiently abundant to maintain the disease intra-specifically, rather there is a need for continued infection from them scavenging tuberculous carrion (Ragg et al. 2000). Consequently, TBfree New Zealand does not usually control ferrets, but instead carries out ferret surveys for disease surveillance purposes. Via their scavenging actions, ferrets provide a reasonably sensitive means of detecting the local presence of tuberculous possums, providing a cost-effective form of wildlife surveillance for some farmland environments, especially where feral pigs are absent. Typically, ferrets are trapped using a variety of kill and live-capture traps (Ragg et al. 2007) and their carcasses are supplied for necropsy, where a selection of predilection site tissues (predominantly gastrointestinal tract lymph nodes) is pooled for bacteriological culture to detect M. bovis. Wild deer In some extensive, rugged and forested areas of New Zealand, there are few or no wild pigs and ferrets are absent. In such areas, surveys of wild deer (normally red deer, Cervus elaphus) are used to detect the presence of TB in possums despite the low sensitivity of detection which this offers in comparison to other wildlife sentinel species (Nugent et al. 2015b Concluding remarks Since the early 1970s, when possums were confirmed as wildlife vectors of TB in New Zealand, the successful management of bovine TB in New Zealand has depended on the application of aerial and ground-based lethal methods to control possum populations. These methods have improved significantly over the last three decades, both in delivering cost-effective possum control and in minimising any adverse non-target and environmental impacts. The improvements in knowledge gained from ongoing research and operational experience are captured and updated in a range of technical guidelines and standard operating procedures produced by TBfree New Zealand, as well as in the National Pest Control Agencies range of best practice manuals. The competitive performance-based contract system implemented in the mid 1990s enabled an industry of independent contractors. To be paid, contractors had to achieve target residual TCI, and this contractual requirement drove a level of innovation in possum control methodology that now supports increasingly ambitious objectives to reduce the incidence of TB, and where appropriate, eradicate the disease from wildlife. The critical pest management developments towards this outcome include the following: aerial application of 1080 baits for controlling possums over extensive areas of rugged and remote forest, at lower bait application rates and with much reduced risk to non-target species; use of groundbased control, e.g. cyanide products, other toxins and traps, for controlling possums over extensive areas of farmland with little risk to livestock, dogs, and people; a national standardised method for monitoring relative possum abundance, enabling a performance-based contract system to be implemented; and a national database and management system (VectorNet) that supports decisions on the type and extent of future control or wildlife surveillance. The first three of these key tools continue to be improved, by incrementally fine-tuning their use, e.g. modifying distribution and sowing rates of aerial 1080 baits), through integration of new tools such as detection devices or adoption of new poisons and means of applying them. Together this has led to improvements in the effectiveness and efficiency of possum control. Application of current research and operational findings are expected to lead to further improvements in both possum control and wildlife surveillance. The currently available suite of tools provides for cost-effective control of possums to very low and even densities (relative abundance of <1% TCI) over the whole or major parts of each VRA, including extensive areas of forests. Possum populations are now being maintained at low densities over approximately 9 million hectares of the remaining 9.9 million hectares of VRA land (PG Livingstone, unpublished data). Possum control has achieved major reductions in prevalence of M. bovis infection in both livestock and wildlife, and has led to eradication of TB from possum populations in 14 VRA. This success indicates that eradication of TB from possum populations in the current TB eradication areas is a realistically achievable goal, using the tools and systems currently in place. In 2011, TBfree New Zealand's strategy changed from one of reducing the number of M. bovis-infected livestock herds to eradication of TB from possum populations in at least 2.5 million hectares (Anonymous 2012a). This change in focus has driven changes in the possum-control contracting industry, particularly for ground-based contractors. These contractors were originally solely focussed on possum control. Under the new strategy, although still using traps and killing possums, contractors have become collectors of data for input into the proof of freedom utility to support next-step management decisions. Thus modern contractors have to be proficient at using GPS enabled personal digital assistants to collect data on detection devices and possum capture locations, and managing these data sets as well as recovering carcasses for necropsy. Given the costs of control, scenario modelling has indicated that it would likely take until at least 2055 for TB to be eradicated from wildlife in New Zealand (PG Livingstone, unpublished data). Research and operational innovation through adaptive management has the potential to substantially reduce the costs, and therefore the time required to achieve eradication by: (1) looking at ways of accelerating the time to confirm TB eradication, by sampling possum populations and assessing the prevalence of M. bovis infection in the population, and then using this information to determine the level of population control required to eradicate infection; (2) using new technologies to identify possum locations for specific targeted control e.g. aerial infrared technology; (3) exploiting new research findings on possum feeding behaviour in the design of lower cost aerial baiting operations; (4) identifying factors necessary to consistently achieve very high possum kills, thus reducing the need for repeat control effort; (5) using sampling theory to determine the probability that TB has been eradicated from possums over large areas of extensive forest, where there are few sentinel species and where the cost of sampling possums themselves would otherwise be prohibitive (Anderson et al. 2015). The outcomes of these new research and adaptive management objectives should become available within the next 5-7 years, which, together with outcomes of research currently being undertaken, are expected to significantly reduce the costs and time to eradicate TB, from both wild and domestic animals in New Zealand.

v3-fos

2018-01-25T21:52:20.068Z

2015-11-05T00:00:00.000Z

1991674

s2

Himalayan dock (Rumex nepalensis): the flip side of obnoxious weed Himalayan dock (Rumex nepalensis) was evaluated for forage value and antinutrients under three, five and seven weeks cutting intervals in the temperate environment. Dry matter (DM) content was measured for each cutting interval. Forage quality parameters such as Crude Protein (CP), Acid Detergent fiber (ADF), Neutral Detergent Fiber (NDF), Calcium (Ca) and Phosphorus (P) were analyzed. Plants with seven weeks cutting interval gave higher DM yield. CP and P content were significantly higher for three weeks cutting intervals. Average CP contents were 31.38 %, 30.73 % and 27.32 % and average P content 0.58 %, 0.52 % and 0.51 % for three, five and seven weeks cutting intervals, respectively. Ca content did not differ significantly between cutting intervals. The average Ca content were 0.91 %, 0.90 % and 90 %, for three, five and seven weeks cutting intervals, respectively. Tannin and mimosine contents were not significantly different between cutting intervals. Average tannin contents were 1.32 %, 1.27 % and 1.26 % and mimosine 0.38 %, 0.30 % and 0.28 % for three, five and seven weeks cutting intervals, respectively. The study concluded that R. nepalensis could be a potential source of protein for livestock. The study also suggests seven weeks harvesting interval to provide plants with high dry matter yield, high forage quality and very low levels of anti-nutrients. Background Rumex nepalensis or the Himalayan dock belongs to the family Polygonaceae. It is commonly sighted at higher altitudes and grows between 900-4000 m on moist as well as dry slopes, under shades, and even in plains. R. nepalensis have broad ecological tolerances. It is a common weed in pastures and the plants are known to become dominant and outcompete desirable pasture species and degrade pasture quality. It regenerates from tap roots and establishes quickly as seedlings. Once established, tough tap roots become difficult to remove and are not readily killed by tillage. These characteristics have often made farmers to regard this species as the most difficult weed [1]. The general issues of Rumex spp are that they are weeds of grassland, particularly on disturbed areas with high fertility. However, available literatures suggest that this species possess other characteristics. From the forage perspective, Humaira et al. [2] noted the moderate palatability of leaves of R. nepalensis to goat. At the young phenological stages of growth, the plants of R. nepalensis are leafy and Al Haj Khaled et al. [3] positively associated leafiness to higher crude protein (CP) and digestibility. While the leaf CP of matured R. nepalenses is about 14 % [4], the leaf CP of young plants of this species is over 32 % [5]. These reports highlight difference in CP content of this species according to phenological growth stages besides indicating that the young plants of R. nepalensis could be a good source of protein. Despite the higher CP content, R. nepalensis has been least studied for its forage value. Further, there is also a lack of information on the anti-nutritive properties of R. nepalensis. Antinutritional factors in animal feed are known to reduce the availability of one or more nutrients to the animal. Feed represents the largest single production expense for cattle production. Protein is a critical dietary nutrient in all cattle feed, yet the high cost of commercial protein supplements is one of the main limitations to efficient animal production by smallholders [6]. On the contrary, the pasture based cattle production is relatively cheaper, however, the cattle farmers must ensure that the forages are supplemented with adequate protein and energy to meet the nutritional demands of animals. In such scenario, the use of protein rich plant sources can reduce demand for protein ingredients from expensive commercial feed sources. Thus, protein from plant resources can reduce the overall production cost. To generate estimates of forage quality and anti-nutritive factors, a study was conducted with the objectives to evaluate difference in forage value and anti-nutritive properties between three phenological growth stages of R. nepalensis in the Himalayan highland. Experimental site The study was conducted in Bumthang valley in central Bhutan. Bumthang lies at an altitude of 2700 m and is one of the focal districts for pasture development in Bhutan. The valley is situated in the north central temperate region with an area of about 2715 km 2 [7]. Most pasture fields are on rugged terrain and normally spread across slopes with gradients ranging from 0-35° [8]. The climate is dominated by the Indian monsoon with high levels of precipitation between June and September followed by cold and dry winter. The mean maximum precipitation of 152 mm is recorded in the month of July and August, and the mean maximum temperature of 22°C is recorded in June and the mean minimum temperature of −2.7°C in January [8]. The vegetation growing period commences from May and peaks in August with gradual decline in temperature from September. Winter is cold and dry from November to March. Description of species under study R. nepalensis is a perennial herb. Roots are large and stems erect. The plants are 50-100 cm tall. Basal leaves have petiole length of 4-10 cm. Leaf blade is broadly ovate. Inflorescence is paniculate and flowers are bisexual. The plants grow in the altitude range of 900-4000 m and are commonly seen on grassy slopes, moist valleys and along ditches. Experimental design and treatments The experimental design was a randomized complete block. The treatment was cutting interval of three types, three, five and seven weeks intervals with five replications. The experiment was established by demarcating plots on existing vegetation of R. nepalensis. The individual plot size was 1 m 2 . Spacing were 50 cm between plots and 70 cm between replications. The plants were grown under natural conditions without fertilizer application. The treatments were applied on June 25, July 8 and July 22 for three, five and seven weeks cutting intervals, respectively. Estimation of DM, forage value and anti-nutrients Plants in each plot were clipped and weighed for fresh weight. The clipped samples were processed and oven dried at 60°C for 48 h. Dried sample weight was recorded and the DM per hectare area was estimated. Following DM estimation, the dried samples were processed and analyzed for total nitrogen (N). The total nitrogen content in the sample was determined with Kjeldahl method and CP was estimated as % N × 6.25. The estimate was compared with the CP content of other high quality forages. ADF was estimated following the procedures described by Goering and Van Soest [9] and NDF with methods of Van Soest [10]. Ca and P were estimated with methods of AOAC [11]. The anti-nutrients estimated were mimosine and tannin. Mimosine content was estimated with the calorimetric method described by Matsumoto and Sherman [12] while tannin content was estimated using vanillin hydrochloric acid method of Burn [13]. Data analysis The dataset was tested for normalcy and homogeneity of variances using Shapiro-Wilk and Levene's tests, respectively. Wherever required, data was logarithmically transformed to meet the assumptions of ANOVA. Differences in the means of morphological traits, CP, Ca, P and anti-nutrients between cutting intervals were assessed with several one-way ANOVAs. Differences between means were considered significant if P values were lower than 0.05. The entire dataset was analysed with SPSS 22 [14]. Results Morphological traits, Crude protein and anti-nutrients DM content was significantly higher for plants receiving 7 week cutting interval (Fig. 1a). CP in actively growing plants of R. nepalensis varied among cutting intervals. CP declined with increasing cutting interval. CP content was significantly lower for the plants subjected to seven weeks cutting interval than those under three and five weeks cutting intervals (Fig. 1b). Ca level was not significantly different between cutting intervals (Fig. 1c). However, P was significantly greater for R. nepalensis under three weeks cutting intervals (Fig. 1d). Both ADF and NDF were significantly higher for plants under seven weeks cutting interval ( Fig. 2a and b). Although non-significant, the tannin and mimosine content were higher for the plants subjected to three weeks cutting interval ( Fig. 3a and b). Comparison with other important feed resources revealed R. nepalensis to possess greater level of CP, Ca and P than white clover and willow leaves (Table 1). However, the CP, Ca and P contents were comparable with the Karma feed, a commercial feed concentrate known to be of higher quality. CP, fiber and mineral content The present study shows that cutting intervals below five weeks provide CP over 30 %, which is closer to the leaf CP content of 32 % reported by Abbasi et al. [5]. Although high in CP, the short intervals are disadvantaged by low DM yield. Amongst cutting intervals, the seven weeks interval appears to provide optimum CP and higher DM yield. From forage standpoint, seven weeks seem to be an optimal cutting interval for R. nepalensis, from which a balance between forage yield and quality can be expected. CP content of seven weeks interval is about 27 %, which translates to 270 g CP kg −1 DM. Upreti and Shrestha [15] categorize feeds and forages with CP 20 % and above as high quality roughages and R. nepalensis falls in this category. This evidently highlights the potential of R. nepalensis to make valuable contributions to the nutritive value of forage-based diets. According to Topps [16], if forage legume is used as protein source for milk production in grass based diet the legume should contain not less than 160 g CP kg −1 DM to obtain a desirable protein level of 100 g CP per kg DM in the final diet. Ruminants require minimum of 150 g CP kg −1 DM for lactation and growth [17]. For the early lactating cows, Linn [18] recommends ration containing 18-19 % CP or 180-190 g CP kg −1 DM. The CP of R. nepalensis is much higher than the required level and in excess in the range of 80-110 g CP kg −1 DM, which strongly suggests that R. nepalensis can be a protein rich source to support milk production. However, harvest beyond seven weeks interval may cause further decline in CP content below 20 %, which will categorize plants as moderate quality roughages [15]. The CP percentage of R. nepalensis is higher than the other important temperate forage resources such as white clover, willow leaves and grass-clover pasture, but compares well with Lucerne hay and Karma feed, the popular concentrate feed in Bhutan. This further reiterates that R. nepalensis if harvested at early stages of growth could be a protein rich supplement in cattle feed. Thus, R. nepalensis has potential to provide alternative low-cost protein to meet the nutritive requirements of cattle in smallholder farms. However, owing to low DM yield, it is likely that the CP yield in terms of kg ha −1 , might be lower than other feed resources. This could be a disadvantage when the demand for dietary protein is high. Martens et al. [6] asserts that, in the smallholder farms, fast growth rates demand an optimum supply of dietary protein, which may not be met by the locallygrown feeds. Both ADF and NDF content increased with increasing cutting intervals with highest content under seven weeks cutting interval. However, the values were below 31 % and 40 % for ADF and NDF, respectively. According to Upreti and Shrestha [15], forages with ADF below 31 % and NDF below 40 % are categorized as roughages with good digestibility. This highlights that R. nepalensis can still have good digestibility even when harvested at seven weeks interval. However, similar to CP, the cutting interval greater than seven weeks may be less desirable as it is likely to cause decline in digestibility below the desired level. The calcium and phosphorus content under seven weeks cutting interval were about 0.90 % (9 g kg −1 DM), 0.52 % (5.2 g kg −1 DM), respectively. For grasses and forage legumes, Upreti and Shrestha [19] categorize Ca content below 2 % as low and P content above 0.5 % as high. Therefore, the Ca content of R.nepalensis must be considered low and inadequate to be included in the ration of a lactating cow that requires a minimum of 0.75 % Ca whereas P level is high enough [18]. Anti-nutrients Tannin and mimosine were two main antinutrients present in R. nepalensis. Tannins has been found to reduce the digestibility of protein and carbohydrates including starch and fibers, and has bitter and astringent taste, which in many cases reduces palatability and can depress growth [6]. In this study, the tannins in R. nepalensis under all cutting intervals were below 1.3 %, which translates to 13 g kg −1 DM. The amount is far below than the total tannin content of oak leaves (Quercus semecarpifolia) (78 g kg −1 DM) [20], an important tree fodder in the Himalayan region. According to Barry and Manley [21], forage containing more than 5 % tannins are tannins rich forages. Tannins with 5 to 10 % in the feed are antinutritive and toxic [22]. The tannin level of R. nepalensis must be considered very low. In contrast to the toxic effects of high tannin level, the low tannin level has been reported to have beneficial effects. Low tannin level protects protein of forages and allows a high efficiency of feed [15] utilization by the animal [23]. This likely explains why there has been no field reports of animal poisoning by this species. Mimosine is the second antinutrient found in R. nepalensis. Under all cutting intervals, the mimosine level was below 0.38 %, which translates roughly to 3.8 g kg −1 DM. The toxic effects are found to manifest when levels of mimosine in diets exceed 0.015 % of animal's body weight [24]. In terms of DM weight, the toxic amount calculates to 45 g per 300 kg body weight, which is the standard live body weight of one livestock unit consuming 6 kg dry matter in the Bhutan Himalaya [25]. If one livestock unit feeds on R. nepalensis vegetation, the amount of mimosine likely to be consumed estimates to just over 20 g per 6 kg DM. This is far below the lethal amount and very unlikely to cause toxic effects. Conclusion R. nepalensis has high CP content under all cutting intervals but seven weeks interval provides high dry matter yield. High CP, low fiber and P content except Ca qualify R. nepalensis to be graded as a prime feed and a potential source of protein. Level of antinutrients is very low and plants are safe for use as protein supplement. The results of this study have value for farmers and development workers. Although high in overall quality, there is a need for further studies on the effects of feeding R. nepalensis on milk production. Further, the combination of this species with other feeds should also be studied to establish optimum levels of inclusion to maximize feed intake. Finally, rather than viewing R. nepalensis as difficult weed, much would be gained if it is utilized as a protein supplement for livestock.

v3-fos

2017-05-31T23:14:06.838Z

2015-03-19T00:00:00.000Z

7818364

s2

Determining Antioxidant Activities of Lactobacilli Cell-Free Supernatants by Cellular Antioxidant Assay: A Comparison with Traditional Methods Antioxidant activity of lactic acid bacteria is associated with multiple health-protective effects. Traditional indexes of chemical antioxidant activities poorly reflect the antioxidant effects of these bacteria in vivo. Cellular antioxidant activity (CAA) assay was used in this study to determine the antioxidant activity of cell-free supernatants (CFSs) of 10 Lactobacillus strains. The performance of the CAA assay was compared with that of four chemical antioxidant activity assays, namely, DPPH radical scavenging, hydroxyl radical scavenging (HRS), reducing power (RP), and inhibition of linoleic acid peroxidation (ILAP). Results of the CAA assay were associated with those of DPPH and ILAP assays, but not with those of RP and HRS assays. The inter- and intra-specific antioxidant activities of CFS were characterized by chemical and CAA assays. L. rhamnosus CCFM 1107 displayed a high antioxidative effect similar to positive control L. rhamnosus GG ATCC 53103 in all of the assays. The CAA assay is a potential method for the detection of antioxidant activities of lactobacilli CFSs. Introduction Increasing scientific evidence suggests that oxidative stress is involved in the pathogenesis of various disorders and diseases, such as alcohol-induced liver injury, non-alcoholic fatty liver disease, ageing, and cancer [1][2][3][4]. Oxidative stress is a result of an imbalance between production and elimination of reactive oxygen species (ROS) and free radicals, which are primarily removed by the endogenous antioxidant defense system [5]. Consumption of antioxidants, which can quench free radicals and ROS, may be beneficial to human health. Synthetic antioxidants are effective in slowing oxidation, but pose concerns in regard to the safety and toxicity of the antioxidants [6,7]. Solution Preparation A: Linoleic acid emulsion (20 mL Preparation of lactobacilli CFS Lactobacilli CFSs were prepared as described by Chen and Wang [12,18] with slight modification. Cultures of the 10 Lactobacillus strains were adjusted to approximately 10 9 CFU/mL. Subsequently, the aliquots of the culture were transferred to 5 mL polypropylene tubes and centrifuged (10,000g, 10 min, 4°C). The pH value of the supernatant was continuously neutralized with 1 M NaOH [33]. The resulting supernatant was filtered (0.22 μm pore size). LGG CFS was used as a positive control. MRS broth medium (pH 7.0, filtered with a 0.22 μm pore size filter) served as a negative control. Chemical Assays to Determine AAs of Lactobacilli CFSs DPPH Radical Scavenging Activity Assay. The scavenging effect of the CFSs of 10 Lactobacillus strains on the free radical DPPH was measured in accordance with the slightly modified method of Lin and Chang [28]. A Sample (CFS, MRS broth, 1 mL) and a freshly prepared DPPH solution (0.2 mM, 1 mL, Sigma-Aldrich, St. Louis, MO) were mixed. The mixture was vigorously shaken and left to react for 30 min in the dark at room temperature. The control sample contained deionized water instead of the sample solution. The scavenged DPPH was then monitored by determining the absorbance at 517 nm using SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale CA). The radical scavenging activity was quantified as where ABS C and ABS S are the absorbance of the control and test samples at 517 nm, respectively, and S is the volume (mL) of the sample. HRS Activity of Lactobacilli CFSs. The HRS activity of the CFSs was analyzed as previously described modified method [13,19]. A sample (CFS, MRS broth, 1 mL), 1, 10-phenanthroline (2.5 mM, 1 mL; Sigma-Aldrich, St. Louis, MO), PBS (pH 7.4, 1 mL), and FeSO 4 (2.5 mM, 1 mL) were mixed. The reaction was initiated by adding H 2 O 2 (20 mM, 1 mL) and incubating at 37°C for 90 min. HRS activity was monitored by identifying the increase in absorbance at 536 nm by using an SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale CA). HRS activity was calculated using the following equation: where A S is the absorbance of the sample, A C is the absorbance of the control solution (deionized water was used instead of the sample at the same amount), and A b is the absorbance of the solution without samples and H 2 O 2 . RP of Lactobacilli CFSs. The CFS reducing activity of the CFSs was determined as described by Lin and Yen [35] with slight modification. A sample (CFS, MRS broth, 0.5 mL) was briefly mixed with potassium ferricyanide (1%, 0.5 mL) and PBS (pH 6.6, 0.5 mL). Subsequently, the mixture was heated at 50°C for 20 min and allowed to cool. Upon cooling, 0.5 mL of 10% trichloroacetic acid (TCA) was added to the mixture and then centrifuged at 3000g for 5 min. The upper layer (1 mL) was mixed with ferric chloride (0.1%, 1 mL) and allowed to react for 10 min. The absorbance of the mixture was obtained at 700 nm by using an Spectra-Max M5 microplate reader (Molecular Devices, Sunnyvale CA). Higher absorbance of the mixture indicated higher reducing activity. The reducing activity of cysteine served as the standard. ILAP of Lactobacilli CFSs. The anti-lipid peroxidation activity of the CFSs was assessed by using the thiobarbituric acid (TBA) method [22] with slight modifications. PBS (pH 7.4, 0.5 mL), linoleic acid emulsion (1 mL), FeSO 4 (0.01% w/v, 0.2 mL), ascorbic acid (0.02%, w/v, 0.2 mL), and sample (CFS, MRS broth, 0.5 mL) were mixed and then incubated at 37°C for 12 h. Subsequently, 2.0 mL of the reaction mixture was mixed with butylated hydroxytoluene (0.4% w/v, 0.2 mL), TCA (4% w/v, 0.2 mL) and TBA (0.8% w/v, 2 mL). The mixture was incubated at 100°C for 30 min and then allowed to cool, 2 mL of chloroform was then added for extraction. The upper extract was obtained and absorbance was determined at 532 nm by using an SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale CA). The samples were substituted with deionized water in the control group. The inhibition rate was calculated by using the following equation: where A S is the absorbance of the sample, and A C is the absorbance of control solution that sample solution, in which the sample solution was replaced with same amount of deionized water. Assessment of Antioxidant Effects of Lactobacilli CFSs using CAA Assay Cell Culture. Human hepatocellular carcinoma HepG2 cells (Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) were grown in HyClone Dulbecco's modified Eagle's medium (DMEM) with high glucose (GE Healthcare, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), penicillin (100 U/mL), streptomycin (100 μg/mL) (Sigma-Aldrich, St. Louis, MO), and 10 mM 4(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Sigma-Aldrich, St. Louis, MO). The cells were maintained at 37°C in an incubator with 5% CO 2 . The cells used in this study were at passage 10 to 20. Cell Cytotoxicity Assay. Cytotoxicity of the lactobacilli CFSs was measured according to the modified methylene blue assay [36]. Briefly, HepG2 cells were seeded at a density of 4×10 4 cells/well on a 96-well microplate in 100 μL of DMEM for 24 h at 37°C. After washing with PBS, HepG2 cells were treated with 100 μL of the sample (CFS, MRS) or deionized water (control) for up to 24 h at 37°C. To assess cell viability, the cells were washed with PBS and then incubated with 50 μL/well methylene blue (98% HBSS, 0.67% glutaraldehyde, and 0.6% methylene blue) for 1 h at 37°C. After the incubation, the cells were washed with PBS until the PBS was clear, and 100 μL/well elution (49% PBS, 50% ethanol, and 1% acetic acid) was then added, the microplate was then placed on a table oscillator (Thermomixer Comfort, Eppendorf AG, Hamburg, Germany) for 20 min. The absorbance was obtained at 570 nm by using an SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale CA). Different samples were compared with the control. A CFS with more than 10% less absorbance than the control, was considered cytotoxic [37]. CAA Assay. CAA assay was used to evaluate the lactobacilli CFSs as previously described [26,38,39]. Briefly, HepG2 cells, the most commonly used in CAA assay [26], were seeded at a density of 6×10 4 cells/well on a black 96-well microplate (with transparent bottoms) in 100 μL of DMEM for 24 h at 37°C. After rinsing with PBS, HepG2 cells were treated with 100 μL of the sample (CFS, MRS broth), which includes 25 μM of DCFH-DA for up to 1 h at 37°C. The cells were washed with PBS and then treated with 100 μL of 600 mM ABAP solution. Fluorescence was obtained using an SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale CA) for 13 cycles at 5 min intervals (λex = 485 and λem = 538). After blank subtraction from the fluorescent readings, the area under the curve of fluorescence versus time was integrated to calculate the CAA value of each sample as follows: Where SA is the area of the sample, and CA is the integrated area in the control curve. Statistical Analysis All of the tests were performed in triplicate. Data were presented as mean ± standard deviation (SD). One-way ANOVA was performed with SPSS (Version 13.0, SPSS Inc., Chicago, IL), followed by Fisher's least significant difference, to verify significant differences between samples. The results were considered significant when p < 0.05. The Pearson correlation test was conducted to determine correlation between variables. Results and Discussion Chemical AA Assay of Lactobacilli CFSs Radical Scavenging Activities. Results of two radical scavenging methods (i.e., HRS and DPPH) showed that the 10 lactobacilli CFSs can inhibit the formation of the two radicals (Fig. 1). The CFSs of the 10 lactobacilli CFSs showed remarkably weaker HRS activity than DPPH radical scavenging activity. Furthermore, the AAs of the 10 lactobacilli CFSs were higher than that of MRS broth in scavenging the DPPH radical. This finding is consistent with the report of Shen et al. [19]. In addition, the HRS method provided similar results on the AAs of the CFSs (Fig. 1). Significant differences in AA values were observed between MRS broth and CFSs, except for CCFM 8661, CCFM381, and CCFM 424 in scavenging hydroxyl radicals ( Fig. 1, p < 0.05). Both the HRS and DPPH methods indicated that the CFSs of CCFM 1107 and CCFM 7469 exhibited strong AAs. The CFSs of CCFM8661 exhibited strong DPPH radical scavenging activities (75.94 ± 1.05 U/mL) that were not significantly different from that of the positive control LGG (77.29 ± 1.51 U/mL) (p > 0.05). However, CCFM8661 demonstrated significantly weaker HRS activity than LGG in scavenging hydroxyl radical (p < 0.05). Furthermore, the effect of L. casei 2W on DPPH and hydroxyl radical contradicted that of CCFM8661. Intact cells and intracellular cellfree extracts from L. casei subsp. casei SY13 and L. delbrueckii subsp.bulgaricus LJJ also differ in DPPH and HRS activities [13]. The different mechanisms involved in the radicalantioxidant reactions may explain the different in scavenging potentials of the compounds [40]. Moreover, the DPPH and hydroxyl radical activities of both L. rhamnosus CCFM 1107 and CCFM 7469 were significantly stronger than those of L. fermenti CCFM381 and CCFM 424 (p < 0.05). Liu et al. [8] reported that 12 Lactobacillus strains exhibit varying capabilities in DPPH radical scavenging. Therefore, a certain degree of inter-specific difference in radical scavenging activities could exist among the 10 tested Lactobacillus strains. RP of Lactobacilli CFSs. An earlier report showed that AAs and RP are directly related [41]. The RP of lactobacilli CFS is based on kinetics of the reduction of Fe 3+ to Fe 2+ to prevent the oxidation reaction and control transition metal ions [41]. The RP activities of lactobacilli CFS are shown in Fig. 2. The CFSs of LGG, CCFM1107, CCFM7469, CCFM14, CCFM137, CCFM419, CCFM381, and CCFM424 showed significantly higher RP values than that of MRS broth (p < 0.05). Meanwhile, the CFSs of L. casei 2W and CCFM 8661 increased, but not significantly, compared with that of MRS broth (p > 0.05). In all of the tests, CCFM1107 showed a high reducing activity that was close to that of the positive control LGG and equivalent to that of 62.68 μM L-cysteine. The results are consistent with the findings of a previous report [8], which revealed that 12 strains show varied abilities in reducing RP activity and that L. acidophilus BCRC 14079 displays six-fold stronger activity than B. infantis BCRC 14602. Therefore, an inter-specific difference exists in the RP of the test CFSs. ILAP of Lactobacilli CFSs. ILAP is commonly used to analyze AAs. Linoleic acid has been used as a source of unsaturated fatty acids [42]. In the present study, the AAs of lactobacilli CFSs were measured by ILAP assay. As shown in Fig. 3, the inhibitory rates of test lactobacilli CFSs on linoleic acid peroxidation ranged from 5.1 ± 0.22% to 67.93 ± 1.22% (Fig. 3). The inhibitory rates of the lactobacilli CFSs were significantly higher than those of MRS broth (p < 0.05), except for CCFM137, CCFM419, and CCFM424 (p > 0.05). The CFSs of CCFM1107, CCFM7469, and CCFM 8661 showed high inhibitory effects on ILAP compared with the positive control LGG (p > 0.05). Meanwhile, L. rhamnosus CCFM1107 and CCFM7469 demonstrated significantly stronger activities on ILAP than did L. fermenti CCFM381 and CCFM424. These observations indicated an inter-specific difference in ILAP among the lactobacilli CFSs. Moreover, the higher inhibitory effect of CCFM381 CFS than of CCFM424 CFS indicated an intra-specific difference in ILAP between these two CFSs. These results agree with the findings of an earlier report [42], which revealed that the inhibitory rates of six strains of L. acidophilus on ILAP range from 34.9% to 46.3%. Correlations among the Four Chemical AA Assays. Results indicated that the lactobacilli CFSs, which were measured by using chemical methods, exhibited certain antioxidant properties. Among the tested CFSs, LGG and CCFM1107 had the highest AA in all of the assays. In addition, lactobacilli CFSs exerted different AAs in various chemical antioxidant models. For example, the CFS of CCFM8661, CCFM7469, and L.casei 2W displayed different AA values in radical scavenging, RP, and ILAP. In a previous study, Zhu et al. [43] found that the water extracts of okara koji and the water extract of soybean koji show different AAs according to different in vitro antioxidant models. In this case, four chemical assays were used and compared to determine the antioxidant capacities of the CFSs, and their correlations are shown in Table 2. A significant association was found between scavenging of DPPH and hydroxyl radicals (r = 0.511, p < 0.01, n = 11). Meanwhile, the DPPH radical scavenging of the CFSs correlated significantly with ILAP (r = 0.604, p < 0.01, n = 11). HRS also had a significant association with RP (r = 0.494, p < 0.01, n = 11). However, ILAP showed no significant correlation with HRS and RP because of the diverse chemical aspects of potential antioxidant compound(s) explored [25]. The lack of correlation between results obtained through these different assays can be attributed to reasons related to instrument limitations, mechanisms, endpoint, quantification method, and biological relevance [44]. Therefore, the use of a single chemical method to screen strains with high AAs is difficult due to the different mechanisms of CFSs in vivo. CAA Assessment of the AAs of Lactobacilli CFSs Cell Cytotoxicity. Modified methylene blue assay is a typically fast and easy method of detecting cell death. Studies have shown that HepG2 cells could be used to demonstrate cell toxicity [36,45]. However, the toxicity of CFS on HepG2 cells has not been evaluated. In the present study, CFS and MRS broth were used to react with HepG2 cells and methylene blue was used to stain the HepG2 cells. Results showed that incubation with CFS and MRS broth inhibited less than 10% of HepG2 cells (Fig. 4). This result indicates that both CFS and MRS broth do not show significant cytotoxicity to HepG2 cells after 24 h incubation [37]. Previous research showed that feijoada whole meal, apple extracts, and vegetables inhibit less than 10% of HepG2 cells [39,46,47]. Therefore, we deduced that the CFSs are not cytotoxic, which is a typical observation for such functional products. Moreover, Maudsdotter et al. [48] found that the CFS of LGG can reduce cell cytotoxicity caused by Streptococcus pyogenes by producing lactic acid. Li et al. [49] showed that the CFSs of L. acidophilus not only display non-cytotoxicity, but also stimulate proliferation of embryonic, endothelial, and inflammatory cells in vivo. These observations suggested that the CFSs of the Lactobacillus strains are non-cytotoxic. CAA Assay. In the CAA assay developed by Wolfe and Liu [26], the ABAP-induced oxidation of dye DCFH-DA to fluorescent DCF and the fluorescence intensity of DCF were measured to represent the oxidation rate, thereby enabling the CAA assay to measure the antioxidation capacity of the antioxidants [26,50]. In the present study, we applied the CAA assay to determine the AAs of lactobacilli CFSs. The positive control showed fluorescence intensity of ABAP oxidation over time. The negative control group contained the cells without the addition of ABAP. These cells were used to illustrate the conditions devoid of any oxidation inducer. The CFSs of all 10 Lactobacillus strains inhibited oxidation, with higher fluorescence intensities than the negative control (p < 0.05) (Fig. 5A). However, these intensities were lower than that of the positive control over time, except for the MRS broth, which showed similar intensities to the positive control. The trends of inhibiting DCFH-DA to DCFH of CFS are similar to those of pure compounds, such as theacrine, sugarcane molasses, and Crataegus azarolus [51][52][53]. Puertollano et al. previously reported that concentrated supernatants from L. plantarum reduce ROS accumulation in HL-60 cells [54]. In addition, the present results showed that the ROS in HepG2 cells could be reduced by lactobacilli CFSs. Similar to other methods used for AA assessment, the CAA method reveals the total antioxidative capacity of the analyzed sample rather than the capacity of the individual components of the system. However, the CAA also differs in other aspects. It is the only method capable of predicting antioxidant response at the cellular level. It also allows the analysis of samples activity to change the redox cellular state. Moreover, the participation of different component cells is critical to develop an antioxidant response [25]. CAA units were calculated on the basis of the area under the curve of the fluorescence intensities of the CFS and ABAP-treated cells over time. A smaller area denotes higher CAA units and higher AAs of the sample [51]. All of the lactobacilli CFSs possessed significantly higher CAA values than the MRS broth (p < 0.05) (Fig. 5B). The cellular AA of quercetin was also determined by CAA assay in HepG2 cells (S1 Fig.) as a standard. The calculation results of CAA values (units) of the CFSs of Lactobacillus strains to the equivalent amount of quercetin with the same AA (μM) present more intuitive view of the AA of the CFSs of the Lactobacillus strains compared with this established antioxidant with known clinical efficacy (S1 Table). The CAA values of the CFSs ranged from 69.14 ± 4.87 to 95.55 ± 2.99 CAA units. The positive control LGG also exhibited a strong antioxidant property (95.55 ± 2.99 CAA units) in the CAA assay, which was stronger than that of Crataegus azarolus aqueous extract (79.62 CAA units in 800 μg/mL) [53]. The difference essentially depends on the bioavailability of the specific mixture of the available compounds and their synergistic interactions to yield final antioxidant responses at the cellular level [25]. CCFM1107, L. casei 2W, and CCFM8661 showed the highest AAs (95.40, 94.06, and 93.48 CAA units, respectively). No significant difference was found represents ILAP values. * and ** mean statistically significant difference at p < 0.05 and p < 0.01, respectively. Abbreviations: HRS, hydroxyl radical scavenging; RP, reducing power; ILAP, inhibition of linoleic acid peroxidation; CFSs, cell-free supernatants; CAA, cellular antioxidant activity; r DPPH , correlation between the CAA and DPPH radical scavenging activity assay; r ILAP , correlation between the CAA and ILAP assay; r HRS , correlation between the CAA and HRS assay; and r RP , correlation between the CAA and RP assay. with LGG (p > 0.05). However, CCFM419 provided the lowest CAA value, which was significantly different from that of the other strains (p < 0.05). Consequently, the 10 Lactobacillus strains showed significant interspecific differences in CAA. L. rhamnosus showed a higher CAA value than that of L. fermenti. CCFM1107 and CCFM7469 originating from the same L. rhamnosus, but the two strains had significantly different CAA values (p < 0.05). Similar results were observed between L. fermenti CCFM381 and L. fermenti CCFM424. These observations indicate intra-specific differences of test CFSs in the CAA assays. Various researchers have reported similar findings which were obtained using different methods [19,55] that is, intra-and inter-specific differences exist for both intact bacteria and CFS. Correlations between CAA and the Four Chemical AA Assays. The AA values of lactobacilli CFSs assessed with the four chemical assays did not correlate well with their abilities to inhibit the radical-mediated damage on HepG2 cells (CAA index). The CFSs of several Lactobacillus strains that exhibited low chemical AAs showed high responses in the CAA assay (Fig. 6). For instance, the CFSs of L. casei 2W and CCFM8661 showed weak AAs in HRS and RP, but were proven efficacious in the CAA assay. CCFM419 had low antioxidant efficacy in the CAA assay, but exhibited high HRS activity. No significant association was found among the results of the HRS, RP, and CAA assays (r HRS = 0.125, r RP = −0.195, p > 0.05, Fig. 6A). This observation agrees with the report of Huang et al. [56], in which the antioxidant values of Chinese bayberry obtained from ABTS, FRAP, DPPH, ORAC, and CAA assays do not correlate significantly with one another. In this case, the results are not surprising because the CAA assay monitors oxidative stress in cells that are related to cellular uptake, distribution, and metabolism of antioxidants. In chemical assays, antioxidants directly react with radicals [44]. Conversely, CCFM8661 has a high value in CAA, DPPH, and ILAP. The CAA assay revealed a significant association with DPPH and ILAP (r DPPH = 0.471, r ILAP = 0.826, p < 0.01, Fig. 6B). In addition, LGG and CCFM1107 exhibited high AAs in the four chemical assays and CAA assay, whereas MRS showed a low AA. Considering the inter-and intra-species difference among numerous bacteria strains, the AAs evaluated by CAA assay show accordance with that got by the four traditional chemical assays to some extent. For the mammalian cells engaged in CAA assay, the AAs evaluated by this assay may be more correlated with the actual situation in organisms than that got with chemical assays. Thus, the CAA assay is a potential method for the detection of AAs of lactobacilli CFSs. Conclusion This study shows that lactobacilli CFSs exhibit AAs that can be assessed quantitatively in HepG2 cells. The CAA method is a relatively reliable and sensitive method compared with the four chemical assays in screening the AAs of CFS. Oxidative stress is involved in numerous chronic degenerative diseases. CFSs that show AAs can be evaluated by the CAA method as promising candidates in the prevention and control of several free radical-related disorders. Future studies will be conducted to elucidate the possible mechanisms of CFS and to verify the specific functions of CFSs screened by CAA assay in animal models or human studies. Table. CAA values of the CFSs of the 10 Lactobacillus strains and MRS broth. The CAA value is expressed as an equivalent amount of quercetin (μM). Different letters indicate statistically significant differences at p < 0.05. (DOCX) Author Contributions Conceived and designed the experiments: JX GW QZ. Performed the experiments: JX ZG. Analyzed the data: JX ZG QZ. Contributed reagents/materials/analysis tools: HZ GW YQC WC. Wrote the paper: JX GW XL QZ.

v3-fos

2018-04-03T04:01:11.855Z

2015-12-30T00:00:00.000Z

18495634

s2

Molecular cloning and expression analysis of KIN10 and cold-acclimation related genes in wild banana ‘Huanxi’ (Musa itinerans) Banana cultivars may experience chilling or freezing injury in some of their cultivated regions, where wild banana can still grow very well. The clarification of the cold-resistant mechanism of wild banana is vital for cold-resistant banana breeding. In this study, the central stress integrator gene KIN10 and some cold-acclimation related genes (HOS1 and ICE1s) from the cold-resistant wild banana ‘Huanxi’ (Musa itinerans) were cloned and their expression patterns under different temperature treatments were analyzed. Thirteen full-length cDNA transcripts including 6 KIN10s, 1 HOS1 and 6 ICE1s were successfully cloned. Quantitative real-time PCR (qRT-PCR) results showed that all these genes had the highest expression levels at the critical temperature of banana (13 °C). Under chilling temperature (4 °C), the expression level of KIN10 reduced significantly but the expression of HOS1 was still higher than that at the optimal temperature (28 °C, control). Both KIN10 and HOS1 showed the lowest expression levels at 0 °C, the expression level of ICE1, however, was higher than control. As sucrose plays role in plant cold-acclimation and in regulation of KIN10 and HOS1 bioactivities, the sucrose contents of wild banana under different temperatures were detected. Results showed that the sucrose content increased as temperature lowered. Our result suggested that KIN10 may participate in cold stress response via regulating sucrose biosynthesis, which is helpful in regulating cold acclimation pathway in wild banana. Electronic supplementary material The online version of this article (doi:10.1186/s40064-015-1617-z) contains supplementary material, which is available to authorized users. Background Banana (Musa spp.) is one of the most important nutrient-rich crops, staple foods and ornamental plants cultivated in tropical and subtropical regions where temperature is relatively high. Nonetheless, considerable interests still exit in exploring banana cold-resistant genes and developing cold tolerant banana cultivars due to the chilling or freezing injuries they might experience at some of their cultivated regions ). Up to now, however, no effective method has yet been developed to effectively solve the cold injury problem. Wild banana germplasm resources are abundant in China, where various studies have been conducted over the past 20 years (Liu et al. 2007(Liu et al. , 2012Lai et al. 2007). Wild banana species are more cold resistant than cultivated ones and can grow under relatively lower temperatures (Lai et al. 2007). The discovery of beneficial wild banana gene resources is consequently of great usefulness for cold-resistance breeding of cultivated banana. Cold acclimation can dramatically increase freezing tolerance of plants and is very important for extending their adaptation areas (Zhang et al. 2009). It was reported that sucrose can enhance cold hardening of plants by regulating expression of cold-acclimation-associated genes such as CBF (C-repeat/DRE-binding factor), ICE1 (inducer of CBF expression 1), HOS1 (high expression of osmotically responsive gene 1) and so on (Palonen and Junttila 2002;Rekarte-cowie et al. 2008). The expression of CBF1, was identified to be induced under cold stress (Lee et al. 2001). And its CBF AP2 motif combines with the CRT/ DRE element in the COR promoter to enhance COR expression and ultimately plant cold tolerance (Chen et al. 2014). In Arabidopsis, three CBF genes, CBF1, CBF2, and CBF3, were found (Stockinger et al. 1997; Open Access Gilmour et al. 1998;Medina et al. 1999) and their functions in cold acclimation were also identified (Novillo et al. 2007). ICE1 is a transcription factor that can activate the expression of CBFs, thus function in regulating the cold-induced transcriptome and freezing tolerance (Chinnusamy et al. 2003). HOS1, a functional E3 ligase targeting ICE1 for ubiquitination-mediated ICE1 degradation, is the negative regulator of plant cold responses and its expression is regulated by osmotic potent changes (Dong et al. 2006). Sucrose can significantly influence the osmotic potential of plant cells (Palonen and Junttila 2002). Interestingly, the bioactivity of SNF1-related protein kinase catalytic subunit alpha 10/11 (KIN10/11), the central integrator of transcription networks in plant stress and energy signaling (Baena-González et al. 2007), can be inhibited by high concentrations of sucrose (Thalor et al. 2012). KIN10/KIN11, in reverse, can phosphate the key enzyme of sucrose biosynthesis (sucrose phosphate synthase) (Thalor et al. 2012). These correlations suggest that KIN10/KIN11 should function in cold hardening of plants. Thus far, however, studies of banana KIN10/KIN11 and cold-acclimation related genes were rare due to the lack of sequence information. Wild banana is widely distributed in all prefecture-level cities in Fujian Province, China (Lai et al. 2007). Among various germplasm resources, a wild banana population recently found in Huanxi, Fuzhou City, China, was found to be very tolerant to cold (Liu et al. 2012), making it very nice gene resources for cold-resistant genes and germplasm resources for cold-tolerant banana breeding. The release of Malaysian wild banana (Musa acuminata) genome data will be surely helpful for identification and characterization of these genes (D'Hont et al. 2012). So, in this study, KIN10, HOS1 and ICE1 (the target gene of HOS1) genes were identified by searching the Malaysian wild banana genome data and were cloned by RT-PCR and/or RACE technologies from cold-resistant wild banana 'Huanxi' (Musa itinerans). Their expression patterns under different temperature, together with the sucrose content in leaves, were also analyzed. Our study could be helpful in understanding the cold acclimation responses of wild banana under different temperature and in exploring the function of KIN10 in cold response. Identification, characterization and bioinformatic analysis of KIN10 genes from cold-resistant wild banana 'Huanxi' Multiple-sequence BLAST search revealed that KIN10-1, KIN10-2 and KIN10-3 had similar ORF sequences that were 95.51 % identical to the KIN10 of Malaysian wild banana (Musa acuminata) (GSMUA_Achr10 G09220_001). But the ORF sequences of KIN10-4, KIN10-5 and KIN10-6 shared lower identity (only 76.58 %). These sequence variations may be due to differences between genes or species. Bioinformatics prediction result revealed that all the 6 KIN10s were basic, hydrophilic, and unstable proteins possessing transmembrane domains with predicted location in the nucleus or in membranes. Moreover, 21-26 phosphorylation sites were found in KIN10s (Table 1). Observed variations in the number and position of these phosphorylation sites suggest that some of their potential functions may be different. The KIN10s possessed 10-13 conserved domains, most of which were protein kinase domains (Additional file 1: Table S1). Phylogenetic analysis of KIN10 sequences generated the tree shown in Additional file 2: Figure S1. Besides the Malaysian wild banana KIN10, Phoenix dactylifera KIN10 and Elaeis guineensis KIN10 showed the closest relationship with wild banana 'Huanxi' KIN10s. Identification, characterization and bioinformatic analysis of HOS1 from cold-resistant wild banana 'Huanxi' The HOS1 cDNA was 2926 bp long and contained a 2904 bp ORF encoding 967 amino acids. Multiplesequence BLAST comparison showed HOS1 from 'Huanxi' shared high similarity (93.95 %) with the Malaysian wild banana HOS1 (GSMUA_Ach1G14640_001). The major difference between the two species was the presence of a 140 bp insertion in the upstream region of the 'Huanxi' . On the basis of bioinformatics prediction analysis, HOS1 was shown to be a nuclear-localized, hydrophilic unstable protein without signal peptide. And 57 phosphorylation sites and a specific ELYS-like conserved domain were found in HOS1 (Table 1). Phylogenetic analysis of HOS1 sequences generated the tree shown in Additional file 3: Figure S3. Besides the Malaysian wild banana HOS1, Phoenix dactylifera HOS1 showed the closest relationship with wild banana 'Huanxi' HOS1. Identification, characterization and bioinformatic analysis of ICE1 genes from cold-resistant wild banana 'Huanxi' Multiple-sequence BLAST search showed that the cloned ICE1-1-ICE1-4 genes shared higher identity (97.52 %) with Malaysian wild banana ICE1 (GSMUA_ Achr10 G18380_001) compared with ICE1-5 and ICE1-6 (92.08 %). A 75 bp sequence, which was almost exactly the same size as that of ICE1 introns in Malaysian wild banana, was missing from the middle region of ICE1-1-ICE1-4 in wild banana 'Huanxi' . Other missing sequences in wild banana 'Huanxi' were a 16 bp sequence absent from the upstream region of ICE1-3 and ICE1-4 and a 19 bp sequence deleted from the termination codon region of ICE1-1 and ICE1-3. ICE1-5 and ICE1-6 were 9 bp longer in wild banana 'Huanxi' . Interestingly, compared with the Malaysian wild banana ICE1, ICE1-5 of wild banana 'Huanxi' contained one more intron and one fewer exon and ICE1-6 possessed two additional introns, which might be results of alternative splicing in evolution (Keren et al. 2010). According to bioinformatics prediction, the first four wild banana 'Huanxi' ICE1s encoded similar numbers of amino acid residues, whereas the number of amino acid residues encoded by ICE1-5 and ICE1-6 was quite different (Table 1). ICE1-1, ICE1-2, ICE1-5, and ICE1-6 were acidic proteins and ICE1-3 and ICE1-4 were basic. All six ICE1s were hydrophilic proteins with a specifically conserved basic-helix-loop-helix domain (IPR011598) as well as a non-integrated domain designated as PTH31945:SF0 and were predicted to be located in the nucleus. ICE5 and ICE6 featured an additional non-integrated conservative domain SSF55021 (Additional file 4: Table S2). The number of phosphorylation sites in ICE1s also varied from 20 to 30 among these 6 ICE1s (Table 1). ICE1s were predicted to be located in the nucleus and contained trans-membrane domains. Phylogenetic analysis of ICE1 sequences generated the tree shown in Additional file 5: Figure S2. Besides the Malaysian wild banana ICE1, the closest relationship was found between wild banana 'Huanxi' ICE1s and Elaeis guineensis ICE1. qRT-PCR analysis of KIN10, HOS1 and ICE1 genes in wild banana under low-temperature stress As shown in Fig. 1, the expression levels of KIN10, HOS1 and ICE1 genes differed significantly under different temperatures. At the banana critical temperature of 13 °C, all these genes showed the highest expression levels. The expression levels of KIN10 decreased significantly at 4 and 0 °C compared with control. Although the up-regulation level decreased, the expression level of HOS1 at 4 °C was still higher than that of the control. At 0 °C, however, the HOS1 expression level dropped to their lowest levels at 0 °C, a temperature at which ICE1 expression levels were obviously increased. Sucrose contents in wild banana changed significantly under low-temperature stress Sucrose contents of samples extracted from plants subjected to 0, 4, 13 and 28 °C for 36 h were shown in Fig. 1. Sucrose content rose markedly as the temperature dropped. Notably, the sucrose content in 'Huanxi' leaves at 4 °C was only about 1.16-fold higher than control. At 0 °C, however, the sucrose content was even 1.5-fold higher than that of the control. Discussion Although banana cultivars grow in tropical and subtropical regions, they also suffer chilling or freezing damage at some part of its cultivated areas such as in Fujian, Guangdong, Guangxi and Yunnan provinces in China. Wild bananas are more tolerant to cold and thus are nice materials for exploring cold-resistant genes and clarifying the cold-acclimation mechanism. Totally, 6 KIN10s, 1 HOS1 and 6 ICE1s genes were successfully obtained. Sequence variations were found among KIN10 and ICE1 genes, which may be a consequence of evolutionary adaption to the environment: the Malaysian wild banana is distributed in tropical regions, whereas the wild banana 'Huanxi' is found at the northern margin of the southern subtropical region, and winter temperatures differ significantly between these two regions. Further research is needed to determine what, if any, functional differences are caused by the observed sequence differences. Bioinformatic analysis also showed that all these genes are rich of phosphorylation sites, which suggest that phosphorylation may function a lot in plant cold resistance as phosphorylation was identified to play important role during the acquisition of freezing tolerance (Monroy et al. 1993;Komatsu et al. 1999;Schulze et al. 2012). Sequences analysis showed that protein-kinase domains of KIN10s were highly conserved, which might be the structural basis of the 'master molecular switch' in plant stress regulation (Baena-González et al. 2007). Observed variations in the number and position of these phosphorylation sites suggest that some of their potential functions may differ. The high number of conserved domains in wild banana KIN10s is unusual and reveals the diversity of these proteins' unique functions. At the same time, many of these conserved KIN10 domains are protein kinase domains, the structural basis of the 'master molecular switch' in plant stress regulation. Notably, the evolutionary alternative splicing events were identified in ICE1 genes. As these genes are cold resistance-related functional genes from wild banana 'Huanxi' , which is located in northern Fuzhou and often subject to low-temperature stress, we hypothesize that this alternative splicing phenomenon may be related to evolutionary adaptation to the environment to improve wild banana cold resistance. Whether any functional changes have occurred due to alternative splicing remains to be determined. KIN10/KIN11 is an important regulator in various plant stress responses and its ability can be inhibited by high concentrations of sucrose (Baena-González et al. 2007;Thalor et al. 2012). The function of sucrose in plant cold acclimation (Palonen and Junttila 2002;Rekartecowie et al. 2008) indicated that KIN10/KIN11 might be also involved in plant cold acclimation. To prove this proposition, a new-found cold-resistant wild banana 'Huanxi' was used for cloning of the stress-related gene KIN10 and cold-acclimation related genes, HOS1 and ICE1. To compare their expression under different temperatures and to elucidate their function in cold-acclimation, expression levels of these genes at the optimal temperature (28 °C), critical temperature (13 °C), chilling temperature (4 °C) and freezing temperature (0 °C) were detected. Banana is basically a tropical crop growing at temperatures ranging from 13 to 38 °C. Below 13 °C or above 38 °C, banana growth ceases and 13 °C is thought to be the critical low temperature for banana growth (Nelson et al. 2006). Our study revealed that the relative expressions of KIN10s, HOS1 and ICE1s all reached the highest levels at the banana critical temperature. KIN10 could promote catabolic processes and suppress anabolic processes (Thalor et al. 2012), the up-regulation of KIN10, therefore, could be helpful in maintaining the energy balance and thus improve the cold resistance of wild banana at critical temperature. The expression of KIN10 was significantly suppressed at both 4 and 0 °C. It was reported that once the cellular sucrose reached certain level, the activity of KIN10 would be suppressed (Baena-González et al. 2007;Thalor et al. 2012). At 4 °C, the sucrose content in 'Huanxi' leaves was about 21.2 mg/g fresh weight, which might be the threshold for sucrose content of wild banana 'Huanxi' . KIN10 was also reported to be able to phosphorylate the key enzyme of sucrose biosynthesis, sucrose phosphate synthase (Thalor et al. 2012), so we inferred that KIN10 could participate in cold responses or cold-acclimation of wild banana through regulating sucrose biosynthesis. The expression level of HOS1, an osmotically responsive gene, was still significantly higher than that of the control at 4 °C, which indicated that non-freezing lowtemperature could induce the expression of HOS1. The sucrose content in leaves was higher at 4 °C than at 28 °C, suggesting that the up-regulation of HOS1 may be a result of increasing osmotic potential in plant cells. HOS1 is a negative regulator of low temperature signal transduction that mediates the ubiquitination and degradation of ICE1 (Dong et al. 2006), which is a regulator of freezing tolerance in plants (Chinnusamy et al. 2003). At freezing temperature (0 °C), expression of HOS1 was suppressed and expression of ICE1 was induced. The downregulation of low temperature negative regulator and up-regulation of freezing tolerance regulator reflected well the increase of cold tolerance. Conclusion In conclusion, we successfully cloned several genes of KIN10, HOS1 and ICE1 from cold-resistant wild banana 'Huanxi' . Expression analysis showed that expression of these genes and sucrose synthesis were significantly influenced by low temperature (Fig. 2): the expression of ICE1 was induced by low temperature; the expression of HOS1 was induced at non-freezing temperature but was suppressed at freezing temperature; the expression of KIN10 was influenced by low temperature and it might participate in cold-response by regulating sucrose biosynthesis. Our study could provide clues for improving banana cold resistance and for cold-tolerant banana breeding. Plant materials and treatments Plants of cold-resistant wild banana 'Huanxi' (Musa itinerans) were obtained from the Wild Banana Germplasm Nursery of the Institute of Horticultural Biotechnology, Fujian Agriculture and Forestry University, Fuzhou, China. For temperature treatments, 25-day-old plantlets growing at 28 °C were subjected to 0, 4 and 13 °C for 36 h with plantlets growing at 28 °C as control. Gene cloning and sequence analysis Homologous gene sequences in GenBank database (http://www.ncbi.nlm.nih.gov) and in the Malaysian wild banana (Musa acuminata) genome-wide data (http:// banana-genome.cirad.fr) were downloaded and used as reference sequences for determination of the conserved regions and for primer design. Information of all the used primers was listed Additional file 6: Table S3. The gene cloning processes were shown in Additional file 7: Figure S4. The obtained cDNA sequences were all submitted to the GenBank database (http://www.ncbi.nlm.nih.gov). Bioinformatics analysis of these obtained genes and their deduced protein sequences were performed according to Tian et al. (2015). Amino acid sequences of KIN10s, ICE1s, and HOS1 were deduced and analyzed with the ExPASy Protparam tool (Wilkins et al. 1999 Fig. 2 The involvement of KIN10, HOS1, ICE1 and sucrose in the cold acclimation pathway of the cold-resistant wild banana 'Huanxi' . HOS1 high expression of osmotically responsive gene 1, ICE1 inducer of CBF expression 1, KIN10 SNF1-related protein kinase catalytic subunit alpha 10, OP osmotic potential searches were performed using the NCBI server. Multiple alignment and structural analysis of deduced proteins was performed using DNAMAN 6.0. Signal peptide and protein subcellular localization information were predicted using SignalP4.0 Server (Petersen et al. 2011) and PSORT (Nakai 2000) software, respectively. Protein transmembrane regions and orientations were predicted using the Tmpred program. Conserved domain fields were identified with InterProScan (http://www.ebi.ac.uk/interpro/ scan.html) (Mitchell et al. 2015) and phosphorylation sites were predicted using the NetPhos 2.0 Server (http://www. cbs.dtu.dk/services/NetPhos/) (Wong et al. 2007). Phylogenetic analyses were performed using neighbor-joining in MEGA 5.02 (Tamura et al. 2011). qRT-PCR expression analysis of KIN10s, ICE1s and HOS1 To explore the relationship of these cold-resistance genes and the cold stress response mechanism in 'Huanxi' wild banana under low-temperature stress, we conducted a gene expression analysis using qRT-PCR. After low temperature treatment, leaves were sampled for total RNA extraction. qRT-PCR analyses were performed as described by Lin and Lai (2013) on Roche LightCy-cler480 (Roache, Basel, Switzerland) with 18S rRNA as the internal control. Conserved regions of KIN10s, HOS1 and ICE1-1-ICE1-4 were used as templates for primer design. Three biological and technical replicates were made for each treatment. Primers used were listed in Additional file 8: Table S4. Sucrose content determination The same extracts obtained from leaves exposed to 0, 4, 13 and 28 °C (control) for 36 h and used for the qPCR analysis were analyzed for sucrose content. The sucrose contents in leaves were measured by using the method of (Xue 1985).

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2019-04-04T13:12:00.495Z

2015-01-01T00:00:00.000Z

93026384

s2

Synthesis Optimization and Characterization of Microencapsulated N-P-K Slow-Release Fertilizers Abstract Slow-release microencapsulated N, P, and K fertilizer synthesis was optimized and the products exhaustively characterized. Four NPK formulations with nutrient mass ratios of 1:0:0, 1:1:0, 1:1:1, and 2:1:1 were characterized following European Council Regulation 2003/2003 for fertilizers as well as ICP-AES, 1H-NMR, FT-IR, HPLC, elemental analysis, XPS, SEM, dynamic light scattering, thermogravimetry, differential thermogravimetry, activity index, and leaching rate. These fertilizers have good slow-release properties and decrease waste and crop contaminants, improving environmental protection. Compared to classic granulated fertilizers, plant nutrient availability was improved allowing reduced application. Nutrient leaching parameters describing four different kinetic models were evaluated. Graphical Abstract Introduction Chemical fertilizers have been crucial to the growth of world agricultural production by approximately 50% over the last 40 years [1][2][3]. According to the UN Food and Agriculture Organization, 1 kg of nutrient (N, P, K) yields an average harvest of 8.8 kg corn, 7.3 kg wheat, 8.5 kg rice, 5.5 kg soy, etc. Moreover, according to American specialists, the harvest is 41% controlled by chemical fertilizers and less by herbicides (15-20%), soil quality (15%), hybrid seed (8%), or irrigation (5%). However, about 20-70% of the applied nitrogen is lost to the environment, causing serious pollution and increasing costs [4][5][6][7]. The nitrogen effectively used is 50-70% in the first year, for potassium it is 50-60%, while for phosphorus it is around 15% [8]. A further 1-2% per year may be used over the following decades. An important quantity is lost by immobilization, denitrification, volatilization, or leaching. These losses are most important for nitrogen, and cause ground water and soil pollution with nitrates, nitrosamines, or other harmful compounds [9,10]. These appear in foods, affecting health [11,12]. Improved fertilizer utilization can reduce the amounts used. Fertilizer design considers two parameters: solubility over time, influenced by interaction with the soil, and plant uptake. Slow/controlled release allows the nutrients to be completely absorbed. These fertilizers contain at least one nutrient that either delays its availability after application or is available for significantly longer than a standard "quickly available fertilizer" [13]. According to standard SR EN 13266/2001, a slow-release fertilizer must release no more than 15% after 24 h, no more than 75% in 28 days and at least 75% overall. An ideal fertilizer: i) gives optimal development with a single application for the season; ii) gives maximum nutrient absorption; iii) has minimal side effects on soil, water, and air [36][37][38]. The present study examines urea-formaldehyde as a nitrogen fertilizer. Environmental decomposition of ureaformaldehyde resin is due to microbial action. Thus, N release depends strongly on biological activity, controlled by soil clay content, pH, moisture, wetting and drying, and temperature. The main objective of this work is the preparation and characterization of a slow-release fertilizer with microstructures that release nutrients when plants need them, and with minimal environmental effects. An ureaformaldehyde polycondensate was prepared by interfacial condensation in a microemulsion. Synthesis method Urea-formaldehyde resins form by reaction of formaldehyde with excess urea at controlled pH, temperature, mole ratio and reaction time, yielding polymethylene urea polymers with different molecular weights [39]. Fertilizer preparation was by an initial pre-polymerization followed by a polycondensation. In the pre-polymerization, urea reacts with basic formaldehyde forming mono-, di-, and trimethylolureas. This is a water miscible liquid, stable in weak base. This pre-polymer solution was brought to a pH around 5 and monoammonium phosphate and potassium chloride were subsequently added. The pre-polymerization product underwent polycondensation-crosslinking in acidic medium using linear alkylbenzenesulfonic acid (LABS-H) catalyst in organic solvent. Polycondensation for 2 hours at 50-70°C yielded products with medium molecular weights. Water was removed by azeotropic distillation, followed by drying at 70-80°C to remove residual solvent. The fertilizers were treated with polymeric agglomerants and extruded to 3 mm diameter extrudates. The NPK slow-release fertilizers were differentiated into three fractions. F1: the cold water soluble fraction (CWSF -soluble in water at 25°C) is mainly urea, dimers, and short U-F chains and is immediately available. F2: the hot water soluble fraction (HWSF -insoluble in water at 25°C, but soluble at 100°C) contains methylene ureas with medium-length U-F chains, and represents slow-release nutrients. F3: the hot water insoluble fraction (HWIFcomponents insoluble in cold or hot water) is mainly long U-F chains containing the extremely slow release and unavailable components. Equipment and characterization methods The fertilizer N, P 2 O 5 , and K 2 O contents were determined according to European Council Regulation 2003/2003 for fertilizers [40]. Nitrogen was determined by the Kjeldahl method with acid catalysis on a Kjeldahl Behrotest system and a K 350 distillation unit. After Kjeldahl digestion, the ammonia released by base addition was absorbed in 2% boric acid and titrated with standard 0.1 M H 2 SO 4 to a bromocresol endpoint. Phosphorus was determined by extraction as monobasic ammonium phosphate followed by acidic precipitation of quinoline phosphomolybdate. Potassium was determined by precipitation with sodium tetraphenylborate in weakly alkaline solution. Because tetraphenylborate precipitates both potassium and ammonium, the ammonium interference was removed by converting it to the unreactive hexamethylenetetramine with formaldehyde. Fertilizer samples were mineralized with a Berghof microwave digester in nitric acid and hydrogen peroxide. The resulting solution was analysed using inductively coupled plasma atomic emission spectrometry (ICP-AES) on a Varian Liberty 110 instrument with V-groove nebulizer, Fassel torch, and Czerny-Turner monochromator. Phosphorus was identified at 214.914 nm and potassium at 769.896 nm. The LOD was greater than 0.4 mg kg -1 . 1 H-NMR spectra were acquired with a Varian Gemini 300 BB operated at 300 MHz. The samples were dissolved in dimethyl sulfoxide (DMSO) and analysed at room temperature with a 12.9° pulse and 32 scans. Fourier transform infrared analyses were performed on a Perkin Elmer GX FT-IR spectrophotometer from (10,000 -20) cm -1 . Liquid chromatograms were recorded on a HPLC with a diode array detector at λ = 200 nm, on a Metacarb 67 H column. The mobile phase was 0.05 M aqueous H 2 SO 4 at 0.6 mL min -1 and 86 atm. Carbon, hydrogen, nitrogen, sulphur, and oxygen were determined by elemental analysis using a Perkin-Elmer 2400 Series II with thermal conductivity detector, at 975°C combustion temperature and 500°C reduction temperature. A Thermo Scientific K-Alpha instrument gave X-ray photoelectron spectra with a Al Kα (1486.6 eV) source, working at 200 eV and 2 × 10 -9 mbar. Product morphology was determined using a FEI Quanta 200 SEM. Activity index (AI) The activity index (AI) characterizes the slow-release performance: The effects of temperature, U/F ratio, and pH on the AI were studied. Slow release experiments Slow release tests of NPK 111 fertilizer were performed in a glass column (L = 600 mm, D = 200 mm) filled with 1-2 mm sand. NPK 111 (20 g) was incorporated 20 cm from the top. The column was washed with 50 mL distilled water at 8 day intervals and the leachate (P1-P8) analysed for N, P, and K. The degree of leaching was determined for each sample P1-P8. Results and Discussion Synthesis of urea-formaldehyde resins to specified degrees of polycondensation is difficult and the resulting mixture of three methylolureas increases the difficulty of analysis. Morphologically, the products are almost spherical with monolithic compact structures, as observed from the SEM images. However, the sphericity is altered by local heating during scanning. The particle dimensions measured by SEM are confirmed by DLS analyses; mean diameters are 2.1-2.7 μm for all samples, with no variation correlated with synthesis parameters. Both SEM and DLS showed particle agglomeration. The elemental analyses are in Table 1. The results obtained by classic methods are confirmed by faster and easier modern XPS and ICP-AES. The correlations between classic and modern methods are good; their complementary nature contributes to a fuller characterization. XPS showed intense signals for carbon (C1s) at ~285 eV, nitrogen (N1s) at ~400 eV, and oxygen (O1s) at ~531 eV. Lower intensity signals appeared for phosphorous (P2p) at ~134 eV and potassium (K2p) at ~292 eV. To determine the nitrogen surface and internal distributions these results were compared with the EA results and expressed as the N/C ratio. EA gave a higher N/C ratio than XPS. The highest N/C ratios were observed for NPK 100 and NPK 211, suggesting that N is concentrated inside the particle rather than at the surface. The presence of C inside the particles suggests that polymerization occurs throughout the particle, forming a network that incorporates the fertilizer elements. P and K, which are not part of the polymer, are concentrated mainly inside the particles. K is not even detected at the NPK 211 surface. Very low concentrations of Ca (in NPK 100) and Si (in NPK 100, NPK 110, and NPK 111) are detected. CaO is added for pH correction in the last synthesis step, while Si may originate from small amounts of Mg 3 Si 4 O 10 (OH) 2 (talc). In agreement with earlier work [38], 1 H-NMR spectra for NPK 211 gave signals at ~2.5 ppm for DMSO, ~3.15 ppm for OH; ~4 ppm for NH and H 2 O; ~4.5 ppm for CH 2 ; and ~5.6 ppm for NH 2 . These signals also appear in the other systems as shown in Figs. 6a, b and c. Increasing formaldehyde increases the CH 2 amplitude and decreases that of NH 2 , confirming the synthesis reaction proposed. Thus, for a 1:1 U/F ratio the CH 2 amplitude is highest and the NH 2 lowest. A high U/F ratio leads to highly cross linked products with high molecular weight. The resulting low solubility diminishes the activity index and the slow-release properties. Since the aldehyde signal (~9-10 ppm) is absent and the amine signal is strong, mono-methyl urea and dimethyl urea preponderantly form, without residual formaldehyde. HPLC chromatograms show that all the products elute simultaneously after 6 minutes, with a maximum at min, suggesting a high reactivity and association tendency among components. If the concentrated solution is diluted 1:1 with water the chromatogram shows at least three components with elution times of 6.0, 6.4, and 8.2 min, corresponding to mono-, di-, and trimethylolurea. Since MetaCarb 67 H is a metal sulfonated polystyrene resin which separates the compounds by ion-exchange, ligand-exchange, sizeand ion-exclusion mechanisms, the order of elution is monomethylolurea (6.0 min), dimethylolurea (6.4 min), and trimethylolurea (8.2 min). This order is strongly correlated with both the molecular weights and the available hydroxyls which may interact with the stationary phase metal ion, a strong interaction increasing the retention time. Based on the peak areas a semi-quantitative estimate shows a decrease from mono-to trimethylolurea. FT-IR shows a broad peak at 3344-3336 cm -1 , shifting to lower frequency with increasing urea content, characteristic of polymeric hydroxyl [38] as well as NH and NH 2 stretching in urea. Other characteristic peaks were the C=O stretch at (1651 ± 2) cm -1 , the (1545 ± 3) cm -1 amide NH 2 deformation combined with the C-N stretch, and the (1004 ± 1) cm -1 C-O stretch in aliphatic chains, as Thermal analysis characterises the thermal stability and thermal behavior. The first thermal event occurs at 150-250°C, with a maximum at 220.4°C for NPK 100, 238.9°C for NPK 110 and NPK 111, and 210.2°C for NPK 211. In this temperature range the polymer urea residues decompose to ammonia, CO 2 , and nitrogen oxides. The decomposition is gradual and depends on the degree of polycondensation. The next thermal event occurs at 250-400°C, with different maxima for each system. Carbonization of the polymer remnant takes place above 400°C, representing 10-15% of the initial mass. The residue at 700°C is lowest for NPK 100 (4.152%), followed by NPK 110 (17.42%), NPK 111 (29.78%) and NPK 211 (32.34%). Thermal stability beyond 50°C guarantees stability during transport, manipulation, and application. Fig. 10 shows the effect of synthesis temperature, U/F ratio and pH on the activity index. Optimum values are shown. A temperature of around 60°C gives a good polycondensation and slow-release fertilizers. Below this temperature reaction initiation is delayed, while above 60°C fast polycondensation forms large blocks with low workability. The optimum U/F ratio of around 1.8 leads to almost spherical aggregates, easily separated by filtration. At lower ratios when formaldehyde predominates, the product is sticky and viscous, almost liquid at U/F equal to 0.3. At U/F ratios higher than 1.8, the product agglomerates in blocks due to rapid polycondensation. The highest AI occurs around pH 5. In more acidic conditions the polycondensation reaction is very fast, exothermic, and uncontrollable, which leads to block products; an almost neutral environment gives a very low reaction rate, an increased reaction time, and a low yield. NPK 111 fertilizer leaching was compared with standard fertilizers. Nitrogen leaching was 44.6% for ammonium nitrate and 42.2% for urea. NPK 111 nitrogen leaching (37.56%) is reduced in comparison with these classic fertilizers. Nitrogen release in 64 days of intensive irrigation is comparable to that of commercially available slow-release fertilizers like Osmoform. P 2 O 5 and K 2 O also showed release rates substantially lower than the classic granular fertilizers ammonium phosphate and potassium chloride, as shown in Table 2. Nutrient release kinetics (Fig. 11) were fitted to linear, power, and exponential equations [41][42][43], and also with a new type, the sigmoidal model ( Table 3). The R 2 values show that the results are best fitted by different kinetic models. Nitrogen leaching is best described by an asymptotic exponential, potassium leaching seems to follow a linear model, while phosphorus leaching is best described by a sigmoidal model. Further studies will be conducted to determine the NPK nutrient leaching mechanisms. Column: L = 600 mm, D = 200 mm. *) Fertilizer incorporation in sand 20 cm from the column top. **) Percent leaching = quantity leached during P 1 -P 4 and P 5 -P 8 Figure 11: Release kinetics of N, P, and K fitted to different models. Conclusions Synthesis of encapsulated fertilizer in urea-formaldehyde matrices was optimized based on maximizing the activity index. Optimum temperature, pH, and urea/formaldehyde mass ratio were 60°C, pH ≈ 5 and U/F = 1.8. All the products obtained had an AI greater than 40%, the minimum required for slow or controlled release fertilizers. These ensure more efficient nutrient uptake and allow decreased application without productivity loss as well as reducing pollution. Leaching tests of NPK 111 suggest that this product can compete with commercially available fertilizers in providing longer nutrient availability than urea or ammonium nitrate. The activity index meets the specifications for "slow-release" fertilizers. The parameters for nutrients' leaching were determined by regression for four different kinetic models.

v3-fos

2019-03-28T13:42:57.618Z

2015-01-23T00:00:00.000Z

196607144

s2

Effects of chitosan and Aloe Vera gel coating on quality characters of pistachio The traditional concept of a packaging is to preserve the quality of the product with a minimal product/packaging interaction, however, in recentyears, a wide variety of packages have been employed for interaction with products to provide desirable or beneficial effects.1 Active packaging technology is a relatively novel concept beneficial for extending the product shelf-life, maintaining its nutritional and sensory quality, as well as contributing to the microbial safety.2 The ability of edible film or coating as a type of active packaging to carry some products additives such as antioxidants, antimicrobials, colorants, flavors, fortified nutrients and spices are being studied.3 Introduction The traditional concept of a packaging is to preserve the quality of the product with a minimal product/packaging interaction, however, in recentyears, a wide variety of packages have been employed for interaction with products to provide desirable or beneficial effects. 1 Active packaging technology is a relatively novel concept beneficial for extending the product shelf-life, maintaining its nutritional and sensory quality, as well as contributing to the microbial safety. 2 The ability of edible film or coating as a type of active packaging to carry some products additives such as antioxidants, antimicrobials, colorants, flavors, fortified nutrients and spices are being studied. 3 Chitosan, a natural carbohydrate copolymer [-(1-4)-2-acetamidod-glucose and -(1-4)-2-amino-d-glucose units], which is yielded from deacetylation of chitin [poly-(1-4)-N acetyl-2d-glucosamine], is harmless to humans, animals and, and the environment; and has been studied for efficacy in inhibiting decay and extending shelf life of fruits. Chitosan and its derivatives have been shown to inhibit the growth of a wide range of fungus, 4,5 so one of interest application of this biopolymer is products preservation because of its ability to be used as coating materials to extend the shelf life of different products. 6,7 Recently, the use of A. vera gel as an edible coating has been reported to prolong the shelf life and to delay the changes in the parameters related to deterioration of quality of products. 8,9 A. vera, a cactus-like plant, is a perennial succulent belonging to the Liliaceae family which grows in hot and dry climates. 10 The plant has triangular, fleshy leaves with serrated edges, yellow tubular flowers and fruits containing countless seeds. For centuries, the yellow latex (exudate) and clear gel (mucilage), exuded from the large leaf parenchymatic cells of A. vera, has been employed for medical and pharmaceutical purposes such as anti-inflammatory effects, treatment of skin burns, protection of the skin against UV and gamma radiation damage, treatment of frostbite and psoriasis, supporting and enhancing the immune system, antiviral and antitumor activity, laxative effects, and, last but not least, wound healing. 11 However, the main use of A. vera gel is mainly in the cosmetology and medication; More recently, it has found its application in the food industry as a source of functional foods in ice-cream, drinks and beverages, 12 and, due to antifungal activity of A. vera gel, as an unique edible coating (plain or in combination with other components) to extend the postharvest storage of arctic snow, 4 apple slices, 13 sweet cherry, 8 papaya fruits 14 and table grapes. 1,9,15 A. vera gel based edible coatings have been shown to prevent loss of moisture and firmness, control respiratory rate and maturation development, delay oxidative browning, and reduce microorganism proliferation in fruits such as sweet cherry, table grapes and nectarines. 6,8,12 There are no reports presently on the post-harvest application of Aloe gel and chitosan coating on pistachio; therefore, the objective of this research was to elucidate the role of A. vera gel edible coating on the storage life of pistachio in comparison to chitosan coating. Preparation of coating solutions Chitosan and A. vera gel coatings were prepared according to the method of Marpudi et al. 3 A. vera gel was ground in blender. After pasteurization at 70 0 C for 45min, gel was cooled to an ambient temperature and ascorbic acid (1.9-2.0g L -1 ) was added to gel for stabilizing it; citric acid (4.5-4.6g L -1 ) was then added to maintain the pH at 4. Sample preparation and coating application Fresh pistachios were purchased from a local producer in Iran, and then randomly distributed into five groups with three replicates. First group immersed in water as the control (F0), four other groups immersed in solutions of chitosan 0.5% (F1), chitosan 0.05% (F2), A. vera gel 100% (F3) and A. vera gel 50% (F4) for 5min. After 5 min; pistachios were dried at 25 0C until their coatings became non-sticky to touch. Each of Fifteen groups were placed into sterile plastic bag and were stored at 4˚C for one month. Physiological water conservation (PWC) After one month, the samples were dried in an oven. Then water conservation was calculated in terms of PWC by the following equation: Where, A is the initial weight of Fresh pistachios after the storage period and B is oven dried pistachios . Fruit disease index (FDI) The differently coated pistachios were visually observed for fungal spoilage. The number of pistachios infected was recorded to assess the effect of the different coating on retarding fruit spoilage. Degree and Rate of Fruit Spoilage was reported as percentage disease index and calculated as follows: Oil extraction Each sample of coated pistachio was ground in a mill, and then oil extraction was carried out using a Soxhlet extraction system with hexane as solvent. An extraction time of 8 hours was chosen. After extraction, the extracts were evaporated on a rotary evaporator, and the samples were stored with the exclusion of light. Peroxide value (PV) PV was determined according to the thiocyanate method. The sample (0.01-0.30 g, depending on the extent of peroxidation) was mixed in a disposable glass tube with 9.8mL chloroform-methanol (7:3 v/v) in a vortex mixer for 2-4seconds. Ammonium thiocyanate solution (50μL, 30% w/v) was added and the sample was then mixed in a vortex mixer for 2-4seconds. Then, 50μL of iron (II) chloride solution ([0.4 g barium chloride dihydrate dissolved in 50 mL H2O] +[0.5 g FeSO4•7H2O dissolved in 50 mL H2O]+2 mL 10M HCl, with the precipitate, barium sulfate, filtered off to produce a clear solution]) was added, and the sample was mixed in a vortex mixer for 2-4seconds. After a 5minutes incubation at room temperature, the absorbance of the sample was read at 500 nm against a blank that contained all the reagents except the sample using a spectrophotometer (Jenway 6105 UVVIS). The entire procedure was conducted in subdued light and completed within 10minutes. All PV analysis results were expressed as meq O2 kg-1 oil. 16 Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA) using Excel and SPSS version 13 software. Duncan test was used to determine the difference at 5 percent significance level. All tests were done in three replicates. Physiological water conservation (PWC) The effect of selected edible coatings on PWC can be seen in Figure 1. PWC was found to be significantly (P<0.05) different among five different coatings. PWC was observed to be 85.43%, 86.68%, 90.26%, 85.93% and 88.08% for F0, F1, F2, F3 and F4, respectively. Among the coated groups, F2 had significantly highest water preservation, and then PWC of F4 was higher than other groups, followed by F1, F3 and F0. Effects of F1 and F3 on water preservation approximately were similar and the difference was to a lesser extent in these two coating groups compared to control (F0). A. vera gel based edible coating have been shown to prevent loss of moisture and firmness. 1,4,14 Marketability Marketability values were found to be 2.5, 3, 4.5, 1.25 and 3.25 for F0, F1, F2, F3 and F4, respectively (Figure 2). At the end of one month marketability was found to be better for F2 coated samples, followed by F4, F1, F0 and F3 coating. It is thought that bark firmness maintenance related to higher PWC. These results are in agreement with other studies. Sensory analysis Color and firmness, the major sensory attributes, were scored by panel members (Figure 3) Color was evaluated based on the bark color with 5 as a score for bright to a score of 1 for complete dark. Bright color of bark changed to dark during storage in both control and coated fruits, but degree of darkening was varying in samples. The F3 had shown a greater degree of darkness. Color values were given as 1.25, 1.75, 2.75, 3.5 and 4.25 and 2.5 for F3, F0, F1, F4 and F2, respectively. Slow changes in physiochemical changes of coated samples revealed the more effective of maintaining ability. 14 Fruit disease index (FDI) FDI was used as a measure to indicate the effect of selected coatings on the microbial quality of product. FDI was observed to be 6.125, 4.125, 0.92, 1.135 and 1.565 for F0, F1, F2, F3 and F4, respectively ( Figure 4). Coated groups had lesser extent FDI compared to control (F0), but among them, F1 had higher FDI than other coating groups, followed by F4, F3 and F2. Thus effect of F2 for retarding pistachio spoilage is greater than other coatings. In the present study, .05% chitosan and A. vera gel coatings showed the low disease index. Antimicrobial activity of A. vera gel against bacteria has been reported previously. 18,19 These results are in agreement with reported results of vena gel based coating in reduction of microorganism proliferation in Sweet cherries. A. vera gel based coatings have been reported earlier to reduce microorganism proliferation in sweet cherries and table grapes. 15 Peroxide value (PV) The PV shows the degree of oxidation in the substance and measures the amount of total peroxides as a primary product of oil oxidation. As be seen in Figure 5, samples of F3 had Highest PV (even more than F0), followed by F0, F1, F4 and F2. These samples of F2 had least PV. Investigation of both primary andsecondary oxidations along with peroxide value (PV) 11 is requested on study of oxidative stability of vegetable oil as one of the key factors. 20 Peroxide value (PV) is an index exhibiting the amount of primary oxidation. 21 Figure 5 depicts that least oil oxidation of coated pistachios is related to 0.05% chitosan, followed by 50% A. vera gel, 0.5% chitosan, control and 100% A. vera respectively. PV of 100% A. vera gel coated pistachio was even rather than control. Since surface coating causes to a reduction in oxidation, gas permeability and modifies international atmosphere as well, it can be thought that this kind of coating method being more practically affects on shelf life of coated food, 22 which has been considered together with the peroxide value, as a classical index of primary oxidation products. 11 Peroxide value (PV) is one of the most frequently determined quality parameters during oil production, storage, and marketing. The PV shows the degree of oxidation in the substance and measures the amount of total peroxides as a primary product of oil oxidation. 21 As be seen in Figures least oil oxidation of coated pistachios is related to 0.05% chitosan, followed by 50% A. vera gel, 0.5% chitosan, control and 100% A. vera respectively. PV of 100% A. vera gel coated pistachio was even rather than control. These results showed that since surface coating increase resistance of fruit skin to gas permeability, creating modified internal atmosphere [22][23][24] and reducing the respiration rate; maybe can said that each coating that reduced oil oxidation is more suitable for coating, rather than other coatings. Conclusion The results have proved the ability of chitosan and A. vera gel coatings used in the present study to extent the post-harvest quality preservation of pistachio. At the end of storage time, results showed that 0.05% chitosan coating had the maximum effect in retarding the change in quality of pistachio (bark Color darkening, lessen of marketability, decay and oil oxidation), to a greater extent than 50% A. vera gel, 0.5% chitosan and 100% A. vera coatings respectively. Since 100% A. vera reduced quality of pistachio is not suitable for coating of pistachio; maybe due to its high concentration that can't penetrate in bark of pistachio. At the other hand, .05% chitosan and 50% A. vera since had greatly delayed the physiological changes, are suitable for coating and can be used for transport of fresh pistachio for long distance.

v3-fos

2018-04-03T00:40:34.317Z

2015-02-28T00:00:00.000Z

17130874

s2

Development and Application of a Method for Rapid and Simultaneous Determination of Three β-agonists (Clenbuterol, Ractopamine, and Zilpaterol) using Liquid Chromatography-tandem Mass Spectrometry β-agonists are anabolic compounds that promote fat loss and muscle gain, and their administration to livestock may provide economic benefits by increasing growth rate and feed efficiency. For these reasons, β-agonists are also commonly added to livestock feed as growth promoters. This can introduce a significant risk of secondary human poisoning through intake of contaminated meat. A new method for the simultaneous determination of three β-agonists (clenbuterol, ractopamine, and zilpaterol) was developed in this study and applied to various meat samples. The limits of quantification, derived through a validation test following Codex guidelines, were 0.2 μg/kg for clenbuterol and zilpaterol, and 0.4 μg/kg for ractopamine. The average recoveries for clenbuterol, ractopamine, and zilpaterol ranged from 109.1% to 118.3%, 95.3% to 109.0%, and 94.1% to 120.0%, respectively. The recovery and coefficient of variation (CV) values fell within the acceptable range according to the Codex guidelines. This method reduced the analysis time without decreasing detection efficiency by modifying the pretreatment steps. This method could be utilized to manage the safety of imported meat products from countries where zilpaterol use is still permitted, thereby improving public health and preventing β-agonist poisoning due to secondary contamination. Introduction Administration of high doses of β-agonists, which were originally developed to treat human bronchial diseases and premature birth, could lead to improved weight gain and carcass yield in livestock (Blanca et al., 2005; Lawrence et al., 2011). Consequently, this approach has long been used to increase livestock productivity. Toxicity studies have demonstrated that long-term administration of clenbuterol, a member of the β-agonist group, might lead to serious adverse effects in the cardiovascular and nervous systems (Juan et al., 2010;Martinez-Navarro, 1990). Several cases of adverse effects in humans have been linked to the consumption of clenbuterol-contaminated meat products (Brambilla et al., 2000). This led to the prohibition of clenbuterol use in over 150 countries, including European countries (Commission of the European Communities, 1996). Ractopamine and zilpaterol belong to the same drug class and have effects similar to those of clenbuterol. These compounds are still used in some countries to promote livestock growth. Although ractopamine is prohibited in most European Union (EU) countries (Blanca et al., 2005), it is still officially permitted in 27 countries worldwide, including the United States and Canada (Health Canada, 2014; U.S. Food and Drug Administration, 2014). Likewise, zilpaterol is authorized for use in South Africa, Mexico, the United States, and Canada (Delmore et al., 2010). Given Korea's high dependence on meat imported from areas that still use ractopamine and zilpaterol, analysis of the three commonly used β- A method that improved upon individual assays was developed in South Korea in 2013, and has been used for the simultaneous determination of clenbuterol and ractopamine (Cho et al., 2013; National Institute of Food and Drug Safety Evaluation, 2013). However, this technique does not include measures for analyzing zilpaterol, which has seen increasing use due to its superior growth-enhancing effects. Policies regarding permission to use zilpaterol vary among countries, and these differences are becoming increasingly problematic. International safety management efforts for zilpaterol are still lacking. In contrast, clenbuterol is prohibited in most countries and is strictly controlled in countries that permit its use. Similarly, the grounds for safety management of ractopamine were established through the adoption of maximum residue limits (MRLs) by the Codex Alimentarius Commission to ensure human health and safety. The limits shown in Table 1 were based on a risk assessment published by the Joint FAO/WHO Export Committee on Food Additives (JECFA) in July 2012 (Food and Agriculture Organization of United Nations, 2014). Therefore, a method that simultaneously measures all three β-agonists would be beneficial. To that end, a new method that met Codex guidelines and had a high detection efficiency was developed for the simultaneous detection of clenbuterol, ractopamine, and zilpaterol in muscle tissue, the primary source of meat. The method was subsequently validated using a collection of meats and meat byproducts. For each of the three standards and three internal standards, a 100 mg/kg stock solution was prepared in methanol based on the known purity and molecular weight of each substance. From these stock solutions, working standard solutions of clenbuterol, ractopamine, and zilpaterol at concentrations of 100, 200, and 200 µg/kg, respectively, were prepared by dilution in methanol. The same procedure was followed for clenbuterol-d 9 , ractopamined 3 , and zilpaterol-d 7 to yield solutions of 100, 200, and 200 µg/kg, respectively. Stock and working standard solutions were stored at -20 o C and diluted to individual concentrations by using 0.1% formic acid(v/v), as required. Sample treatment and extraction Frozen chunks of beef, pork, and beef byproducts imported from several countries were used as samples. Prior to analysis, the samples were thawed and then ground using a mixer (HMF-560/HK, Hanil, Korea). Five grams of each homogenized, refrigerated sample was placed into 50 mL centrifuge tubes. The working standard solution of each internal standard (50 μL), 1 mL of 4 M K 2 CO 3 , and 5 mL of distilled water were added to each sample, and the mixture was stirred for 10 min. Ten milliliters of ethyl acetate was then added and the mixture was shaken for 10 min. Next, 10 g of anhydrous sodium sulfate was added and mixed for 10 min to absorb residual water. The solution was centrifuged for 10 min at 4,000 rpm and -4 o C, and the supernatant was transferred to a new 50 mL centrifuge tube. Then, 10 mL of ethyl acetate was added to the solution, and the resulting supernatant was isolated using the same extraction procedure. The combined supernatants were gently enriched with nitrogen at 40 o C to obtain a final volume of 2-3 mL. Ten milliliters of acetonitrile and 15 mL of n-hexane were added to the concentrate and vortexed for 10 min. The mixture was centrifuged for 10 min at 4,000 rpm and -4 o C to separate the organic layer (acetonitrile). The isolated organic layer was gently enriched with nitrogen at 40 o C and evaporated to dryness. The residue was dissolved in 1 mL of 0.1% formic acid, which served as solvent A for the chromatographic analysis, and 0.5 mL of the solution was filtered through a 0.2 μm polyvinylidenedifluoride syringe filter prior to injection. Conditions and experimental set-up for chromatography and mass spectrometry LC-MS (API 4000, AB SCIEX, USA) was used to develop a simultaneous determination method for clenbuterol, ractopamine, and zilpaterol. Separation was performed with X Bridge C 18 (2.1 mm × 150 mm, 3.5 μm; Waters, Ireland), and the column temperature was main-tained at 35 o C. The analysis was carried out with 0.1% formic acid (solvent A) and acetonitrile (solvent B) as the mobile phase solvents. Initial mobile conditions were set to 90% A and held for 1 min. The fraction of A was linearly decreased to 10% over 7 min and held at 10% for 3 min. At 10.10 min, the compositions were returned to 90% A and held there until 15 min. The flow rate was maintained at 0.4 mL/min, and the injection volume was 10 μL. Positive electrospray ionization mode was selected, and the analysis was performed in multiple reaction monitoring mode. The source temperature was set at 600 o C. The precursor ion, product ions, collision energy, and dwell time for each substance are listed in Table 2. MS/MS optimization Conventional analysis methods for clenbuterol and ractopamine were used to determine the optimal assay conditions for mass spectrometry. Since ions at m/z 203 and 259 have been used in published literature regarding clenbuterol, the collision energy shown in Table 2 was applied to the product ion to obtain ions at these m/z ratios zilpaterol-d 7 was used as an internal standard for zilpaterol to improve efficiency and reproducibility in the present study. Validation Linearity, recovery, limit of quantification (LOQ), and reproducibility tests were conducted to validate our method according to the Codex guidelines (Codex Alimentarius Commission, 1993). Three imported samples each of beef, pork, and beef byproducts were used for these validation experiments. Three different concentrations of each sample were tested. Calibration curves were prepared at concentrations of 0.2-16 µg/kg for clenbuterol and zil-paterol, and 0.5-32 µg/kg for ractopamine, for which MRLs have been established. The correlation coefficient (R 2 ) for each calibration curve was calculated, and the LOQ with a signal-to-noise ratio greater than 10 was obtained for each substance. Coefficients of variation (CV) were calculated as the ratio of the standard deviation to the mean. Fig. 1 shows the chromatogram for each substance after injection at the LOQ level. To evaluate the accuracy and precision of our method, the recovery rate was also determined using concentrations that were 0.5, 1, and 2 times the MRLs (0.005, 0.01, and 0.02 mg/kg) for ractopamine, and 1, 2, and 4 times the LOQ values (0.2, 0.4, and 0.8 µg/kg) for clenbuterol and zilpaterol. Peak retention times in the chromatogram were compared and adjusted according to the internal standards to determine recovery rates using the content values of clenbuterol, ractopamine, and zilpaterol. Each concentration was analyzed in triplicate to verify the experimental methods. Results and Discussion Comparison with current β-agonist detection methods A method for the simultaneous determination of clenbuterol and ractopamine was developed in 2013 in South Korea and is currently used as a Food Code analysis method (KFDA, 2013). . We used 4 M K 2 CO 3 to adjust the pH of the solution to strongly basic, and 5 mL of triple distilled water to promote dispersion of the sample in the pretreatment solvent and to increase the recovery rate. We also used acetonitrile and hexane in place of saturated methanol and hexane to increase the liquid-liquid distribution effect (Juan et al., 2010), and maintained the temperature during nitrogen decompression at 40 o C, rather than 55 o C, to minimize evaporation Mauro et al., 2014). Finally, we used 0.1% aqueous formic acid and acetonitrile as the mobile phase instead of buffered ammonium acetate to ensure no overlapping retention times, encourage optimal peak shape, and minimize run time (Fig. 2). Applicability In our validation tests, R 2 values for the clenbuterol, ractopamine, and zilpaterol calibration curves were 0.9992, 0.9998, and 0.9979, respectively. The LOQ was 0.2 µg/kg for clenbuterol and zilpaterol, and 0.4 µg/kg for ractopamine. The average recovery rates were 109.1-118.3% for clenbuterol, 95.3-109.0% for ractopamine, and 94.1-120.0% for zilpaterol. The CV values were less than 10.58% for all three compounds. These results met the Codex criteria for linearity, recovery rate, LOQ, CV, and reproducibility (Table 3). Accordingly, these findings confirmed the applicability of our method. International trends in β-agonist drug permissibility Based on safety concerns, β-agonist drugs are now con-sidered more controversial than any other veterinary drug internationally. Unlike clenbuterol, which is prohibited from use in livestock in most countries, ractopamine is still authorized for use in over 27 countries, including South Korea. The Codex Alimentarius Commission, the United Nations food standards body, established ractopamine MRLs in 2012, proposing residue levels that have no known impact on human health (Food and Agriculture Organization of the United Nations, 2014). Unfortunately, not all countries comply with these Codex MRLs. Although all β-agonists are prohibited for use as growth promoters in the EU (Commission of the European Commu- Application of the new method Using our method, we performed residue analysis for levels of the three β-agonists in the following 299 imported meats and byproducts samples: 154 beef, 57 pork, and 88 beef byproducts. Our results revealed that ractopamine was detected in two beef samples, and zilpaterol was detected in one beef and one beef byproduct sample (Table 4). Based on these results, we believe that there is non-negligible contamination of imported meat and meat byproducts. In particular, a high detection rate was observed for zilpaterol, for which MRLs have not yet been established, indicating a need for the continued surveillance of this drug. Other less frequently used β-agonists that still pose a health risk, such as cimaterol and salbutamol, could be assessed using similar simultaneous determination method in future studies. In addition, the scope of the method may be extended to include processed meat products. Conclusion A new method for the simultaneous determination of three β-agonists, including zilpaterol, was developed. The pretreatment method was modified to reduce the analysis time while enhancing detection efficiency. Moreover, the validation test results met the international Codex guideline standards, verifying that the method is applicable for actual practice. This method should enable rapid and efficient analysis of β-agonists and was applicable to muscle samples, making it appropriate for further studies of βagonists in meat. Also, it had the advantage of determining zilpaterol, which is not included in the Food Code method, at lower levels than those detectable using the US FSIS method. Additional analyses of zilpaterol may facilitate continued management of the drug and prove to be useful for assessing the risk and establishing MRLs for zilpaterol in the future. Furthermore, it is hoped that this method could be utilized to manage the safety of imported meat products from countries where zilpaterol use is still permitted, thereby improving public health and preventing β-agonist poisoning due to secondary contamination.

v3-fos

2017-06-28T04:00:59.634Z

2015-07-07T00:00:00.000Z

15519484

s2

Response and inbreeding from a genomic selection experiment in layer chickens Background Genomic selection (GS) using estimated breeding values (GS-EBV) based on dense marker data is a promising approach for genetic improvement. A simulation study was undertaken to illustrate the opportunities offered by GS for designing breeding programs. It consisted of a selection program for a sex-limited trait in layer chickens, which was developed by deterministic predictions under different scenarios. Later, one of the possible schemes was implemented in a real population of layer chicken. Methods In the simulation, the aim was to double the response to selection per year by reducing the generation interval by 50 %, while maintaining the same rate of inbreeding per year. We found that GS with retraining could achieve the set objectives while requiring 75 % fewer reared birds and 82 % fewer phenotyped birds per year. A multi-trait GS scenario was subsequently implemented in a real population of brown egg laying hens. The population was split into two sub-lines, one was submitted to conventional phenotypic selection, and one was selected based on genomic prediction. At the end of the 3-year experiment, the two sub-lines were compared for multiple performance traits that are relevant for commercial egg production. Results Birds that were selected based on genomic prediction outperformed those that were submitted to conventional selection for most of the 16 traits that were included in the index used for selection. However, although the two programs were designed to achieve the same rate of inbreeding per year, the realized inbreeding per year assessed from pedigree was higher in the genomic selected line than in the conventionally selected line. Conclusions The results demonstrate that GS is a promising alternative to conventional breeding for genetic improvement of layer chickens. beef cattle, swine, broiler and layer chickens), studies on the impact of empirical results of GS on genetic gain are not available. In addition to increasing accuracy of selection at young ages, GS is expected to reduce rates of inbreeding per generation because GS provides additional information on Mendelian sampling terms of selection candidates [4,5]. GS is predicted to allow a reduction in size of breeding programs, without increasing the rate of inbreeding. The reduced costs in management and performance recording for a smaller population size should help offset the considerable investment in genotyping that is required. Alternative or additional strategies could also involve preselection of candidates for genotyping. The objectives of this study were to design, simulate, implement and retrospectively evaluate a GS program for layer chickens, which could potentially double the response to selection per year, while maintaining the same annual rate of inbreeding, compared to a typical pedigree and performance-based layer selection program. The selection lines for the GS and conventional breeding programs were derived from the same foundation line, and multiple-trait performance was compared over the same time period in the terminal generations. Methods This research was conducted in four steps as follows: (1). Design of a genomic selection program for layer chickens using deterministic prediction models based on selection theory that would double the response to selection per year but maintain the same rate of inbreeding per year compared to a conventional selection program based on pedigree and phenotypic information. Our aim was to achieve these goals while minimizing genotyping and phenotyping costs by changing the population structure. (2). Evaluation of the performance of this GS strategy by stochastic simulation. (3). Implementation of the GS and conventional breeding strategies, in which both selection lines were derived from the same foundation generation of a real elite purebred layer chicken population. (4). Retrospective evaluation of the realized responses to selection for the two breeding programs. Design of the genomic selection strategy using deterministic prediction Responses to selection and rates of inbreeding were predicted by deterministic methods for a conventional BLUP (best linear unbiased prediction) selection program and for a range of GS programs in order to identify a strategy that could double the response to selection per year with the same rate of inbreeding per year as the conventional program. To reflect selection for egg laying traits, we considered a trait with a heritability of 0.3 and phenotypes available only on females at 1 year of age. The program SelAction [6] and procedures of [5] were used to predict asymptotic rates of response and inbreeding for the conventional BLUP and GS strategies. The conventional BLUP strategy assumed selection and mating of 60 males and 360 females per 56-week long generation cycle. The 60 males were selected from 1080 selection candidates (three sons raised per hen) based on their BLUP estimated breeding values (EBV) from phenotypic data on female ancestors and sibs (individual phenotypes or progeny with phenotypes were not available at the time of selection). The 360 females were selected from 2880 selection candidates (eight daughters per hen) based on BLUP EBV from the phenotypes of female ancestors and sibs, as well as individual phenotypes. Deterministic predictions of this program using SelAction showed an expected response of 0.48 phenotypic standard deviation per generation and a rate of inbreeding of 1.38 % per generation. Using the response to selection and rate of inbreeding of the conventional program as targets, predicted responses and rates of inbreeding per generation were evaluated for a large number of GS strategies; the number of sires selected was varied from 25 to 60, the number of dams selected from 40 to 120, and the number of male and female offspring per dam that were genotyped was varied from 3 to 12. The initial accuracy of genomic EBV that were assumed to be available on all selection candidates was set equal to 0.7, which resulted in an asymptotic accuracy after accounting for the Bulmer effect between 0.58 and 0.60 for all GS strategies. Based on deterministic predictions, a GS breeding program with 50 males and 50 females that were selected at each generation from 300 selection candidates per sex (six male and six female progeny from each single sire-dam mating) was predicted to result in a similar rate of response per generation as the conventional BLUP program (0.43 vs. 0.48 phenotypic SD), with less than half the rate of inbreeding per 28-week generation cycle (0.59 versus 1.38 %), while minimizing the number of individuals genotyped per generation. Thus, with the generation interval for the GS program being half that of the conventional 56-week program, this GS program was predicted to nearly double the response to selection per year, with a slightly lower rate of inbreeding per year. Evaluation of the genomic selection strategy by stochastic simulation The conventional and GS programs described above were compared by stochastic simulation to validate these deterministic predictions. A genome that comprised 20 chromosomes of 37.5 cM each, for a total of 750 cM, was simulated. In the foundation generation of the base population (g0), 6001 equally spaced SNPs per chromosome were simulated for 500 individuals, with allele frequencies of 0.5 and in linkage and Hardy-Weinberg equilibrium. To generate mutation-drift equilibrium, the subsequent 1000 generations (g1-g1000) were simulated with random mating, mutation (rate = 2.5*10 −5 ), and recombination, with effective population sizes of 500 for g1-g900 and 100 for g901-g1000. To create the training data, the population was expanded to 1000 individuals in g1001. Breeding values were generated by designating 200 random segregating (minor allele frequency ≥ 0.1) SNPs as quantitative trait loci (QTL). Each QTL was assigned an effect that was drawn from a Gamma distribution with shape parameter 0.4 and inverse scale parameter 1.66, following Hayes and Goddard [7]. The QTL effects were scaled such that the genetic variance in g1001 was 3/7. Each phenotype was simulated by adding a random environmental effect drawn from a standard normal distribution, assuming heritability to be equal to 0.3. Another 6000 segregating SNPs across the whole genome were sampled in the same base population and used as markers starting in g1001, which resulted in an average of eight SNPs per cM and is equivalent to 24 000 segregating SNPs for typical livestock genomes. These SNPs were used for training the genomic prediction models to reflect the real situation in which it is likely that causal mutations are not included in the SNP panels but markers with different levels of LD with the causal loci are. A total of 1000 females with phenotypic data were available to provide training data in g1001. Starting in g1002, the conventional phenotype-based BLUP selection program (generation interval of one year) and the GS program (generation interval of 0.5 year) were simulated. Response and rate of inbreeding were evaluated over four generations of conventional selection and eight generations of GS because the generation interval was reduced by 50 % in GS. For conventional selection, EBV were estimated at each generation by fitting the conventional animal model to available phenotypic data [8,9], using a numerator relationship matrix based on pedigree going back to g1001, with generation as a fixed effect. Heritability was set equal to the true heritability of 0.3 in the base population. For GS, the BayesB method of [1] was used, by fitting the following model to the phenotype of individual i in the training data: where μ is the generation effect, summation Σ j is over all genotyped SNPs, X ij is the number (0, 1 or 2) of copies of allele 1 that individual i carries at SNP j, g j is the allele substitution effect for SNP j, and e i is a random residual. Allele substitution effects g j were assumed to be normally distributed with mean 0 and variance σ gj 2 with probability 1-π = 0.05 or to be null with probability π = 0.95. Preliminary analyses found that the choice of π had a limited impact on the results. The prior for the variance of the substitution effects σ gj 2 was χ −2 (4.234, 0.0429). Estimates of allele substitution effects for each SNP, ĝ j , were obtained as posterior means from 1000cycles of a Gibbs chain, of which the first 100 cycles were discarded as burn-in, combined with 10 cycles of a Metropolis-Hastings algorithm within each Gibbs cycle to obtain samples of σ gj 2 . Preliminary analyses showed that these numbers of cycles led to converged estimates of EBV. For selection candidates, EBV were computed based on their SNP genotypes as: GS was evaluated with and without retraining. Without retraining, the SNP effects that were estimated based on g1001 were used for all eight cycles of selection. With retraining, SNP effects were re-estimated at each generation, after adding phenotypes of the 300 female candidates from the previous generation to the training dataset. Responses to selection were evaluated based on the mean true breeding value of selection candidates at each generation based on 48 replicates of the simulation. The level of inbreeding at each generation was based on the average pedigree-based inbreeding coefficients of selection candidates, using g1001 as the founder generation. Standard deviations of response and inbreeding were evaluated across the 48 replicates. Implementation of genomic selection Based on the simulation results, a GS program was implemented in an experimental pure-bred commercial brown egg layer chicken line that was previously under conventional BLUP selection. To allow side-by-side comparison of the conventional and GS programs, the line was split into two sub-lines (later denoted as genomic and pedigree lines) by random splitting of full-sib families. The pedigree line underwent two subsequent generations of conventional selection, whereas the genomic line underwent four generations of GS. All birds were produced, reared, and managed in facilities of the Hy-Line breeding program by Hy-Line staff, using standard protocols implemented by Hy-Line International. Population structure and selection strategy Different population structures were implemented for the two sub-lines, with numbers of contributing sires and dams and the total number of selection candidates shown on Fig. 1. In the pedigree line, 1000 male and 3000 female candidates were produced at each generation from 60 male and 360 female parents that were selected on a multi-trait index of phenotype-BLUP EBV, after records on female candidates were collected, with a restriction on the number of full-sibs selected. Selected males and females were mated in a hierarchical manner (six females per male), with some restriction to avoid matings between full-or half-sibs. The GS program that was implemented was a slight modification of the one used in the simulation because it applied two-stage female selection. No changes in selection strategy were implemented on the male side: 50 males were selected at a young age on a multi-trait index of genomic EBV (GEBV). In the first stage of female selection, 150 of the 300 female candidates were selected at a young age based on a multi-trait index of GEBV. All 150 females were mated to produce progeny for the next generation and before being recorded for phenotypes, which resulted in a generation interval of 28 weeks. A partially cross-classified mating design was implemented to reduce rates of inbreeding and capitalize on the ability of the GS procedure to assign parentage based on genotypes. For this mating design, the 50 selected males and 150 females were divided into five mating groups of 10 males and 30 females. Within each mating group, each male was mated to a different set of three females by artificial insemination. Males were rotated between the sets of females within a mating group. Parentage of the chicks was established based on SNP genotypes. In the second stage, at 42 to 46 weeks of age, after most individual phenotypes had been recorded, the 150 females were re-evaluated based on a combination of GEBV and own performance. From the best 50 dams 300 male and 300 female progeny (now 14 weeks of age) were selected for genotyping and phenotyping. The remaining progeny were kept only for phenotyping. This two-stage selection strategy made it possible to reduce the generation interval by 50 %, while increasing the accuracy of female selection, although at the cost of doubling the number of progeny that were produced and reared. Using progeny that could be uniquely assigned to the parents and excluding animals which had no progeny, each sire had on average 12.4 progeny (ranging from 1 to 31) and each dam 10.8 progeny (ranging from 1 to Selection criteria All phenotypes used in this study were obtained from routine data collection of Hy-Line International. Both sub-lines were selected based on the same multi-trait index combining 16 production and quality traits measured at early (e) or later (l) ages: age of sexual maturity (eSM, d), body weight at late age (lBW,g), shell color (based on an index obtained from the l, a, b Minolta® Colormeter system) for the first three eggs (eC3), at early (eCO) and late age (lCO), egg weight (g) for the first three eggs (eE3), at early (eEW) and late age (lEW), puncture score at early (ePS) and late age (lPS), albumen height (mm) at early (eAH) and late age (lAH), and yolk weight (g) at early (eYW) and late age (lYW). Egg production was expressed as ePD and lPD, which are the egg production rates (ratio of the number of saleable eggs to number of days in lay). Early measurements for egg quality traits were taken at 26 to 28 weeks of age. Late measurements for egg quality traits were taken at 42 to 46 weeks of age on birds not culled after early measurements. In addition, egg number (eEN and lEN, which are the total numbers of eggs (regardless of saleability) laid in weeks 1 to 10 and in weeks 11 to 20 of production, respectively) were monitored for correlated response. Complete records for late egg production were not available on GS females at the final stage of selection. Early and late egg quality measurements were averages of three to five eggs. Observations that deviated from the within-hatch generation mean by more than three standard deviations were excluded from breeding value estimation and treated as outliers, but all biologically feasible values were retained for line comparison in the final generation. Breeding values of selection candidates in the pedigree sub-line were estimated using the pedigree-based multitrait BLUP that is used for routine evaluation in the Hy-Line International breeding programs. Hatch-bygeneration was used as the only fixed effect to account for contemporary group effects. Selection candidates in the genomic sub-line were genotyped using a custom high-density Illumina SNP panel which provided 23 356 segregating SNPs (minor allele frequency > 0.025; maximum proportion of missing genotypes < 0.05; maximum mismatch rate between parent-offspring pairs < 0.05; parentage probability > 0.95). The same panel was also used to establish the training population for the first generation, which consisted of all selected parents from the previous five generations: 2708 genotyped animals, of which 1563 were females with individual phenotypes and 1145 were males without phenotypes. In addition, phenotypes of 11 486 progeny of the genotyped individuals were included as progeny means in the training data. In subsequent generations, phenotypes and genotypes of the 300 genotyped female selection candidates were added to the training population, i.e., the retraining option for GS was used. In the final generation, the GS line was expanded to produce enough progeny to make comparisons with the pedigree sub-line valid. A total of 2318 progeny were hatched, of which 1977 were assigned to 49 sires and 148 dams based on low-density SNP genotypes (71 SNPs). Animals that were not genotyped or that had one or more parents not matching the mating scheme were excluded. At each generation, the accuracy of several genomic evaluation methods was evaluated by using phenotypes of the last available generation for validation and the method with the highest accuracy was used for training on a dataset that included the phenotypes of the last generation for each trait. The genomic evaluation methods that were used included: univariate and bivariate GBLUP (using the early and late phenotypes for a given trait), GBLUP with a modified genomic relationship matrix [10], univariate BayesA, BayesB, and BayesCPi [11]. BayesCpi and GBLUP tended to have the highest accuracy and were therefore the predominant methods used. Variance components were estimated from the data using a multitrait animal model. Because not all individuals were genotyped, both individual performance and progeny means were used for genomic evaluation by applying methods described by [12]. Bayesian analyses were performed using the GenSel software [13], while conventional BLUP and GBLUP analyses, which estimated variance components simultaneously, were done using ASReml [14]. Because the scale of GEBV can differ from the scale of the observed phenotypes, the GEBV of each trait were rescaled at each generation by multiplying by the coefficient of regression of adjusted (for hatch effects) phenotypes on GEBV obtained from the latest validation analysis. For the second stage selection of females, the rescaled first stage GEBV were combined with each hen's adjusted own phenotypes, using a simple index of GEBV and own phenotype for each trait. The weights assigned to own phenotype (b 1 ) and GEBV (b 2 ) in this index were derived using selection index theory [5], based on the accuracy of GEBV obtained in the latest validation analysis (r) and the associated estimate of pedigree-based heritability (h 2 ): Evaluation of response to selection In the final generation of the experiment, the two sublines were hatched and housed together for a direct comparison of performance. Since the environmental conditions were the same, least square means for the line from a model including effects of line and hatch were assumed to reflect genetic differences resulting from the two methods of selection. Calculations were performed with SAS [15]. In order to have a reference point for response to selection since the start of the experiment, the data from the pedigree line were analyzed with a multi-trait pedigree-based animal model, with variance components estimated from the data, and response to selection in the pedigree line was estimated based on the difference in average EBV in the last and first generations of the experiment. Estimates of the line difference from the final generations were then added to responses to selection for the three generations in the pedigree line to obtain estimates of response to selection in the genomic line. Responses were expressed in genetic standard deviations. Evaluation of inbreeding rate Pedigree-based inbreeding was evaluated in both lines using the CFC software [16]. For the genomic line, genomic measures of inbreeding were calculated using PLINK [17], including average homozygosity, number and size of homozygosity runs of at least 50 consecutive SNPs, and inbreeding coefficients based on expected vs. observed homozygosity. Simulation results Observed average responses to selection and inbreeding rates based on stochastic simulation for the conventional and GS breeding programs are in Fig. 2. Results are shown on a per year basis and account for the fact that the generation interval for GS was reduced by 50 % compared to that of the conventional BLUP selection. The simulated conventional BLUP-based breeding program resulted in responses to selection that were similar to those predicted by SelAction. Two main scenarios are summarized in Fig. 2: genomic selection with retraining (GS-all) and without retraining (GS-1). For both GS-1 and GS-all, the accuracy of GS-EBV in year 0 (the generation following training) was equal to 0.77, which was slightly higher than the starting accuracy used to obtain . Accuracy in year 0 was the same for GS-1 and GSall because retraining in a particular generation was done before females from that generation had their own performance records. Accuracy remained fairly constant for GS-all through year 2.5 (results not shown) and then gradually dropped to 0.73 by year 4. For GS-1, accuracy gradually dropped to 0.34 in year 4. Resulting responses to selection for GS-all were similar to those predicted by SelAction. For GS-1, observed responses were similar to those predicted by SelAction through year 1.5 but dropped off after that because of the decline in accuracy. Consistent with the target, the rate of inbreeding on an annual basis was of the same order of magnitude for GS and BLUP approaches (Fig. 2). Observed rates of inbreeding were, however, greater than predicted by SelAction for all programs, but in particular for BLUP and GS-all. For BLUP, the observed rate of inbreeding was 1.95 % per year when ignoring the lower rate in year 1, compared to a prediction of 1.44 % by SelAction. For GS-all, the observed rate was equal to 0.89 % per generation compared to that of 0.58 % predicted by SelAction. For GS-1, the observed rate was equal to 0.69 % when the substantially higher rates in the first two generations were ignored, which was only marginally greater than the rate of 0.58 % predicted by SelAction. The higher rates for GS-all and for GS-1 in the first two generations are likely caused by the implicit prediction of pedigree when data from recent ancestors are used for training. Standard deviations of the cumulative response across replicates increased over generations for all strategies as a result of drift (Fig. 2). Standard deviations of the response were similar for GS-1 and GS-all in the initial generations but increased slightly faster for GS-all than for GS-1 and reached 0.43 and 0.37 phenotypic standard deviation units for GS-all and for GS-1, respectively. When strategies were compared at the same generation of selection, the standard deviation of the response for BLUP was similar to that for GS-1, despite the much greater level of inbreeding at a given generation for BLUP compared to GS. Experimental results Estimates of heritability and genetic correlations between traits using all data from the pedigree selected line are in Table 1. All traits, except PD and PS, had moderate to high heritability estimates. Estimates of genetic correlations between measurements of the same trait at two ages (early versus late) were high. Egg quality traits were in general positively correlated with each other but negatively correlated with egg production. The standardized responses to selection by the end of the experiment are in Fig. 3. On average, trait means were changed in the desired direction by the end of the experiment for all traits. For most traits, the genomic line significantly outperformed the pedigree line, with a doubled response to selection for some traits, such as EW and YW. Body weight increased for both lines, with Table 1 Pedigree-based estimates of heritability and correlations a between traits based on data from the pedigree sub-line Egg weight for first three eggs (eE3), at early (eEW) and late age (lEW), shell colour for first three eggs (eC3), at early (eCO) and late age (lCO), albumen height at early (eAH) and late age (lAH), yolk weight at early (eYW) and late age (lYW) puncture score at early (ePS) and late age (lPS), ePD and lPD are egg production rates (ratio of the number of saleable eggs to number of days in lay) body weight at late age (lBW), age at first egg (eSM) a larger response in the genomic line. This reflects selection for a revised objective, i.e., in the past, layer chicken lines were selected for lower BW, while, more recently, selection has aimed at increasing BW at a young age to allow pullets to develop adequately. For egg production rate (ePD and lPD), the pedigree selection line showed a positive response, while the genomic selection line showed a negative response to selection. It should be noted that, for this trait, phenotypes were available for the pedigree selection line at the time of selection but not for the genomic line. However, these results were not supported by the results of egg production measured by egg number (eEN and lEN), for which both lines showed a positive response, with a greater response for the genomic line. This difference in responses in egg production rate versus egg number was explained by both a higher frequency of egg defects in the genomic line (5.7 vs 4.0 %) and earlier age at sexual maturity of birds in the genomic line (141 vs. 145 days). In the first 1.5 years of the selection experiment, inbreeding rate per year was similar between the two lines ( Table 2), although the genomic line had twice as many generations and a smaller population size. However, in the final generation, the genomic line had a higher level of inbreeding than the pedigree line. The recorded pedigree of both lines traces back to 131 founders. In the pedigree line, all founders were represented in all three generations, with a variance of contributions in the final generation of 8.2E-05. In the genomic line, four of the founders were lost after six generations of selection but the variance of contributions at the end of the experiment was equal to 7.6E-05, which according to optimal Fig. 3 Responses to selection in the experimental breeding program, as deviation of trait means and expressed in genetic standard deviation units. Responses to selection are based on deviations of trait means from trait means at the start of the selection experiment, expressed in genetic standard deviation units of each trait; trait abbreviations: egg weight for first three eggs (eE3), at early (eEW) and late age (lEW), shell color for first three eggs (eC3), at early (eCO) and late age (lCO), albumen height at early age (eAH), yolk weight at early (eYW) and late age (lYW) puncture score at early age (ePS), egg production rates at early (ePD) and late (lPD), egg numbers at early (eEN) and late (lEN) age, body weight at late age (lBW), and age at first egg (eSM) contributions theory [18] should help to control inbreeding. At the end of the experiment, the most influential founder contributed 7 % of its genes to both lines. At the genomic level, the average homozygosity and number of homozygous segments increased over generations (Table 2), but the size of the homozygosity runs and genomic inbreeding dropped in generation G0, which was the first generation of genomic selection. Until generation G0, genomic inbreeding reflected the homozygosity level in the selected parents, in contrast to the following generations, in which all selection candidates were genotyped. The rate of inbreeding (based on pedigree and all genomic estimates) increased in generation 4 of the genomic line. Genotypes were not available for the pedigree line but it was possible to compare changes in allele frequencies from generations −5 to 0 when pedigree selection was practiced with the changes observed with subsequent genomic selection. The average change in allele frequency was close to zero for both pedigree and genomic selected generations. The maximum average change in frequency per generation was equal to 0.062 for the pedigree selected generations and 0.097 for the genomic selected generations. Loci with the greatest changes (>5 SD) in allele frequencies were located on chromosomes 8, 9, and Z (six loci) for the pedigree selected generations and on chromosomes 6, 9, and 12 (four loci) for the genomic selection generations. Although the SNPs with the greatest change in frequency differed between the pedigree and genomic selected generations, several regions showed substantial changes in a consistent direction across the pedigree and genomic selected generations. Discussion Optimizing breeding programs with genomic selection Use of marker information removes many of the limitations of conventional phenotype-based selection programs, as has been argued and demonstrated by many simulation studies [19], because phenotypic records on selection candidates and/or their close relatives are not required to estimate breeding values. This feature is even more pronounced for GS and provides opportunities to substantially change the structure of breeding programs [3,20,21]. In this study, we investigated the various opportunities that GS offers to improve breeding programs of layer chicken by changing their structure. Results clearly demonstrate that GS provides interesting opportunities to reduce generation intervals and the size of breeding programs, which impacts the number of animals that need to be raised and phenotyped on a routine basis. Maximizing response per year for a given rate of inbreeding per year was used as the objective for comparing alternative strategies for GS. This has been suggested as a reasonable objective for balancing short-and longterm responses to selection [22]. Even when reducing the generation interval by 50 %, the much lower rate of inbreeding per generation that resulted from GS because of the lower within-family correlations of EBV, allowed a substantial drop in the number of parents selected with GS for the same rate of inbreeding per year. Furthermore, with GS-EBV of equal accuracy for males and females and no limitations on reproductive rates, this resulted in equal numbers of selected males and females as being optimal. Removing the hierarchical mating restriction of one male per female that was used here in the simulation and allowing for factorial or crossclassified mating designs is expected to further reduce rates of inbreeding [23] or could reduce the number of parents needed for the same rate of inbreeding. The simulated GS breeding program required much smaller numbers of selection candidates (500 males and 500 females per year for GS compared to 1080 males and 2880 females per year for the conventional program) to achieve rates of genetic gain per generation that approached those of the conventional program. This was due to the greater accuracy of EBV with GS, in particular for males, which had EBV based on pedigree and sibs in the conventional program. In addition, the numbers of individuals that were phenotyped were substantially smaller for the GS program, even with retraining (500 females per year for GS, compared to 2880 per year for the conventional program). Lower rearing, housing, and phenotyping requirements would substantially reduce Further analyses that consider specific prices, requirements and limitations are needed. The optimal strategy for retraining, including how often and on which animals new phenotypic data should be generated, also requires further investigation and will depend on the species used and the goals of the breeding program. The GS program was successfully implemented, but the short generation interval was not very practical in a commercial breeding program setting. Reproducing females at a very young age increases selection pressure on sexual maturity, and requires using suboptimal sized eggs and hatching them over multiple hatches, which complicates optimization of animal management, especially the lighting program. When selection is initiated, the late maturing females have not started laying yet or produce eggs that are too small to hatch good quality chicks. A more feasible approach would be to use young genomic selected males on older females, which would also improve accuracy of selection because the females would already have own records. A novelty in the implemented GS breeding program was the use of crossclassified mating, in which females are given the opportunity to leave progeny from more than one male parent. Parent assignment with a high level of accuracy based on the number of opposing homozygotes in parentoffspring pairs was possible for almost all offspring from the multi-sire matings. The use of cross-classified mating improves population structure and creates more opportunities for varying chromosomal combinations in the progeny and thus, results in higher effective rates of recombination. Genomic prediction models In the stochastic simulation, the Bayes-B method of [1] was used to develop the prediction model for GS. This method has been found to give greater accuracies than other methods in many simulation studies [1,24]. The method was, however, not optimized with respect to the prior probability π that a SNP has zero effect; a value of π = 0.95 was used throughout, since preliminary analyses suggested that results were robust to choice of this parameter. In the analyses on real data, we found only small differences between genomic evaluation models [25] and some variation in their ranking between generations, but on average they were consistently better than pedigreebased EBV. This lack of differences between methods is often interpreted as evidence that the number of QTL is large but could also result from genetic relationships and within-family effects having large impacts on GEBV [12]; in contrast to historic linkage disequilibrium, which is short-range, genetic relationships and within-family effects do not require markers that are close to the QTL and, therefore, the effects of QTL can be spread across markers surrounding the QTL. Impact of genomic selection on the initial accuracy of prediction Analyses of real data showed that accumulation of information across generations improved the accuracy of genomic predictions on average [25]. In general, the trends observed with real data agree with those of the simulations but, for most traits, the accuracy of prediction estimated based on real data was lower than expected for a given heritability estimate, which suggests that the genetic architecture of traits is more complex than the simple additive model used in the simulations. This would also be in line with the poor consistency of estimates of small QTL over generations [26]. However, for some of the traits with a lower heritability, decreasing the weight on genomic information from very distant relatives of the selection candidates was shown to be advantageous [10]. Across traits, adding phenotypes from distant relatives (more than four generations apart from that of the selection candidates) did not improve the accuracy of predictions (Weng personal communication). Lourenco et al. [27] reported similar observations for cattle and pig data. Previous analyses with this data showed a substantial decline in accuracy of GEBV over generations without retraining, since the selection candidates were more distant from the animals in the training population [25]. Although the decline in accuracy of GEBV was lower than expected based on the decline in genetic relationships and as observed in pedigree-based EBV, the average accuracy of GEBV in grand-progeny of training animals was similar to that of pedigree-based EBV in progeny and thus suggested a need for retraining every generation. Retraining resulted in increased accuracy of GEBV in subsequent generations [25] for all traits. Any decrease in the accuracy due to selection was outweighed by an increase in size of the training population. Impact on inbreeding and loss of alleles Figure 2 clearly demonstrates the ability of GS to reduce rates of inbreeding. Sonesson and Meuwissen [28] observed even larger reductions in rates of inbreeding from GS because their conventional breeding program consisted of sib-testing for both sexes. The main reason for the reduction of rates of inbreeding with GS is that SNP genotypes provide information on Mendelian sampling terms, which reduces the emphasis placed on family information and, as a result, reduces correlations of EBV among family members and probabilities of co-selection of relatives, as demonstrated by [4]. This unique feature of marker genotype data, i.e., to increase accuracy of EBV and response to selection, while not increasing or even decreasing rates of inbreeding, is one of the main advantages of GS. Rates of inbreeding in GS programs will be greater if the selection candidates have strong genetic relationships with individuals in the training population because the information on relationships that is captured by genomic prediction will then result in greater correlations of EBV among selection candidates. This explains, in part, the greater rate of inbreeding for GS-all than for GS-1 and also the greater rate of inbreeding for GS-1 in the initial compared to later generations. Heidaritabar et al. [29] observed more directed and localized selection pressure on specific regions of the genome when using genomic information compared to pedigree-based selection, which agrees with our result that changes in allele frequencies had a greater range after implementation of genomic selection. The effects of such local inbreeding are not yet well understood but could represent signatures of selection as a result of large effect QTL. Conclusions This study demonstrates the advantages of using genomic selection combined with cross-classified mating in layer chicken breeding programs, in terms of increased accuracy of prediction relative to pedigree-based parent average, providing the potential to shorten generation intervals and thereby increase response to selection per year. The advantages were reflected in the phenotypic superiority of the genomic sub-line for almost all traits included in the selection index. Genotyping also allowed sire assignment and relaxed traditional constraints on hierarchical matings. Using multiple sire matings improves the population structure and is expected to decrease inbreeding rate. Nevertheless, a sustainable breeding program still requires an effective population of sufficient size. In view of the rapid changes in genotyping technologies and costs, breeding programs based on genomic selection need to be re-evaluated and optimized on a regular basis.

v3-fos

2022-12-22T14:58:49.818Z

2015-10-12T00:00:00.000Z

254934404

s2

Effects of Endogenous Salicylic Acid During Calcium Deficiency-Induced Tipburn in Chinese Cabbage (Brassica rapa L. ssp. pekinensis) By cultivating tipburn-susceptible plants in modified Hoagland’s medium containing of gradient exogenous calcium (Ca2+), we have shown that Ca2+ deficiency is one of the main causes of tipburn in Chinese cabbage (Brassica rapa L. ssp. pekinensis). The effect of endogenous plant Ca2+ concentrations on tipburn was also studied in a doubled haploid (DH) population consisting of 100 individuals, but no correlation was found. We then examined the expression of 12 Ca2+ transporter genes that function in cytosolic Ca2+ homeostasis in both tipburn-susceptible and tipburn-resistant plants under normal and tipburn-inducing conditions. Expression patterns for most of these genes differed between the two types of plants. Salicylic acid (SA) accumulated in response to conditions of calcium deficiency in our study, and both total SA and SA β-glucoside (SAG) in tipburn-susceptible plants was ∼3-fold higher than it was in resistant plants following Ca2+ deficiency treatment. Also, the changes observed in SA levels correlated well with cell death patterns revealed by trypan blue staining. Therefore, we speculate that the cytoplasmic Ca2+ fluctuation-induced downstream signaling events, as well as SA signaling or other biological events, are involved in the plant defense response to tipburn in Chinese cabbage. Introduction Tipburn is one of the irreversible physiological disorders that causes large economic losses in the production of crops such as cabbage, Chinese cabbage, lettuce, cauliflower, strawberry, and tomato (Nieuwhof 1960;Bradfield and Guttridge 1979;Kuoa et al. 1981). Typical symptoms of tipburn initially appear as necrosis at the leaf tips and margins, followed by browning and shriveling of the entire leaf as the disease progresses. Also, tipburn always develops together with pathogen infections, which further reduces yield and quality. The causes of tipburn are both complex and controversial; however, it is generally accepted that lack of available calcium (Ca 2+ ) is a contributing factor (Barta and Tibbitts 1986). For plant, Ca 2+ is absorbed from the soil by apoplast and cation channels and transported through the xylem by transpiration (White 2001). In the plant cell, Ca 2+ is mainly stored in the plasmalemma, the vacuole, and the endoplasmic reticulum (ER), but it is the cytosolic Ca 2+ that shows significant concentration changes (Ca 2+ oscillations) and plays central roles in the host response to various stress signals (Berridge and Taylor 1988;Kaplan et al. 2006). Modulation of cytoplasmic Ca 2+ levels provides a rapid response to environmental stimuli that is achieved by a system of Ca 2+ -transport and storage pathways. The best characterized Ca 2+ transporters are tonoplast-localized ACA (autoinhibited Ca 2+ -ATPases) and CAX (Ca 2+ /H + antiporters) proteins, and ER-localized ECA (P 2A -type Ca 2+ -ATPases) proteins, which catalyze Ca 2+ efflux Tongbing Su and Shuancang Yu contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s11105-015-0949-8) contains supplementary material, which is available to authorized users. to dampen cytoplasmic Ca 2+ concentration changes (Robertson 2013). ACA4 and ACA11 have been shown experimentally to be important for removing excess cytoplasmic Ca 2+ to the vacuole. When the expression of ACA4 and ACA11 is knocked out, groups of cells in the mesophyll begin to undergo programmed cell death (PCD), with the strong appearance of scattered macrolesions around the leaf, especially at the leaf margin. These growth defects can be suppressed by exogenous Ca 2+ (Boursiac et al. 2010). CAX1 and CAX3, which are 77 % identical at the amino acid level, can exchange two vacuolar protons for one cytoplasmic Ca 2+ ion to reduce cytoplasmic Ca 2+ levels, and the cax1/cax3 double deletion displays necrosis of the leaf tips and shoot apex (Cheng et al. 2005). ECA1 and ECA3 are important for Ca 2+ and Mn 2+ homeostasis between the cytoplasm and the ER in the plant cell, and growth of the eca1 and eca3 mutants are sensitive to Ca 2+ and Mn 2+ , respectively (Mills et al. 2008;Wu et al. 2002). All of these results imply that the ACA, CAX, and ECA genes maybe involved in the process of calcium deficiency-induced tipburn. The expression patterns and functions of these Ca 2+ transporters, as well as other Ca 2+ -metabolism related genes, have been recently analyzed in cabbage and tomato (Lee et al. 2013). The results show that expression of these genes respond differently to abiotic stresses in cultivars with different tipburn resistance. In addition, it has been reported that overexpression of some of the proteins, such as CAX1 and CRT (a Ca 2+ -binding protein), can result in an enhanced resistance to calcium-limiting conditions and/or improved stress resistance under such conditions in Arabidopsis, tobacco, and tomato (Li and Komatsu 2000;Pittman and Hirsch 2001). Salicylic acid (SA) is required for local and systemic disease resistance responses in higher plants, and SA-dependent cell death has been reported to correlate closely with Ca 2+ homeostasis (Van Doorn 2011). Indeed, crosstalk between the SA and Ca 2+ signaling pathways has been reported in some Ca 2+ -metabolism mutants. Overexpression of calreticulin protein (CRT2), which binds a 50-fold excess of Ca 2+ and may facilitate Ca 2+ transport across plasmodesmatal ER, causes a dwarf phenotype with an increased level of SA in transgenic Arabidopsis plants (Qiu et al. 2012). The result, as well as the finding of SA-dependent PCD in aca4/aca11 mutants (Boursiac et al. 2010), implies that there is an interaction between the SA and Ca 2+ pathways in the process of tipburn. To further understand the relationship between Ca 2+ deficiency and tipburn incidence, we firstly demonstrated the correlation between tipburn severity and plant exogenous, intrinsic Ca 2+ levels, and SA concentrations in Chinese cabbage. In addition, the transcriptional changes of the Ca 2+ transporter genes, and SA biosynthesis and response genes were also studied under different Ca 2+ concentration or infection conditions. Our study will provide valuable information to understand the mechanism of tipburn incidence in Chinese cabbage. Plant Materials A population consisting of 100 DH lines was derived by microspore culture from an F 1 of the cross between the female parent BOrange Queen^(OQ) and the male parent BQinXiao No. 2^(QX). The inbred line OQ is highly susceptible to tipburn, while QX is highly resistant. Another population consisting of 285 F 1 BC4 individuals was obtained by four generations of backcrossing. The F 1 was generated by crossing the tipburn-susceptible parent BSheng Xiao^(SX) with the tipburn-resistant parent BDaBaiKou( DBK), which was also used as the recurrent parent. Field and In Vitro Evaluation of Tipburn Severity For field evaluation, the DH and F 1 BC4 populations and the parental inbreds were planted following a randomized complete block design with three replications in the experimental field at Beijing Vegetable Research Center (Beijing, China) in the autumn of 2013. For the DH population, three replicates were conducted, with 10 plants per replicate (n=30). A higher nitrate supply and less water compared to normal cultivation were applied to induce tipburn during the crop growing period. The symptoms and disease index (DI) of tipburn were observed when the plant heads reach acceptable commercial firmness. For in vitro evaluation, we used the method described in Zhang and Xu (1994)) with modifications. Seeds were germinated on 0.8 % (w/v) agar medium supplemented with 3 % (w/ v) sucrose and grown until the first two leaves had formed. The seedlings were transferred to a modified liquid medium which consisted of Hoagland's medium at pH ∼6.0 containing four levels of Ca 2+ (0.57, 1.14, 1.17, and 5.7 mM CaCl 2 ). The Hoagland's medium containing 0.57 and 5.7 mM CaCl 2 were defined as Ca 2+ -deficient and normal-condition controls, respectively. The plants were maintained at 25°C under a 16-h photoperiod with approximately 70 μmol m −2 s −1 light for 10 days. Each treatment (five Ca 2+ concentration) consisted of three replicates with 10 plants each in a completely randomized block design. Eight plants were finally selected in each treatment (n=24). Disease severity was evaluated based on the disease rating scale as described by Zhang and Xu (1994). Correlation Analysis Between Tipburn Severity and Plant Endogenous Cations Spearman's rank correlation analysis, implemented in SAS9.3, was used to analyze the correlation between tipburn severity and plant endogenous Ca 2+ , as well as other major macronutrient cations, including Mg 2+ , Fe 2+ , Zn 2+ , Mn 2+ , Na + , and K + . Real-Time PCR Analysis The in vitro-evaluated plants of inbred lines OQ and QX were used for Ca 2+ content, SA concentration, and gene expression analysis. Two leaf discs from two rosette leaves were collected, and samples of 18 individuals (36 discs) were pooled together to conduct these experiments at each Ca 2+ concentration (n=18). A total of 18 F 1 BC4 individuals with three different disease rankings: high resistant (HR, with a DI of 0∼11.1), tolerant (T, with a DI of 33.4∼55.5), and high susceptible (HS, with a DI of 77.8∼100) from the field test (six plants of each), were used for SA concentration and gene expression analysis. Leaves used in the test were harvested and divided into three types: the outer, middle, and inner leaves, as described in Su et al. (2015)). Total RNA was extracted using an RNAeasy pure kit (Tiangen), and cDNA was synthesized using a PrimeScript R RT reagent Kit (Takara). Real-time PCR was performed in a Roche thermocycler (LightCycler480, Roche, Germany). Amplification reactions (10 μL) were prepared by mixing 1 μL of cDNA template (250 ng/μL) with 1×SYBR Green (Roche) and 0.5 μL of each primer at 10 mM. The thermal cycling conditions consisted of an initial denaturation step at 95°C for 2 min followed by 40 cycles at 95°C for 10 s, 60°C for 10 s, and 72°C for 10 min, with a final extension step at 72°C for 5 min. The results were analyzed with Lightcycler version 2.0 (Roche Diagnostics) software. The Chinese cabbage glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal standard to normalize different plant DNA samples (Qi et al. 2010). The PCR primer sequences used to amplify the candidate genes are given in Supplementary Table 1. Ion Measurements Samples from the DH population were harvested using the one-eighth-excision method. Individual heads of Chinese cabbage were divided longitudinally into eight equal parts, one of which was then lyophilized and ground to a fine powder for use in the experiments. Analyses were performed with a Dionex ICS3000 ion chromatograph equipped with a highpressure pump, an ED40 conductometric detector, and a Rheodyne injection valve (25 ml sample loop) following the method of Lee et al. (2013). Salicylic Acid Analyses The aboveground plant parts (∼1 g) were homogenized completely and extracted twice with 3 ml of 90 % methanol. These two supernatants were mixed and dried in a speed vacuum at 40°C. The residue was then re-suspended in 4 ml of water and incubated at 80°C for 10 min. For free SA analysis, 1 mL of the supernatant was resuspended and incubated for 10 min in 2.5 mL of ethyl-acetate/cyclohexane (1:1) after the addition of 50 μL of concentrated hydrochloric acid (HCl). After centrifugation, the organic phase was dried and then dissolved in 0.5 mL of the high-performance liquid chromatography (HPLC) mobile phase (20 % methanol in 20 mM sodium acetate buffer, pH 5.0) for HPLC analysis. For total SA quantification (free+ bound), 1 mL of β-glucosidase solution (3 U mL −1 ) was added to 1 mL of the water extract and incubated for 8 h at 37°C. The sample was then prepared as for free SA. HPLC analysis was performed on an Agilent 1200 HPLC Pump system equipped with a 4.6×250 mm C18 column containing 5-μm particles (Phenomenex) with a flow rate of 0.8 ml min − 1 , and SA was detected and quantified fluorometrically (295 nm exCitation and 370 nm emission) . Trypan Blue Staining Tissue staining with trypan blue was conducted as described by Keogh et al. (1980). Tipburn Severity Increases at Low Exogenous Ca 2+ Concentrations Two inbred lines, OQ and QX, showed highly susceptible and resistant to tipburn in the field (Fig. 1b,d) and were submitted to hydroponic test. Plants of both lines grew well in normal Hoagland's solution (5.7 mmol·L −1 Ca 2+ ) after 10 days of cultivation (Fig. 1a, c). However, for OQ plants that were cultivated in the Ca 2+ concentration series, a high incidence of tipburn was observed, especially in the medium with the lowest calcium concentration (0.57 mmol·L −1 Ca 2+ ). The affected hydroponic OQ plants were stunted and the leaves were tightly wrinkled, and in some newly developing leaves, the leaf margins and tips became yellow and eventually died (Fig. 1a). The affected hydroponic and field-cultivated QX plants showed no noticeable symptoms (Fig. 1c, d), and only shrivel leaves started to appear at 1.14 and 0.57 mmol·L −1 Ca 2+ (Fig. 1c). The DI of plants was then evaluated for the 0.57, 1.14, 1.71, and 5.7 mM Ca 2+ -containing solutions; DI scores were 100, 89.3, 55.6, and 4.7, respectively. The result showed that the incidence and the severity of tipburn increased as the Ca 2+ concentration decreased. Tipburn severity has been reported to be closely associated with the balance between Ca 2+ and other cations, as shown by strong correlations of tipburn incidence with Ca 2+ /K + ratios in strawberries and cabbages (Palencia et al. 2010;Lee et al. 2013). In the current study, correlations between the disease severity and the ratios of Ca 2+ /Mn 2+ , Ca 2+ /Mg 2+ , Ca 2+ /Fe 2+ , Ca 2+ /P, Ca 2+ /K + , and Ca 2+ /Na + were also calculated. However, the tipburn disease severity was not associated with any of these ratios (data not shown). Expression Patterns of Ca 2+ Transporter Genes in Tipburn-Susceptible and Tipburn-Resistant Chinese Cabbage Plants Cytosolic Ca 2+ is a major source of calcium for cell growth and signaling. The homeostasis between cytosolic and storage Ca 2+ is controlled by a system of membrane-localized Ca 2+ pumps and channels, including ECA1-4, ACA4, ACA11, and CAX1. A total of 12 Brassica homologous genes were selected for analysis based on gene annotation of Brassica database (BRAD, http://brassicadb.org/brad/) and amino acid sequence alignments. Expression patterns of the 12 genes were assayed and compared between tipburn-susceptible and tipburnresistant plants of Chinese cabbage (Fig. 2). For plants cultured in calcium deficient artificial solution, relative transcription of all 12 genes increased in both susceptible (OQ) and resistant lines (QX) after Ca 2+ deficiency treatment for 10 days, and there was no clear distinction in the expression patterns between the two lines, except for two CAX1 orthologs (Bra005131 and Bra017134). mRNA for both of the CAX1-orthologous genes accumulated to higher levels in the tipburn-resistant line compared to the susceptible line (Fig. 2a). To examine tipburn under natural conditions in the field, plants from the F 1 BC4 population that were scored as being either high resistant (HR), tolerant (T), or high sensitive (HS) to tipburn were used for analysis. Leaves used in the test were divided into outer, middle, and inner classes, and the DI of the outer leaves (DI=88.7) was much higher than that of the inner leaves (DI=0) (Fig. 5d, e). In the outer leaves, eight of the 12 Ca 2+ transporter genes showed higher expression levels in HS plants compared to HR plants, while four genes, including one ECA2 ortholog (Bra037404), one ECA3 ortholog (Bra031701), one ECA4 ortholog (Bra031593), and an ACA11 ortholog (Bra003276), showed unchanged expression levels (Fig. 2b). We also found that expression patterns for the 12 genes in the outer leaves were quiet consistent to the expression patterns in plants grown in calcium deficient artificial medium (Fig. 2a, b). Transcription of the Ca 2+ transporter genes in the inner leaves was evaluated and compared (Fig. 2c). Interestingly, we found that the expression patterns of the Ca 2+ transporter genes showed almost the opposite expression patterns compared to those in the outer leaves (Fig. 2b, c). Transcript levels from 11 of the 12 genes were much lower in the HS plants; only the ECA2 ortholog Bra037404 showed a stable expression pattern (Fig. 2c). SA Biosynthesis Is Induced by Tipburn Both artificially and naturally diseased plants were used in the analysis of free SA, SA β-glucoside (SAG), and total SA (free SA+SAG) contents. For plants grown in artificial solution, the SA levels in both resistant (QX) and susceptible (OQ) plants showed a generally increasing trend following Ca 2+ deficiency treatment, although the relative magnitude of the increases varied (Fig. 3a). For instance, basal levels of total SA in resistant and susceptible plants grown under normal conditions were 0.18 and 0.36 μg·g −1 FW, respectively. For plants exposed to Ca 2+ deficiency for 10 days, total SA increased to 0.41 and 1.40 μg·g −1 FW, respectively, which was ∼2and ∼4-fold higher than in normally-grown plants. A similar pattern was also observed for SAG in both lines. However, there was no significant change in free SA levels. Moreover, it is worth noting that the total SA and SAG contents in susceptible plants were ∼3-fold higher than in resistant plants after Ca 2+ deficiency treatment (Fig. 3a). For naturally diseased plants, the levels of all three forms of SA in the outer leaves increased from 2.05, 0.76, and 1.6 μg· g −1 FW in healthy HR plants to 6.41, 1.33, and 5.10 μg·g −1 FW in diseased HS plants, respectively (Fig. 3b). A consistent DI disease index, DW dry weight pattern was also found for the inner leaves. Also, it was also easy to find that the contents of the total SA and SAG increased as the disease severity aggravated in inner leaves (Fig. 3c). Two alternative pathways have been proposed for SA biosynthesis, one requiring isochorismate synthase (ICS) and the other requiring phenylalanine ammonia-lyase (PAL) (D'Maris et al. 2011). Here, we analyzed transcription of three ICS1 orthologs (Bra019813, Bra008165, and Bra0037895), two PAL1 orthologs (Bra017210, and Bra005221), and three PAL2 orthologs (Bra006985, Bra003126, and Bra039777) (Fig. 4). For plants exposed to Ca 2+ deficiency treatment, most genes showed stable expression patterns, although single PAL1 (Bra005221) and PAL2 (Bra003126) orthologs did not (Fig. 4a). The levels of Bra005221-specific transcripts increased only in the tipburn-susceptible lines after Ca 2+ deficiency treatment, while Bra003126-specific mRNA levels increased in both tipburn-susceptible and -resistant Chinese cabbage plants. For naturally diseased plants, the expression patterns of the above genes were quite different from those in plants grown on artificial medium. In the outer leaves, mRNA from all the tested genes accumulated to higher (Fig. 4b), while in the inner leaves, only the expression of a PAL1 ortholog (Bra017210) increased, and expression of the other genes was nearly unaffected (Fig. 4c). Also, in both artificially and naturally diseased plants, PAL1-and PAL2-specific mRNA accumulated to much higher levels than did ICS-specific mRNA in susceptible plants. To investigate whether tipburn-induced SA biosynthesis also activates the plant defense response, we examined the expression pattern of PR1, a typical plant defense response marker gene. Five PR1 orthologs, including Bra014635, Bra014636, Bra015873, Bra017313, and Bra017314, were tested. For artificially diseased plants, mRNA specific for all of the five genes accumulated to high levels after Ca 2+ deficiency treatment in both susceptible and resistant lines. However, it is interesting to note that the relative expression of these genes increased more in the tipburn-susceptible line compared to the tipburn-resistant line (Fig. 5a). For naturally diseased materials, the expression levels of the five PR1-like genes in both the outer and inner leaves were measured. Three of the five genes (Bra015873, Bra017313, and Bra017314) showed similar increased expression profiles in both types of leaves, and interestingly, the relative increases in mRNA abundances were higher in the inner leaves compared with the outer leaves (Fig. 5b, c). All these results suggest that the SA signaling pathway was vigorously activated in inner leaves upon tipburn induction. One of the consequences of activated SA signaling pathways in plants is cell death. Therefore, we assayed tissues of the outer and inner leaves of naturally diseased plants for trypan blue retention (Fig. 5h-k). Physiological changes in cells committed to die are believed to result in the uptake of the dye (Keogh et al. 1980). Necrotic patches were observed at the outer leaf tip, margin, and vasculature of the diseased plants (Fig. 5h); in contrast, the healthy plants showed almost no detectable staining (Fig. 5j). This result was quite constant with macroscopic symptom observation prior to staining (Fig. 5d, f). Interestingly, we found that although there were no visual differences for the inner leaves of the two plant types before staining (Fig. 5e, g), the mild but almost identical staining pattern described above was observed in both diseased and healthy plants (Fig. 5i, k). Thus, the cell death pathway is activated when no obvious visual symptoms appeared in the inner leaves of susceptible plants. Discussion Soil Ca 2+ deficiency is a key factor in the induction of tipburn, and it is generally accepted that supplying tipburn-damaged plants with exogenous Ca 2+ can alleviate the symptoms. However, a recently conducted study of leaf tipburn in strawberry suggests that there is no correlation between the level of applied exogenous Ca 2+ and the incidence of tipburn (Palencia et al. 2010). In addition, the effects of exogenous Ca 2+ have also been shown to be complex in Arabidopsis Ca 2+ transporter mutants, such as cax1/cax3, eca3, and aca4/aca11, which show various levels of tipburn-like symptoms (Wu et al. 2002;Cheng et al. 2005;Mills et al. 2008;Boursiac et al. 2010). Cax1/cax3 double mutants show a significant decrease in endogenous Ca 2+ content of 17 %, and interestingly, instead of alleviating the growth defect, exogenous Ca 2+ (b, c, d, e, f, g, h, i, j, k). Diseased outer leaves (d, h) and inner leaves (e, i) from a susceptible plant. Healthy outer leaves (f, j) and inner leaves (g, k) from a resistant plant. In panels h, i, j, and k, leaves were stained with trypan blue to show tissue necrosis. Three replicates were conducted and only the data for a representative experiment was shown. The error bars only represent the variation in technical replicates aggravated the severity of symptoms in the mutant (Cheng et al. 2005). Knockout of ECA3 leads to a weak accumulation of total Ca 2+ content, and supplementing the eca3 mutant with Ca 2+ does not rescue the growth defect (Mills et al. 2008). In our study, we demonstrated a negative correlation between exogenous Ca 2+ concentrations and the severity of tipburn by cultivating a tipburn-susceptible Chinese cabbage line in modified Hoagland's medium containing different concentrations of CaCl 2 . In addition, the plants grown hydroponically in Hoagland's medium displayed typical symptoms of necrosis at the leaf tips and margins. This suggested that the hydroponic method is appropriate for the evaluation of tipburn resistance of Chinese cabbage in vitro and could produce significant increases in speed and efficiency. The correlation between tipburn and plant Ca 2+ has long been a subject of interest. Barta and Tibbitts (1991) demonstrated that the levels of Ca 2+ in tipburn-injured lettuce were lower than in uninjured plants. A recent study showed a correlation between tipburn incidence and cytoplasmic Ca 2+ concentration in cabbage (Lee et al. 2013), but no such correlations were found in strawberry (Palencia et al. 2010). In the present study, a population consisting of 100 DH lines, which could reduce random errors caused by limited plant numbers, was used for further testing. However, the results showed that there was no direct correlation between tipburn symptoms and the total endogenous Ca 2+ level. One of the possible reasons is that although Ca 2+ deficiency is the key inducer of tipburn, it is the Ca 2+ absorption and utilization efficiency of the plant, rather the Ca 2+ in the environment, which plays a role in tipburn resistance. Moreover, cytoplasmic Ca 2+ is the primary source for organ development in plants and that cytoplasmic Ca 2+ oscillations are thought to confer preponderant advantages over a sustained bulk Ca 2+ rise in the activation of Ca 2+ -dependent responses (Berridge 1990;Di Capite et al. 2009). Therefore, we concluded that one possible reason could be that it is the cytoplasmic Ca 2+ , not the total Ca 2+ , which is correlated with tipburn onset. Future research should pay more attention to studying the correlation between cytoplasmic Ca 2+ and tipburn. It is not clear how Ca 2+ moves from areas with high rates of transpiration leaves into the developing leaf tips and meristems due to the developmental immaturity of the vasculature. Previous studies have reported that Ca 2+ transport in plants occurs through two main pathways: the apoplastic pathway and the symplastic pathway (Robertson 2013). The apoplastic route allows long distance transport of Ca 2+ , while the symplastic route functions in cell-to-cell transport. We speculate that the symplastic pathway should play an important role in the final steps of Ca 2+ transport. We assayed gene expression in 12 Ca 2+ transporters involved in symplastic transport during the course of tipburn onset. The results showed that transcription of most of these genes was reduced significantly in inner leaves during the onset of tipburn, while it increased in the older outer leaves and the whole plant involved in tipburn evaluation in vitro. The inner leaves of Chinese cabbage are tightly enveloped in the head, and they do not participate in active transpiration, which is the driving force for long distance Ca 2+ movement. Hence, if Ca 2+ transporter genes are expressed at lower levels in the inner leaves, with the fast growth of these tissues, tipburn would be expected to occur on the inner leaves. There should be a crosstalk between the SA and Ca 2+ pathways in the process of tipburn as summarized in the introduction. Thus, we speculate that SA functions in the onset of tipburn. In our study, SA accumulation was detected in both susceptible and resistant plants under growth conditions in which calcium was limiting, and both total SA and SAG contents in susceptible plants were found to be ∼3-fold higher than in resistant plants after Ca 2+ deficiency treatment. Moreover, we also speculated that the PAL-dependent SA biosynthesis pathway plays a more complex role than the ICS-dependent pathway in tipburn incidence because of the higher accumulation of PAL1-and PAL2-specific mRNA compared with ICS1. Furthermore, it is worth noting that although there was no visual growth defect of the inner leaf in diseased susceptible plants, a remarkable increase in SA levels was still observed. The SA accumulation correlated well with our PR1 gene expression measurements and trypan blue staining experiments, in which the visually healthy inner leaves of the diseased plants showed an elevated SA response and obvious cell death. These results, viewed from different angles, further showed that the onset of tipburn was previously activated in the inner leaves. Many recent studies have described the key role of SA signaling in pathogen-induced disease resistance in plants; however, the function of SA in physiological processes related to cell death is still poorly understood. Our results suggest a new role for SA in the induction and onset of tipburn in Chinese cabbage. We also speculate that the relative increases or decreases in cytoplasmic Ca 2+ levels themselves are not toxic to tipburn-injured plants, but rather it is the Ca 2+ fluctuationinduced downstream signaling events, such as SA biosynthesis and signaling or other biological events, which modulate plant defense responses and subsequently act to initiate cell death.

v3-fos

2017-04-30T07:54:18.534Z

2015-05-26T00:00:00.000Z

12504294

s2

Green Extraction of Antioxidants from Different Varieties of Red Grape Pomace The extraction yield, phenolic content, anthocyanin content and antioxidant activity of extracts from different varieties of red grapes, Cabernet Sauvignon, Merlot, Petit Verdot, Syrah, Tempranillo and Tintilla, using pressurized green solvents have been analyzed. Two techniques were studied and compared: supercritical fluid extraction (SFE) with CO2 + 20% ethanol and pressurized liquid extraction (PLE) with either ethanol, water or an ethanol/water mixture as the extraction solvents. The Petit Verdot variety allowed the highest global and phenolic yield, and antioxidant activity. The best conditios for PLE obtained from the experimental design and kinetic study were 50% ethanol/water as the pressurized solvent at 90 bar, 120 °C, a flow rate of 5 g/min and, an extraction time of 90 min. A statistical analysis of variance has been performed and it was found that temperature is the only variable that has a statistical influence on the extraction yield. The antioxidant activity levels of the extracts are very promising and they are similar to those obtained with the antioxidant tocopherol. Introduction Wine production is of great importance in agro economic activities. The world grape production in 2012 exceeded 69 million tons and Europe was the largest producer of wine, with 66% of the total world production [1]. The solid wastes generated by the wine industry represents between 25%-30% of the material used and it consists mainly of grape pomace (containing seeds, pulp, stem and skin) [2,3]. It is well known that high quantities of valuable compounds like dietary fiber, oils from the seeds, anthocyanins and phenolics compounds still remain within the grape pomace after processing [3,4]. The phenolics, such as resveratrol, have great potential due to their antioxidant capacity and health benefits against coronary diseases by the inhibition of LDL (low-density lipoproteins) and other chronic diseases, like cancer, diabetes and neurodegenerative disorders [3,4]. In addition, from the economic point of view, the market of these compounds have been increased in the recent years by the increasing consumer demand for the use of more natural antioxidant compounds, achieving the value of US$30 billions, based on 2008 grape wine production data [3]. In this sense, the valorization and reuse of these wastes from the wine-making industry would have a significant environmental and economic impact, and this possibility has been studied by several authors [3][4][5][6][7]. In recent years, numerous methodologies for the extraction of compounds of relatively high polarity have been developed in an effort to displace conventional solvent extraction techniques. These novel alternative techniques significantly reduce solvent consumption and increase the speed of the extraction by simplifying the process. Supercritical fluid extraction (SFE) is an efficient technique that is widely applied in the separation of active compounds from herbs and other plants [8]. This technique is appreciated due to the very high solvent power and the distinctive physicochemical properties of supercritical fluids (SCFs). The relatively low viscosity (near to the gas) and the high diffusivity of SCFs help to penetrate the porous solid materials more efficiently than liquid solvents, thus resulting in faster and more efficient extractions. For example, conventional solid-liquid extractions lasting several hours or even days can be achieved in ten minutes on using supercritical fluids [9]. The first scientific work on SFE from grape residues concerned the use of carbon dioxide modified with 5% methanol at 350 bar and 50 °C for the extraction of phenolic compounds [10]. The recovery of resveratrol from grape skin was also optimized by using CO2 + ethanol as the solvent system [11]. Chafer et al. evaluated the SFE of polyphenols from five grape skin varieties and reported that the most suitable conditions were 60 °C, 250 bar and 20% ethanol as a CO2 modifier [6]. It has been shown in different studies that SFE is selective for phenolics, such as gallic acid, catechin, epicatechin and quercetin and have showed high recovery of this polyphenols from grape pomace [6,12,13]. However, this technique cannot be used to extract high molecular weight polyphenols, such as proanthocyanidins, which were more easily extracted by conventional extraction [12]. PLE is based on the use of conventional liquid solvents at subcritical conditions with controlled temperature and pressure. With respect to conventional extraction techniques, PLE has the advantage of using less solvent and the extraction is carried out in a shorter time. PLE is widely used for the extraction of antioxidant compounds from winery residues and other natural products [14][15][16][17]. Piñeiro et al. compared catechin and epicatechin extraction from tea leaves and grape seeds using ultrasound-assisted extraction (UAE) and PLE [15]. In addition, different solvents, such as water, ethanol and methanol, have been evaluated as hot pressurized solvents for the extraction of anthocyanins and phenolic compounds from grape skin [16][17][18]. Several other methods have been applied for extraction of antioxidants from plant matrices, one such novel process being microwave-assisted extraction (MAE). The advantage of PLE and SFE over MAE is the applicability at different scales. PLE and SFE can be applied to systems on various scales, from the laboratory scale (a few grams) to the pilot plant scale (several hundred grams of samples), through to the industrial scale (tons of raw material) [19,20]. In addition, for winery residues, comparative studies have shown that PLE was more efficient than conventional solvent extraction, MAE and UAE for the recovery of high levels of phenolics from grape pomace and grape skin [5,16]. On the other hand, several studies have demonstrated the economic viability of SFE and PLE for the extraction of winery residues [19] and other raw materials [20]. A great variety of phenolic compounds have been extracted under superheated pressurized conditions from white and red grape skins. Phenolic acids (caffeic acid, gallic acid and protocatechuic acid) and flavonols (catechin, epicatechin and gallate derivatives) were detected, but pyroanthocyanin was also tentatively identified [5]. However, there has been a marked increase in the number of crops for the production of red wines in southern Europe. Many different varieties are grown and these include Tempranillo, Syrah, Cabernet Sauvignon and others. The by-products have already been used for the production of antioxidants using high pressure techniques. Campos et al. [21], Tünde et al. [22], and previous studies by Mantell et al. [23] and Casas et al. [24] are some examples. As have been aforementioned, several studies have showed that both techniques, SFE [6,12,13] and PLE [14][15][16][17][18], are successfully used to recovery phenolic compounds from grape pomaces, but there are no comparative studies of these techniques to evaluated the efficiency to extract anthocyanins and phenolics from this raw material. Therefore, the aim of the work described here was to evaluate the effect of different experimental parameters, such as pressure, temperature and extraction solvent on the SFE and PLE from different varieties of red grape pomace (Petit Verdot, Tintilla, Syrah, Cabernet Sauvignon, Merlot and Tempranillo). The extracts were analyzed according to the global extraction yield, total phenolic content, total anthocyanin content and antioxidant activity. In addition, the effects of the extraction time and flow rate for PLE were evaluated at the best extraction conditions. Variety Selection In order to analyze the influence of red grape varieties for the preparation of a product with a high antioxidant capacity, a series of experiments were designed in which the raw material was varied. Two extraction methods, namely Supercritical Fluid Extraction (SFE) with carbon dioxide with 20% of ethanol and PLE with ethanol, were tested and both the extraction yield and antioxidant capacity were analyzed. For SFE, several authors have reported that 20% of co-solvents is efficient enough to the high extraction yields of anthocyanins and polyphenols from red grape pomace and other raw materials [6,23]. Raising the percent of ethanol as modifier to CO2 up to 15%-20% have not showed a significant increase in the yield or the phenolic extraction from grape skins [6,21]. The results obtained in the SFE at 100 bar, 55 °C and flow rate of 25 g/min are shown in Figure 1 and Table 1. It can be seen from this figure that the best extraction yields were obtained with Petit Verdot, followed by Syrah and Merlot varieties. The lowest yields were obtained with Tintilla, Tempranillo and Cabernet Sauvignon, with values of just over half those of the aforementioned varieties. In contrast to the abovementioned, Table 1 shows the SFE from Tintilla and Syrah presented higher recovery of anthocyanins and phenolics than the other grape varieties. The addition of higher concentrations of ethanol as CO2 modifier (20%) favored the extraction yield of phenolic compounds in comparison with previous studies using only 4.5% of ethanol (0.2-0.3 mg gallic acid/g dry grape skin) [25]. The antioxidant activities of the extracts were analyzed and lower EC50 values (associated with a higher antioxidant activity) were obtained for the varieties Petit Verdot followed by Syrah ( Figure 1). Grape variety Extraction Yield EC50 In this case, the antioxidant capacity of the Cabernet Sauvignon samples was the lowest. Comparison of the antioxidant capacities of the extracts obtained in this work with data published previously by other authors shows that the antioxidant capacity of the extracts obtained in this work are lower and are still far from that obtained for the standard (+)-α-tocopherol (EC50 = 6.17 ug/mL) [26]. As a consequence, PLE was explored, as it is considered to be an efficient technique for the extraction of polar or slightly polar compounds from different natural materials [16,26,27].The results obtained in PLE experiments using ethanol for the different varieties of grapes analyzed are presented in Figure 2. Once again, the highest global yields were obtained when Petit Verdot was used, followed by Syrah and Tempranillo. In this case, the lowest global yield was obtained when the extraction was carried out on the Tintilla variety. However, according to the anthocyanins and phenolic recovery, Tintilla, Syrah and Tempranillo showed higher yields of anthocyanins whereas Petit Verdot, Syrah and Cabernet presented the higest phenolic yields (Table 1). As far as the antioxidant capacity is concerned, the highest antioxidant capacities were obtained in the extraction on the Petit Verdot variety. The results obtained with Merlot, Tempranillo, Cabernet Sauvignon and Syrah were very similar and presented values of EC50 close to (+)-α-tocopherol between 7.5 and 9.2 μg/mL. Nevertheless, there are significant differences with the Tintilla grape, which gave the lowest results (higher EC50). Comparison of the yields and antioxidant capacities of the extracts obtained using SFE with carbon dioxide and 20% of ethanol as co-solvent and PLE with ethanol shows that the best results were achieved when the extraction was conducted with PLE. In some cases, double the global yield was obtained in comparison to the SFE method and around 10 fold was increased the yield of anthocyanins and phenolic compounds. The yield of anthocyanins and phenolics was in agreement as reported previously for different grape pomaces' varieties; however, it depends on the origin of the varieties, the genetic variation and the cultivation conditions [7,16]. Similarly, the antioxidant activity results are significantly higher than those shown in Figure 1 and they are comparable to those described by other authors, such asBozan et al. [28], who reported EC50 values of approximately 3 µg/mL for Merlot and Cabernet samples, and Tounsi et al. [29], who reported values of 6.8 µg/mL for Carignan and 30 µg/mL for Syrah seeds, amongst others. Table 2 shows the concentrarion of anthocyanins and phenolic in the extracts obtained by SFE and PLE from the different varieties of grape pomace. In all the cases, the contents of anthocyanins and polyphenols of PLE extracts were higher than those reported for the extracts obtained by SFE. The PLE extracts obtained from Merlot gave the highest concentration of phenolics (254.6 ± 2.6 mg GAE/g extract) followed by Tintilla, Carbernet and Petit Verdot, which also presented high enough phenolic content. However, Tintilla extracts obtained by PLE presented a significant concentration of anthocyanins that was superior to the others grape varieties. However, a direct relationship between the total phenolics and the antioxidant activity of the extracts was not observed. Different authors have also reported similar results considering that the antioxidant activity depends on the quality of phenolic compounds. Besides, the antioxidant capacity of the extracts could also be affected by the synergistic effects caused by the interactions of antioxidant compounds and the presence of other non-phenolic compounds [30,31]. Therefore, considering that Petit Verdot grape pomace showed the highest extraction yield and extracts with high antioxidant capacity as well as adequate total phenolic content, this variety and PLE as extraction technique were selected for the subsequent experiments aimed at identifying the best process operating conditions. Solvent System On the basis of the results obtained in the previous analysis, the highest extraction yield and the best antioxidant activity were accomplished with the same method (PLE) and with the same grape pomace variety (Petit Verdot). PLE is increasingly being used as an alternative to carry out environmentally friendly green extractions since it avoids the use of large amounts of solvents, which also provides significant advantages in process automation and sample preparation. In regard to the solvent employed in the extraction, ethanol (EtOH) has been studied as one of the more environmentally friendly solvents ("green" solvent) and it is recognized as safe according to the European Food Safety Authority (EFSA) and FAO/WHO Expert Committee on Food Additives [32,33]. In an effort to reduce the consumption of organic solvents, reports by Štavíková et al. [15] and Aliakbarian et al. [34] have shown that PLE is favorable for the extraction process on grape skin. Moreover, it has been reported that ethanol/water mixtures are environmentally favorable compared to pure alcohol [35]. In order to select the most suitable solvent system in this work, a study was carried out to evaluate PLE procedures with different solvent composition, namely pure ethanol, ethanol/water (50:50) and water, and the results for the global yield and antioxidant activity are represented in Figure 3. It was found out that the system with the ethanol/water mixture gave the highest extraction yield and the extract with the highest antioxidant activity; for this reason, this solvent was chosen to optimize the extraction method. The performance of this system can be explained because with a dual mixture, particularly a mixture of an organic solvent and water, the extraction efficiency is improved because the organic solvent enhances the solubility of the analyte and water increases the analyte desorption [35]. Furthermore, the analysis of the total phenolic content for the hydroalcoholic mixture (497.32 ± 4.93 mg GAE/mg extract) was superior to that obtained using pure ethanol (204.92 ± 9.43 mg GAE/mg extract) and water (334.61 ± 9.16 mg GAE/mg extract). Other studies have also shown that for PLE, an increase in the percentage of ethanol has a negative effect on the extraction from grape skin or grape pomace and hydroalcoholic mixtures are more favorable for anthocyanins and polyphenols extraction [23,27,36]. Influence of Extraction Parameters Temperature and pressure both play a significant role in the extraction process and, as a result, these parameters were studied in order to select the best operating conditions. The effects of temperature were studied considering that high temperatures favor extraction efficiency [37]; although this might cause degradation of thermo-labile compounds [38,39]. Three extraction temperatures were tested: 80 °C, 100 °C and 120 °C. It can be observed in Figure 4 that higher temperatures gave higher yields and, therefore, the highest extraction yield was obtained at 120 °C, although the best antioxidant activity was achieved with the extract obtained at 100 °C. The use of higher pressure led to an improvement in the extraction because the solvent makes contact with the analyte more easily. Three values were analyzed: 90 bar, 120 bar and 150 bar. It can be deduced that the effect of pressure depends on the temperature; hence at a fixed pressure, both extraction yield and antioxidant activity are determined by the temperature. Nevertheless, there are very few differences between the EC50 values, and extraction efficiency is, consequently, the decisive result. It can be seen in Figure 4 that a considerably higher extraction yield is obtained at 120 °C and, at this temperature, the highest antioxidant activity is achieved at 120 bar. These results are consistent with those of Hawthorne and Miller [40], who first studied PLE with water and showed that temperature has a predominant effect over pressure. The extraction yields and antioxidant activities obtained for different temperatures and pressures were statistically analyzed. Regression analysis was performed on the experimental data and the coefficients of the model were evaluated for significance. It was observed in the Pareto diagram ( Figure 5) that temperature is the only factor that had a significant influence on the extraction yield (p ≤ 0.05). The relationship between temperature and pressure for the global extraction yield is represented by Equation (1). Extraction Kinetics The flow rate is an important variable in the design of extraction processes. Excessive solvent flow rates lead to insufficient loading of the solvent and, in severe cases, compacting of the bed, which results in preferential pathways through the bed and causes inefficient extraction. On the other hand, low flow rates lead to unprofitable processes due to the long extractor residence times. The global extraction yields obtained with PLE from Petit Verdot variety at 90 bar of pressure and 120 °C of temperature with the mixture ethanol/water (50:50) as solvent at flow rates of 5, 10, 15 and 20 g/min are plotted against extraction time and solvent mass in Figure 6. It is remarkable that, in the initial stage of the process, the extraction rate is dependent on the flow. At 15 min, a higher extraction yield was obtained in the process with a solvent flow rate of 20 g/min than that obtained at 5 g/min. This behavior is as one would expect; an increase in the flow rate leads to higher yields of extracts. This effect is due to the presence of a large amount of solvent in the operation, a factor that enhances the extraction yield. For extraction times above 50 min, an increase in the flow did not lead to an improvement in the extraction process, despite the fact that solvent consumption was high. In this second part, the slope of the curve decreased and the extraction rate was reduced until a limiting yield was reached, which depended on the total amount of extractable solutes under the fixed operating conditions. As a result, it is advisable to work at a flow rate of 5 g/min for 90 min. Under these conditions a yield of 87% was obtained after 180 min. This behavior is commonly seen using different raw materials [41]. Samples and Chemicals The materials used in this study were the wastes from the vinification of red wine from different varieties. The varieties analyzed were Cabernet Sauvignon, Merlot, Petit Verdot, Syrah, Tempranillo and Tintilla. All of the raw materials were provided by "Bodegas Luis Pérez" (Jerez de la Frontera, Spain). The grape pomaces were obtained immediately after the vinification process and were dried in an oven at 60 °C to constant weight. Prior to the extraction process, the samples were milled in order to reduce the particle size. Extraction Methods High pressure extraction tests were carried out in a system supplied by Thar Technology (model SF100, Pittsburgh, PA, USA) provided with an extraction vessel (capacity of 100 mL) and two pumps with a maximum flow rate of 50 g/min. In SFE, two pumps were used (one for carbon dioxide and the other for co-solvent) while in PLE only one pump was employed. Pressure was adjusted at the back pressure regulator and solvent pumps. A thermostatized jacket allowed control of the extraction temperature. The cyclonic separator permitted periodic discharge of the extracted material during the process. The operating methodology involved loading the extraction vessel with approximately 35 g of the sample, which had previously been homogenized in order to maintain a constant apparent density in all experiments. The extracts were collected in a cyclonic separator and transferred to glass bottles, which were stored at 4 °C with the exclusion of light. For SFE, the experiments were carried out at a flow rate of 25 g/min for 3 h at 100 bar and 55 °C using CO2 + 20% ethanol as solvent. Previous studies have reported that raising the percent of ethanol as modifier to CO2 up to 15%-20% have not showed a significant increase in the yield or the phenolic extraction from grape skins [6,21]. For PLE, all experiments were carried out at a flow rate of 10 g/min for 3 h. The influence of pressure (P), temperature (T) and ethanol concentration (C) on the extraction process was analyzed. Pressures of 90, 120 and 150 bar, temperatures of 80, 100 and 120 °C, and different concentrations of ethanol in water between 0% and 100% were evaluated. In the case of hydro-alcoholic mixutres, previos studies have showed that 50% ethanol was successful to extract anthocyanins from red grape pomace with similar yields than conventional extraction [35]. The experiments on each sample were carried out in duplicate in order to evaluate the variability of the measurements. The global extraction yields obtained by SFE and PLE were calculated as the ratio of dry extract to dry raw material and the results are expressed as g extract/g raw material. Total Phenolic Content The determination of total phenolic compounds present in the extracts was carried out using an HPLC system 1100 series supplied by Agilent Technologies (Waldbronn, Germany) comprising a degasser, a quaternary pump, an autosampler and a UV/vis detector. The extracts were previously filtered and subsequently 20 µL aliquots were injected and analyzed by HPLC using a Synergi Hydro-RP C18 column (150 mm × 3 mm i.d., 4 μm particle size) (Phenomenex, Torrance, CA, USA) with a C18 ODS guard column Total phenol content was determined as the sum of the peak area of all the phenolic compounds and expressed as mg of gallic acid equivalent (GAE)/mg dry extract based on a calibration curve with gallic acid. Analyses were carried out in triplicate and SD was estimated. Other authors have also used chromatographic methods as an accurate approach to obtain the total phenolic content [31,42]. Anthocyanins Analysis The analysis of the anthocyanins present in the extracts was performed in and HPLC system provided by Agilent Technologies (Palo Alto, CA, USA) 1100 Series chromatograph. 5% Formic acid in water (v/v) (A) and methanol (B) were used as solvents in this HPLC method. The HPLC gradient program was executed as follows: 98% A to 40% A in 60 min, 40% A to 98% A in 5 min. The entire HPLC run time was 70 min using a flow rate of 1 mL/min. The resultant extracts were filtered before HPLC assay using a 0.45 µm PTFE filter (Varian Inc., Palo Alto, CA, USA) and 100 µL of the filtered extract was injected into the column (250 mm × 4.6 mm) C18 Hypersil ODS (5 µm particle size) (Supelco). The total content of anthocyanins was calculated as the sum of the peak area of all the compounds detected at a wavelength of 510 nm. The results were reported based on the calibration curve of malvin chloride and expressed as mg of malvin chloride/mg dry extract. The experiments for each extraction were carried out in triplicate in order to evaluate the variability of the measurements. The method above described has also been used by other authors [43,44]. Antioxidant Assay with DPPH The method used to measure the antioxidant activity of the extracts obtained from the grape pomace was based on the use of DPPH as a free radical. The technique proposed by Brand-Williams et al. [45], and modified by Scherer et al. [46], is based on the use of free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH•), which absorbs at 515 nm. Reduction of this radical with an antioxidant leads to the disappearance of the absorption at this wavelength. Thus, the decrease in the absorbance allows an assessment of the ability of the compound to scavenge free radicals. The 3.9 mL of 6 × 10 −5 mol/L methanol DPPH solution were added to 0.1 mL of extract methanolic solutions at different concentrations. The blank sample consisted of 0.1 mL of methanol added to 3.9 mL of DPPH solution. The absorbance of DPPH was monitored spectrophotometrically at 515 nm at 0 min and every 2 min until a steady state was reached. The DPPH concentration (CDPPH) in the reaction medium was calculated from a calibration curve determined by linear regression with the following equation: The percentage of DPPH remaining was calculated as described in Equation (3). The EC50 (efficient concentration providing 50% inhibition) was calculated graphically using a non-linear calibration curve by plotting the extract concentration vs. %DPPH remaining on the steady state. The experiments were carried out in duplicate in order to evaluate the variability of the measurements. Experimental Design Initially, a preliminary study was conducted to select the most appropriate technique and the best variety of grape pomace. All 6 grape varieties were tested in the two systems studied: supercritical fluid extraction (SFE) with CO2 + 20% ethanol as co-solvent and pressurized liquid extraction (PLE) with ethanol. The use of ethanol as a modifier in the supercritical fluid extraction (SFE) is justified since it increases significantly the solubility of the polyphenols in the supercritical phase and improves their extraction. Moreover, ethanol is the most suitable polar modifier due to its volatility and non-toxicity, in addition to its regular use in the pharmaceutical, medical, cosmetic and food industries [47]. The SFE process operating conditions were: 100 bar, 55 °C, flow rate of 25 g/min and 20% (v/v) of co-solvent. In PLE the extraction tests were performed at 120 bar, 100 °C, flow rate of 10 g/min. In each case, the response variables analyzed were the extraction yield (expressed as g extract/100 g of dry matter) and antioxidant activity of the extracts (AA) (expressed as µg/mL of extract). After selecting the most suitable variety of grape pomace, the PLE process was carried out with different solvents, namely ethanol, water and an ethanol/water mixture (50:50), with the operating conditions listed above kept constant for this technique. In addition, with the variety of grape pomace and the solvent chosen for the PLE, an experimental factorial design 2 2 was performed to study the extraction process and to identify the best operating conditions (according to the objective of the present article). The variables selected for the experimental design were pressure (P), with values of 90 and 150 bar, and temperature (T), with values of 80 and 120 °C. Finally, flow rates of 5, 10, 15 and 20 g/min were tested for PLE under the best operating conditions. All experiments were carried out with an extraction time of 3 h. The results were analyzed using the Statgraphics Plus 5.1 ® (1994-2001, Statistical Graphics Corp., Princeton, NJ, USA) program to evaluate the influence of the factors on the extraction process and to determine significant differences in the samples for each variable. Empirical correlations were developed in order to predict the influence of extraction conditions on both the extraction yield and the antioxidant activity. Significance levels of factors were defined using p = 0.05, so factors and their combinations with a p-value < 0.05 have a significant influence on the extraction process with a confidence level of 0.95. The sign associated with each factor indicates positive or negative effect caused by the variable. Conclusions Two high pressure extraction techniques, SFE and PLE, were evaluated from wine industry wastes. The comparative study using different variaties of grape pomace showed PLE using hydro-alcoholic mixture as solvent was more efficient than SFE using CO2 + 20% ethanol in terms of both phenolic ontent and antioxidant activity. The global yield and the yield of anthocyanins and phenolic compounds was higher for PLE than SFE. The comparison of six different grape varieties showed that Petit Verdot grape pomace provided the highest global and phenolic extraction yield, as weel as strong antioxidant activity using pressurized ethanol as extraction solvent. However, in terms of anthocyanins Tintilla showed the highest yield (49.7 mg/g dry pomace) and total content in the extracts (741.9 mg/g extract). In addion the experimental design and the kinetic study showed that the highest extraction yield for PLE was obtained at 120 °C and 90 bar using hydro-alcoholic mixtures at a flow rate of 5 g/min for 90 min. Furthermore, from the statistical analysis of variance it was found that temperature was the only experimental parameter that has a statistical influence on the extraction yield from grape pomace by PLE. It was demosntrated that this technique is succesfull to extract antioxidant phenolic compounds from grape pomace in order to increase the value of this winery by-product with potential applications in differents industrial sectors like cosmetics or nutraceutics.

v3-fos

2018-12-13T14:10:16.155Z

2015-06-22T00:00:00.000Z

55337984

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Understanding perennial wheat adoption as a transformative technology: evidence from the literature and farmers Abstract Perennial grain crops are an example of a ‘transformative technology,’ in which the functionality and science of the technology differ in a fundamental manner from conventional grain crops. A review of the literature indicates that the motivation for farmer adoption of transformative technologies is complex and poorly understood. At the same time, many studies have found concern and awareness about environmental issues to be significantly and positively correlated with the adoption of no-till agriculture, organic farming and agroforestry. Building on these insights, we conducted an ex ante study of perennial wheat adoption among 11 farmers from Michigan and Ohio. Perennial wheat is not yet commercially available, so a semi-structured interview format was chosen to allow for in-depth discussions of the crop's potential characteristics and uses. Consistent with the literature on transformative technology adoption, farmers who approached us to learn more about perennial grains described soil and environmental quality as their primary motivations for doing so. Farmers suggested a total of ten different uses for perennial wheat, only one of which was mentioned specifically by interviewers. This diversity of proposed uses implied a wide range of criteria for adoption. A striking result was that the ability of perennial wheat to compete with annual wheat on the basis of yield, a focus of researchers, was brought up by only one of the interviewees, as many farmers proposed perennial wheat as a means of solving a problem for which no other crop provided an adequate solution, often by planting perennial wheat on an under-used or marginal area of the farm. This is suggestive that interacting with farmers could alter priorities in perennial grain improvement, as has occurred in other radically transformative agriculture technologies. Perennial grains Perennial grain crops have been proposed as a transformative means to conduct agriculture, one that has potentially radically improved ecosystem services compared with their annual counterparts, including carbon sequestration, reduced nitrate leaching and erosion reduction (Jackson, 2002;Cox et al., 2006;Culman et al., 2013). In the USA, breeding programs focusing on hybridization between annual wheat and its perennial relatives are the most advanced in development of a perennial grain crop (Jaikumar et al., 2012). Perennial wheat is not yet commercially available, but for its potential benefits to be realized, farmers would have to be interested in adopting it. Would American wheat farmers switch from annual to perennial varieties, or grow both types? Would farmers who do not grow wheat currently be interested in a perennial wheat? To date, studies examining these types of questions have focused solely on the economic potential of perennial wheat to replace annual wheat (Bell et al., 2008). The economic competitiveness of perennial grain crops compared with their annual counterparts will certainly be a major driver of adoption, but not the only driver. Restricting famer considerations of adoption potential to one dimension (the expected profit approach) is not consistent with the literature on adoption of transformative agricultural technologies, which suggests that farmers' decisionmaking processes around adoption of these technologies are complex and dynamic. Moreover, farmers often modify agricultural technologies to make them suitable for their own farming system. Replacement of annual varieties may not be the only, or even the primary, use for perennial wheat. As plant-breeding efforts have made progress in the development of perennial wheat and closely related cereal relatives, there have been a number of high-profile reviews calling for major investments in these efforts as a transformative, environmentally friendly form of field crop agriculture. The benefits of perennial grains for reducing nitrate leaching, enhancing the biodiversity of agricultural landscapes, sequestering carbon, reducing erosion, reducing labor and input costs and improving resilience to climate change have all been proposed, and some of these benefits have been demonstrated on the field scale (Glover et al., 2010;Jaikumar et al., 2012;Pimentel et al., 2012;Culman et al., 2013). However, the focus of efforts to develop a new perennial growth form of wheat has privileged grain yield, and to a lesser extent grain quality (Cox et al., 2006;Murphy et al., 2009Murphy et al., , 2010Jaikumar et al., 2012;Hayes et al., 2012), over the potential environmental benefits these crops would provide both on-farm and across the landscape. Our goal was to elucidate the adoption and potential uses of perennial wheat, as a case study of a transformative technology. We conducted a review of the agricultural technology adoption literature for three transformative systems, and conducted a series of semi-structured interviews on perennial wheat potential with farmers in Michigan and Ohio. Our objectives were to understand the criteria farmers might use in deciding whether and how to adopt perennial grains, and the ways in which they envisioned these grains fitting into their farming operation. Agricultural technology adoption studies Studies on the adoption of agricultural technologies can be broadly classified into two, ex ante and ex post. The latter are studies conducted after a technology has been adopted by farmers to determine why a technology was adopted, disadopted or not adopted (Lesser et al., 1999;Babu and Rhoe, 2003;Mercer, 2004;Sirrine et al., 2010). Because perennial grains are a technology that is still under development, in this literature review we focus on ex ante adoption studies. Ex ante studies are conducted prior to the introduction of new agricultural technologies (Pingali et al., 2001). Such studies may be carried out before, during, or after on-farm trials of agriculture innovations (Franzel and Scherr, 2002) or before their commercialization (Babu and Rhoe, 2003). The focal point of ex ante studies is to gain insight into the factors which, hypothetically, may affect the acceptance of new farm technologies and determine their adoption potential (Franzel and Scherr, 2002;Pierpaoli et al., 2013). This may extend to assessing and eliciting information on the farmers' perception of the technology (Pingali et al., 2001), and the technology's likely economic impacts before their introduction (Alston et al., 2003); or to discern and define, ex ante, the agronomic, socio-economic, ecological and biophysical factors that may inform the likelihood to adopt (or not) (Franzel and Scherr, 2002). Ex ante studies are also used to obtain information that improves the performance of the technology, enhances its acceptability, and overall fosters the innovation's successful transference to farmers (Dearing and Meyer, 1994;Franzel and Scherr, 2002); to determine how the innovation potentially fits into farmers' production goals and resource allocation decision-making; and to gather information needed to decide and ensure that the technology in a notional state is appropriate to farmers' specific needs and their farming conditions (Mercer, 2004). Adoption of transformative agriculture technologies Perennial grains are expected to be a 'radical', or transformative technology, meaning that their architecture, functionality, component principles and underlying science markedly depart from existing agricultural systems. This conception of radical agricultural innovations draws on Rennings et al. (2013). Here we examine the literature on adoption of other transformative technologies, to gain insights on how perennial grains might be adopted. No-till cultivation is another example of a radical agriculture innovation (Coughenour, 2003;Huggins and Reganold, 2008), which fits the context of this study. No-till represents a clear departure from traditional farm practices, which require seasonal tilling of the land multiple times, to prepare for planting, for nutrient release and during the growing season for weed control (Huggins and Reganold, 2008). In contrast, no-till technology involves no or very little soil disturbance and uses equipment and chemistry markedly different from traditional agricultural technologies. Organic farming systems and agroforestry are two other radical agricultural innovations (Morgan and Murdoch, 2000;Vanloqueren and Baret, 2009). Organic agriculture is considered a radically altered system because it is based on principles, practices, knowledge systems, values and perceptions about soil, plants, farmers, and environmental relations which are clearly distinct from conventional agriculture's (Morgan and Murdoch, 2000). Agroforestry has also been so identified because it is based on a farming practice which deliberately incorporates perennial woody plants into cropped fields. Like agroforestry systems, perennial grain systems may demonstrate what Jaikumar et al. (2012) describe as 'delayed reproductive investment' (p. 1716). All of these transformative technologies are widely associated with gains in ecosystem services. Having discussed why we chose adoption studies on notillage, agroforestry and organic farming systems for review, we shall now focus on the drivers of their adoption. Studies have investigated how the awareness of soil problems such as soil erosion impact adoption of radical agricultural technologies. Vitale et al. (2011) andAndrews et al. (2013) found that soil erosion is a significant and positive determinant of farmers' decision to adopt notill conservation technology. However, in a study which examined the factors which determine the use of conservation tillage in Australia, D'Emden et al. (2008) found that the awareness of, and the proneness of soils to erosion was insignificant to the adoption of no-till. Similarly, Bultena and Hoiberg (1983) found that perception of, and awareness of soil erosion problems was not a determinant of Iowan farmers' decision to adopt or not to use conservation tillage practices which included no-till. Their study revealed that, 'early adopters and non-adopters both perceived soil erosion as being less of a problem than did late adopters' (p. 283). The authors reasoned that considerable improvements to the soils of early adopters may help explain their findings. Conversely, in their study of farm forestry adoption in Australia, Race and Curtis (2007) found a positive relationship between landholders' decision to adopt agroforestry and soil erodibility concerns. A similar finding has been reported by Valdivia and Poulos (2009), whose studies examined the determinants of farmers' decisions to use agroforestry farming practices in Missouri, USA. Valdivia and Poulos further found the impact of soil problems and environmental problems to be significant, a finding which is akin to Trozzo et al. (2014). In line with this, McCann et al. (1997) found that farmers with a high awareness and concern for soil erosion are likely to adopt organic farming practices. Overall, most studies have found concern and awareness about soil erosion to be significantly and positively correlated with the adoption of no-till agriculture, organic farming and agroforestry. Attitude toward risk and economic situation of producers have also received attention as explanatory drivers in the technology adoption literature. A study by McCann et al. (1997), found that organic farmers were more disposed to the risks associated with their adoption decisions, by, for example, accepting reduced yields and delayed return on their productive investment for future benefits. The willingness to take related risks has also been associated with adopters of no-till (Bultena and Hoiberg, 1983) and riparian buffer agroforestry practices (Trozzo et al., 2014). While organic producers may be willing to accept greater risk, they also are motivated by the potential for higher profit margins associated with organic products (McCann et al., 1997). In a corresponding finding, Peterson et al. (2012) found that the motivations of organic grain farmers in the USA extend beyond organics as a way of life to include profit maximization. The literature on subsidies paints a different story; studies such as Andrews et al. (2013) have found no statistically significant relationship between the provision of economic incentives for environmental services, carbon storage and the adoption of no-till conservation tillage. This contrasts with the findings of Alavalapati et al. (2004), that incentive payments positively and significantly impact the likelihood of adoption of silvopasture agroforestry practices. However, an adoption synthesis study by Prokopy et al. (2008) found the relationship between profitability, incentive payments and the adoption of no-till to have both negative and positive correlations. In contrast, findings by Vitale et al. (2011) indicated that profitability significantly impacts the adoption of no-till. However, the cost of equipment is found to negatively impact the adoption of no-till (Vitale et al., 2011). Similarly, Andrews et al. (2013) found that reduction in labor and fuel cost help drive the adoption of no-till. Overall, a major implication of these findings is that the adopters of organic farming, no-till and agroforestry are not averse to taking risks for future returns on their investments (including returns to soil quality), and that market-based profitability, rather than government-provided incentives, is more likely to drive the economic aspects of farmers' adoption decisions. The relationship of different agronomic and biophysical factors, such as soil type, soil compaction and drainage, slope, plant residue, and insect and disease problems to the adoption of no-till has also been reported. Soil types and soil compaction negatively affect the adoption of no-till in Oklahoma (Vitale et al., 2011). Likewise, Rahm and Huffman (1984) found that the adoption of no-till by Iowan corn farmers significantly depends on soil characteristics. The likelihood of no-till adoption has been found to be greater on rolling, lighter and welldrained soils (Vitale et al., 2011). A related finding has been reported by Andrews et al. (2013). However, Prokopy et al. (2008) found that higher slopes both negatively and positively correlate with the adoption of conservation tillage practices, including no-till. Finally, residue management limits and negatively affects the adoption of no-till, due to the associated difficulties with soil management, plant establishment and insect infestation problems (Vitale et al., 2011). The relationship between environmental concerns, land stewardship, values and the adoption of transformative agricultural technologies has also been investigated. A study conducted on organic and conventional farmers in three West German states found a statistically significant relationship between farmers' environmental concerns and the adoption of organic agriculture (Best, 2010). In this context, the decision to adopt organic agriculture increases with farmers' environmental concerns and the latter helps in framing farmers' adoption decisions. However, the West German study also found that environmental concerns alone are not enough to predict organic agriculture adoption, and a key factor was if farmers found the decision to go organic 'economically very attractive' (p. 464). A related finding has been reported by Peterson et al. (2012) and also by Lapple and Van Rensburg (2011) and others (Läpple, 2010;Kaufmann et al., 2011), who found that environmental concerns positively correlate with the organic agriculture adoption decision. Likewise, Arbuckle et al. (2009) found a strong positive association between environmental concerns and agroforestry adoption decisions of non-operator landowners in two Missouri watersheds. Valdivia and Poulos (2009) also found a significant relationship between these two variables. Prokopy et al. revealed that environmental concerns exhibit both negative and positive correlation with the no-till adoption decision (Prokopy et al., 2008). This is similar to the finding by Lahmar (2010) that environmental concerns were not pivotal to European farmers' decisions to adopt conservation agriculture, including no-till. Ryan et al. (2003) reported that farmers who are good land stewards and are innately attached to their lands are more likely to adopt no-till, while Andrews et al. (2013) found that those who exhibited strong concerns for soil compaction and soil drainage are likely to adopt soil conservation practices, including no-till. However, Vitale et al. (2011) reported no clear association between awareness of environmental consequences and the adoption of soil best management practices, including no-till. Other factors that might influence a farmer's decision to adopt organic agriculture include placing a high value on the quality of farm products (Kallas et al., 2010) or on soil management (Kaufmann et al., 2011) and soil quality; or concern about the ecological and human health effects of agro-chemical use (McCann et al., 1997;Kaufmann et al., 2011). Kaufmann et al. found that positive perceptions about the impact of organic farming methods on human, wildlife and plant health significantly correlates with organic adoption decisions (Kaufmann et al., 2011). Ryan et al. found that among the major reasons farmers in Midwestern riparian zones adopt woody vegetation cover and no-till are the reduction of agrochemical use, the protection of stream health, and to make their farms appear well-managed (Ryan et al., 2003). Trozzo et al. (2014) as well as Strong and Jacobson (2006) have reported that farmers who adopt agroforestry consider it to have offered beneficial environmental impacts such as water quality enhancement, wildlife preservation and soil protection. Thus far, the discussion has focused on a synthesis of some of the factors that determine the adoption decisions for three transformative agriculture technologies we selected for review. The effects of variables such as farm size, adopters' economic orientation, environmental concerns, land stewardship, values, and agronomic and biophysical factors on adoption decisions appear to be complex. One reason for this may be spatial non-stationarity (Taus et al., 2013), meaning that these variables have differential impacts on different actors. Spatial non-stationarity is a 'condition in which a simple "global" model cannot explain the relationships between some sets of variables' (Brunsdon et al., 1996, p. 281). For example, spatial non-stationarity may explain why different studies have reported different findings as to the effect of farm size on transformative technology adoption. In addition, farmers' decisions to adopt transformative technologies may be informed by several motivations interacting with one another across multiple points in time-that is, famers' decisions are both complex and dynamic, as are the circumstances under which they make decisions. For example, Vitale et al. (2011), Lapple andVan Rensburg (2011) and Best (2010), demonstrated that ideological commitments, economic motives and/or environmental concerns taken individually do not adequately predict the decision to adopt transformative technologies. Finally, differences in methodology may also help explain the complexity of the findings on factors contributing to adoption decisions. Knowler and Bradshaw (2007) observed that studies that use logit or probit models (a common tool in econometric analyses) 'are likely to ascribe greater influence to the variables "education" and "farm size"' (p. 40). D' Emden et al. (2008), studying no-till adoption in Australia, found that while logit regression models returned education as a significant determinant of adoption decision, a duration analysis model based on the same data found otherwise. Despite the indeterminate and occasionally contradictory nature of findings from the literature, there is consistent evidence that economic factors, farmers' values and concern about long-term soil health, farm size and attitudes toward risk all may influence the decision to adopt transformative technologies in different and interacting ways (Table 1). Concern about both soil erosion and environmental health are consistently, positively and significantly associated with adoption of organic agriculture and agroforestry. Concerns about soil erosion seem to drive no-till adoption, although the determinants of no-till adoption are complicated by their association with genetically engineered seed. This association likely explains the positive and negative associations with environmental concern which adopters of this technology display. Health concerns play a significant role in organic adoption decisions. The effects of farm size, economic priorities and risk attitudes on the adoption of transformative technologies appear to be more complex and context-dependent. None of these factors in isolation is likely to be sufficient to explain an adoption decision. Moreover, the fact that many of these explanatory factors (soil quality, farmer attitudes and farm size) change over time, partly in response to adoption decisions, highlights the need to adopt research methods that consider farms as adaptive, dynamic systems. The literature review informed our targeted selection of interviewees and interview questions. Overall, early adopters of transformative technologies appear to be concerned about environmental quality, and willing to take the risk of deferred financial gain in order to achieve soil health and long-term investment goals. We therefore felt it was important to recruit farmers to our study who are already experimenting with alternative technologies, and who are therefore likely candidates for early adoption of perennial grains. In addition, we wanted to keep the interviews relatively unstructured, because the sometimes contradictory nature of the literature on transformative technology adoption reveals the need for farmer-led narratives of how adoption decisions are made. Methods: farmer interviews We recruited farmers through conferences and growers' meetings in Michigan between January and March, 2012. We targeted a range of venues to attract conventional wheat growers, pasture-based livestock farmers, small, alternative and family farm producers. At each meeting, members of the research team set up a table with a poster and some information on the perennial wheat breeding program, and invited interested farmers to learn more about the research and to sign up to be interviewed. We emphasized that the perennial wheat crops were in the developmental phase, and would not be ready for on-farm use in the near future. Each meeting yielded a sign-up sheet with names and contact information of farmers who were interested in being interviewed about how they might use perennial wheat. Interviewees were selected from the sign-up sheets to represent a range of farm operation types (large, small, organic, conventional, pasture-based, grain-based and integrated crop/livestock) ( Table 2). A total of 11 interviews were conducted between March and April, 2012. Interviews typically took approximately 1 h, and were conducted by two or three interviewers. Interviewees were compensated US$5 for their time. The interviews were semi-structured, and were recorded with interviewees' permission (see Appendix A for interview guide). Farmers were asked about the size of their farm, crops grown, inputs used, history of farming, priorities for farm management, and financial/personal goals for their farming operation. They were also asked about their interest in perennial wheat, how they might use it on their farms, what characteristics it would need to have in order for them to be interested in planting it, and what benefits or challenges such a crop might provide. The interviews were transcribed in summer, 2012. All interviews were read over by multiple members of the research team to clarify technical information and resolve uncertainties and inconsistencies. A coding table was codeveloped by several research team members to address the research questions (Appendix B). The interviews were then coded using NVivo software in late summer, 2012, and two coded interviews were checked for accuracy by the lead author. Summary documents were prepared containing all potential uses of perennial wheat The 'significant' and 'not significant' columns refer to the statistical significance of the independent variable (e.g., farm size and environmental concern) for the studies summarized in the table (most studies used multiple regression). mentioned by interviewees, and all perceived or desired characteristics of perennial wheat described by farmers. Results Farmers in our study were clearly motivated by concerns about soil and environmental quality on their farms. Farmers' perception that perennial varieties of wheat would exhibit these traits (soil-improving; environmentally sound; compatible with organic farming) motivated them to approach us to learn more about the crop. For example: I2: What other benefits do you think a crop like this might provide for your farm? F1: I think just its perennial nature. Having its roots there all the time. Again, good for the bacteria and fungus, not disturbing the soil. Getting back to the whole, whole biology of the soil aspect is really important, and that's one thing that I see is a really promising aspect of this. F9: When you are organic you need that soil life a LOT more. And that is a huge thing for me; not tilling the soil is good for soil life too. So I see advantages there. This focus on soil health and stewardship ethic on the part of farmers interested in this potentially transformative technology is consistent with the literature review described above (Soltani et al., 2014). Farmers interested in perennial wheat appeared to exhibit characteristics of farmers who adopt both no-till and organic technologies, given their concern for improving soil health, conserving soil and producing healthy foods (Table 1). One farmer mentioned being influenced by a vision of agriculture as mimicking the biology of 'natural' ecosystems, which has also motivated the development of perennial grains: Farmers suggested a total of 11 potential uses of perennial wheat, only one of which (dual-purpose grain/forage) was mentioned specifically by interviewers (Table 3). Along with these potential uses, they described the reason(s) why a perennial grain might be desirable for that use, and the characteristics it would have to have in order for the farmer to be interested in planting it. High grain yield was considered a desirable characteristic for only one potential use (dual-purpose grain/forage). This is in marked contrast to previous studies of the adoptability of perennial wheat, which have assumed that grain yield is a necessary characteristic farmers look for when deciding to substitute perennial wheat for annual wheat (Bell et al., 2008). Only one group of interviewees, a family of farmers growing annual wheat in a corn-wheatsoybean rotation, stated that the total monetary value of the grain and forage produced from perennial wheat would have to be comparable with the value of annual wheat grain yields before they would consider adoption: F7b: But if you could get it to where the yield is such … that the balance between the yield and selling the green matter is equal to or better than annual wheatthen I think you're right on track. Many of the proposed uses did not involve a substitution of perennial wheat for a cash crop already grown on-farm. Rather, they proposed perennial wheat as a means of solving a problem for which no other crop provided an adequate solution, often by planting perennial wheat on an underused or marginal area of the farm. For example, one farmer suggested that perennial wheat could be planted between his organic bean fields and his conventionally grown neighbor's fields as a buffer strip: F9: … put this wheat in the buffer I would have one planting for multiple years so I wouldn't have to clean my planting equipment … cause right now, we don't grow any cash crops on the buffers. In this case, the farmer was using buffer strips to grow forage for his hired workers' goat, but he saw perennial wheat as a potential means to cultivate both grain and forage on this underutilized piece of land. Farmer responses also reflected a growing trend in direct marketing of farm products to consumers and local intermediaries. Several responses indicated an interest in perennial wheat as a mechanism to provide niche products to consumers interested in healthy, locally produced foods. For example: F8: … have you looked into the nutrient contentyou mentioned not having gluten, are there other … something that's really high end or low end or whatever that will help sell the product? Farmers asked multiple questions about the characteristics of perennial wheat. The function of these questions often appeared to be to 'think out loud' about how to fit the crop into their current farming systems For example, the following dialog occurred around whether perennial grains could be planted with a farmer's current equipment: F1: You said the seed is a little bit smaller than annual wheat? I2: A little bit, yeah. F1: So you plant with a grain drill? Or with a seeder, like you'd use with alfalfa and clover seed? Do you know? I2: I think we used a seed drill… F1: … cause if you have a grain drill with a seeder, it usually can handle anything from the tiny clover to the soybeans. Discussion The fact that farmers listed multiple potential uses of perennial wheat, and discussed multiple criteria which could be satisfied in order for them to adopt the crop, is consistent with the complex nature of adoption decisions described in the literature on transformational technology adoption. Only one interviewee described yields comparable with annual wheat as a prerequisite for adoption of the hypothetical grain, and even in that context the total forage production in addition to the grain yields were considered an asset of a perennial wheat. Grain characteristics and growth characteristics were mentioned by all of the farmers in our interviews. Similar findings are widely reported in the participatory variety selection literature, as grain quality traits and growth habit have frequently emerged in farmer assessments of new crop varieties in a developing country context (Virk et al., 2005). Interestingly, in the USA, participatory plant breeding efforts to develop wheat and other crops for organic production systems have recently been implemented (Kandel et al., 2008), and yield has emerged as a key trait of concern for farmers, but other traits are as important. These have included protein grain content, seedling vigor and disease resistance. Although plant breeding efforts over many decades have addressed multiple simultaneous objectives, these have almost always had yield as a primary focus (Glover et al., 2010). Recent efforts to develop perennial wheat and perennial rye involve crossing of annual wheat and rye with wild relatives and selecting for improved grain yield (Acharya et al., 2004;Murphy et al., 2010). Intermediate wheatgrass is currently being domesticated through selection for high yield (Cox et al., 2006). Related agronomic research has also focused on yield (Jaikumar et al., 2012;Hayes et al., 2012). Disease resistance is another key objective of some perennial grain plant breeders (Cox et al., 2005). There is one report that highlights the characteristics of the grain and grain quality in novel perennial wheat lines (Murphy et al., 2009), and perennial regrowth vigor (Murphy et al., 2010), but there are few reported efforts in these areas. The results of this study imply that breeders may wish to consider developing multiple growth forms of perennial grains to address novel market niches, and that not all of these forms may need to be yield-competitive with annual grains in order for farmers to find them desirable (Table 3). Farmers often adapt technologies for their own particular situations, rather than accepting the technology for its 'intended' use (Adegbola and Gardebroek, 2007). There is no reason to expect that perennial grain adoption will be different from other transformative technologies, based on the evidence from these interviews. A breeding program which seeks to optimize one characteristic of perennial wheat (yield, for example), without referencing the multiple potential uses farmers envision, would therefore miss opportunities for more widespread adoption. The multiple potential uses of perennial wheat also shed light on why the technology adoption literature does not reach consensus on major determinants of transformative adoption. Farmers' decisions about technology adoption are highly contextual, and that context is dynamic. A study conducted as a snapshot of a farmer decision-making process will therefore yield different results across different types of systems and at different points in the adoption trajectory. This was highlighted through the dialogic nature of the interviews, in which farmers considered out loud how perennial grains could fit into their current farming systems, and asked questions about perennial wheat as they 'problem solved'. Some recent studies are incorporating methodologies which can accommodate the complex and dynamic nature of adoption decisions, such as agent-based modeling (Berger, 2001) or system dynamics modeling. Because farmers self-selected into our study, only those who showed interest in adopting a perennial grain were interviewed. This necessarily biased our group of interviewees, as we did not include those who would not consider adopting a perennial wheat because its characteristics did not meet their adoption criteria. For example, no one from the conventional wheat growers' meeting ultimately agreed to be interviewed, although some gave us their contact information. The fact that conventional growers were not particularly interested in a perennial wheat is informative for our study in itself. As our literature review demonstrated, early adopters of radically different technologies in general tend to have (1) high environmental concern; and (2) a willingness to take risks of delayed returns in hopes of achieving long-term financial and soil health gains. While conventional wheat growers certainly display a range of these characteristics, it is possible that early adopters of perennial wheat will come from a smaller pool of farmers who are already experimenting with alternative technologies such as polycultures, rotational grazing and organic grain production. Adoption may spread more broadly once the benefits of growing perennial grains (in terms of soil health and possibly reduced labor) are demonstrated by these early adopters. It is therefore not surprising that our group of farmer interviewees was made up disproportionately of experimental farmers, who have used and adopted other transformational technologies on their farms. This type of farmer is likely to be the early adopter targeted for perennial wheat dissemination, so understanding their decision processes is important. Our interviewee pool was small, however, despite widespread recruiting efforts, so the innovative and experimental group of farmers represented in our interviews likely make up a small minority of all wheat farmers. Conclusions This is a novel ex ante study using interviews to gauge interest in and potential uses of perennial wheat among farmers in Michigan and Ohio. Using a semi-structured interview format allowed us to gain insight into farmer decisionmaking around the adoption of a radical new technology. Consistent with other studies of transformative technology adoption (agroforestry, organic agriculture and no-till), farmers mentioned multiple, interacting factors that would contribute both to their interest in learning more about perennial grains, and to their decision about whether or how to use perennial grains. These factors included interest in supporting production of environmental services on the farm, consumer preference, market availability, timing of crop emergence and regrowth, disease/pest resistance, and providing a new crop suited to marginal lands. In only one incidence was direct substitution for conventional wheat and yield mentioned. We conclude that breeding programs for perennial grains should take this complex decision-making into consideration, potentially by developing multiple products with different economic markets and on-farm uses. Furthermore, studies of perennial grain adoption will mischaracterize both potential adopters and potential uses of the crop if they rely purely on reductionist methods that emphasize one adoption criterion independent of other crop aspects.

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Antimicrobial activity of onion and ginger against two food borne pathogens Escherichia coli and staphylococcus Aureus The purpose of this project was to investigate the antimicrobial effect of phytochemicals extracted from onion and ginger (fresh and boiled) at different concentrations, against Escherichia coli and Staphylococcus aureus using different methods (MIC, MBC, Disk and well diffusion). This project was chosen because onion and ginger are very common spices and have been claimed to contain several antimicrobial agents. Introduction Food borne disease is gradually becoming a serious risk to public health with the number of cases increasing yearly. 1 The cause is determined to be from microorganisms, people become infected by either eating food infected with microbes or intoxicated with enterotoxins produced from microbes. Proper control of microbes and effective sanitation will help to reduce the rate at which people become sick from eating food. Spices in general show both antimicrobial and antioxidant characteristics, inhibiting the growth of bacteria and other pathogenic microbes. 1 Scientists in the past have conducted numerous experiments testing food borne pathogens with phytochemicals extracted from the spices. 2 The antimicrobial properties of active compounds extracted from spices and other plants in general based on recent findings have shown to affect the behavior of pathogenic bacteria and fungi in the agro food and medicinal sector. 1 This study investigates the antimicrobial activity of phytochemicals extracted from onion (Allium cepa) and ginger (Zingiber officinale) against E.coli and S.aureus using; Minimum inhibitory concentrations (MIC), Minimum bactericidal concentrations (MBC), and disc and agar well diffusion. Onion and ginger Onion has been revered throughout time not only for its culinary use, but also for its therapeutic properties. Consumption of onion is beneficial to human health as scientific studies show onion contains polyphenol molecules or phytonutrients which includes flavonoids, tannins as well as allicin which possess antioxidant and antimicrobial properties. 3 The main antimicrobial agent in onion isquercetin and allicin (thio-2-propene-1-sulfinic acid-5-allyl-esters), quercetin binds to the bacteria DNA gyrase while allicin inhibits certain thiol containing enzymes in the microorganisms by the rapid reaction of thiosulfinates. 4 On the other hand, ginger has a long tradition of being very effective in alleviating symptoms of gastrointestinal distress. 3 Ginger can also be used as herbal medicine, and modern scientific research has revealed that ginger possesses numerous therapeutic properties that are similar to onion, including antioxidant effects, an ability to inhibit the formation of inflammatory compounds, and direct antiinflammatory effects. 1 The main antimicrobial agent is gingerol a naturally occurring phenol which disrupts the cell wall of bacteria causing cytoplasmic leakage. 5 Mininum inhibitory concentration and mininum bacteriacidal concentration Minimum inhibitory concentrations (MIC) is the lowest concentration of any antibacterial substance that prevents or inhibits the growth of a particular pathogen after an overnight incubation period while minimum bacterial concentration (MBC) is the lowest antibacterial concentration that kills the pathogen after an overnight incubation period of about 24-48hours. 6 Minimum inhibitory concentrations (MIC) are regarded as the standard for determining the susceptibility of organisms to antimicrobials. 6 Disc and agar well diffusion The principle behind disc diffusion is relatively simple and easy to carry out, it involves an antibacterial impregnated disk placed on an agar previously inoculated with the test bacteria, when the disc picks up moisture the antibiotic diffuses outward through the agar producing an antibiotic concentration which produces zones of inhibition, the more susceptible the bacteria is to the antibiotic the greater the size of the zones. 7 The principle behind the agar well diffusion is similar to that of disc diffusion, wells uare bored into in the plate previously inoculated with the test bacteria and even volumes of the various antimicrobial agents are put into the wells which diffuse onto the agar and produces zones of inhibition. 7 Escherichia coli Escherichia coli is a Gram-negative, straight rod bacteria of about 1.1-1.5µm by 2.0-6.0µm occurring either singly or in pairs. They belong to the class Proteobacteria, the order Enterobacteriales, the family Enterobacteriaceae and the genus Escherichia. 7 They are motile by peritrichous flagella and facultatively anaerobic having both a respiratory and a fermentative type of metabolism (Bergey, 2000). Biochemically they are oxidase negative, catalase positive, methyl red positive, Voges-Proskauer negative and usually citrate negative. 8 E.coli occurs as normal flora in the lower part of the intestine, optimal growth for is about 37degrees but they are known to grow at temperatures of about 44.5degrees. 9 E.coli strains containing enterotoxins are known to cause diarrheal diseases due to their invasive and colonization characteristics, they are also a major cause of urinary tract infections and nosocomial infections including septicemia and meningitis. 9 When plated on differential media such as FC agar and Eosin methylene blue agar at 44.5 and 35degrees respectively for about 48hours they produce dark green metallic sheen colonies, when plated on MaCconkey agar at 35degrees they give pink colonies due to the fermentation of lactose. 10 E.coli was used for this study because it is a bacterial food borne pathogen responsible for infecting hundreds of people each year. Staphylococcus aureus Staphylococcus aureus is a gram-positive cocci bacterium that is a member of the Firmicutes group. It belongs to the class Cocci, order Bacillales, family Staphylococcaceae and genus Staphylococcus. 7 The cells are spherical about 0.5-1.5µm in diameter and in clusters or pairs. They are non-motile non-sporing facultatively anaerobic with both respiratory and fermentative mechanisms. 8 Colonies are usually white and cream on general purpose agar such as TSA or yellow on differential media such as MSA. Biochemically they are catalase positive and can grow in about 10% NaCl with an optimum temperature of about 30-37degrees. 9 They are mainly associated with the skin and mucus membrane of warm blooded animal and serves as an indicator of nasal contamination in foods. They are known as opportunistic pathogen in humans causing diarrhea by producing extracellular toxins. 9 S.aureus is an important food borne pathogen causing several incidents of food poisoning which leads to diarrhea. Media MacConkey agar: MacConkey Agar is used for the detection and enumeration of gram negative bacteria based on their ability to ferment lactose. Bile salts and crystal violent serves as the selective agent while phenol red is the indictor. E.coli a lactose fermenter grows extremely well on MAC producing pink colonies with red zones. 10 MR-VP BROTH: MR-VP Medium and MR-VP Broth (Methyl Red-Voges Proskauer Medium/Broth, are used for the differentiation of bacteria by means of the methyl red and Voges-Proskauer reactions. 11 Simmons citrate agar: Simmons Citrate Agar is used from differentiating bacteria which can utilize citrate as the sole source of carbon and inorganic ammonium salt as the sole source of Escherichia coli either do not grow at all on this medium, or grow so sparsely that no change in reaction is apparent as the media still remains green. Sodium Citrate is the sole source of carbon in this medium. Magnesium Sulphate is a cofactor for a variety of metabolic reactions. Bromthymol Blue is the pH indicator. 12 Mannitol salt agar: Mannitol Salt Agar is highly selective and used to isolate coagulase positive Staphylococcus aureus by inhibiting growth of most other bacteria with a high salt concentration of about 7.5% Sodium Chloride. Bacteria that grow in the presence of a high salt concentration and ferment mannitol to produce acid products, turning the Phenol Red pH indicator from red to yellow. Staphylococcus aureus ferment mannitol and form yellow colonies with yellow zones. (Neogen 2015). Mueller hinton broth and agar: Mueller Hinton Broth and Agar are used for performing antibiotic susceptibility tests (MIC, MBC) using a single disk of high concentration. The medium provides excellent growth for most non-fastidious pathogens due to its low concentration of sulphonamide. 13 Tryptic Soy Broth: Tryptic Soy Broth is a general purpose medium. Tryptic Soy Broth is used for the preparation of inoculum used in antibiotic tests. Casein and Soybean Meal serves as the source of nitrogen in TSB. Dextrose is the carbon source which enables the organism to grow. Osmotic balance and buffering agent are maintained by Sodium Chloride and Dipotassium Phosphate. Most clinical tested microbes grow extremely on TSB. 14 Recent research conducted on the antimicrobial effect of ginger and onion showed weak to medium antibacterial properties. 1 The author used agar well diffusion to determine the zones of inhibition. Also results according to Onyeagba et al. 15 also showed both ginger and onion to have significant antimicrobial properties. Sample collection 100grams of onion and 100grams of ginger were purchased from Food basic store along Neilson road. Restreaking on selective media A colony of E.coli obtained from the prep room was streaked on a MaCconkey agar plate and incubated at 35degrees for 48hours; the same was done with a colony of S.aureus on an MSA plate and incubated at 35degrees for 48hours. Restreaking on selective was carried out regularly at the beginning of every week in other to have a fresh supply of bacterial colonies needed for the experiment. It was used for bacteriological analysis and biochemical characterization. Morphological Characterization The isolated microbes from both Mac cultures and MSA cultures were characterised morphologically on the basis of simple gram staining. Biochemical characterization The isolates were characterised by biochemical tests using citrate utilization test, Methyl red test and Voges Proskauer test for E.coli and catalase and coagulase test for S.aureus, this was carried out in order to determine purity of tested micro-organism. A single colony of E.coli was inoculated on citrate agar slant and MR-VP broth for 48hours and incubated at 35degrees after which two drops of MR reagent and VP reagents were added. Also, a colony of S.aureus was placed on a microscope slide and three drops of hydrogen peroxide was added onto it and viewed for the formation of gas bubbles which indicates the presence of catalase. Coagulase test was also carried out on the same colony of S.aureus, which was inoculated and incubated for 48hours at 35degrees. Negative controls were also carried out by incubating the media used without any inoculate for 48hours at 35degrees. Preparation of Inoculate Isolated colonies of E.coli and S.aureus from the Mac and MSA cultures were inoculated in a test tube containing 9ml of TSB and incubated at 35degrees for 48hours. The overnight cultures obtained from the tubes were used to determine the minimum inhibitory concentration of onion and ginger extracts. Preparation of onion and ginger extracts The onion and ginger obtained, was cleaned and washed using sterile distilled water. Using a disinfected knife and chopping board the samples were cut into smaller pieces, crushed in a mortar using a pestle and liquefied in a blender. The extracts were then sieved through a sterile cheese cloth into a sterile Erlenmeyer flask; this extract was considered as the 100% raw extract. The same procedure was repeated and the extract was boiled in a sterile conical flask for 5minutes, this was thus considered as the 100% boiled extract. Determination of Minimum Inhibitory Concentration (MIC) 0ml, 1ml, 2ml, 3ml, 4ml, and 5ml of Mueller Hilton broth was pipetted into different sterile test tubes. After which 10ml, 9ml, 8ml.7ml, 6ml and 5ml of each extract was added into the corresponding tubes containing 0ml, 1ml, 2ml, 3ml, 4ml and 5ml of Mueller Hinton broth. This represented 100%, 90%, 80%, 70%, 60% and 50% concentration of each extract. Using a sterile Pasteur pipette 2drops of each culture was added into each tube respectively. This was done for both the raw and boiled extract for both onion and ginger for each organism and thus a total of 48 tubes was incubated at 35degrees for 48hours and observed for growth by turbidity. Negative controls were also set up using 5ml of both the raw and boiled extract for onion and ginger and incubated at 35degrees for 48hours. Media controls were also set up using 10ml of Mueller Hinton broth and incubated at 35degrees for 48hours. The purpose of the control was to determine the presence of growth and no growth, to determine if the media was contaminated and to eliminate false positives. Determination of Minimum Bactericidal Concentration (MBC) A loop-full from each tube containing the different concentrations of both onion and ginger extract with the respective microbes was plated onto a plate containing Mueller Hilton agar. Each tube was plated onto the same plate with different partitions indicating concentration. In all a total of 8 plates one for each microbe and spice (boiled and raw) was incubated for 48 hours at 35 degrees and observed for growth on the surface of the agar. Negative controls were also done by incubating an empty plate of Mueller Hinton agar for 48 hours at 35 degrees. This was done to check sterility of media. Disc diffusion and agar well diffusion Each tube containing overnight cultures of E.coli and S.aureus were diluted in phosphate buffer to a concentration of 2.5Mcfarland. After which 0.1ml of each culture was spread across the surface of the plates containing MH agar. 24sterile discs were used for each organism and each spice which was impregnated in various concentrations ranging from 100-50% and placed on the surface of the agar which was incubated for 35degrees at 48hours and examined for zones of inhibition. Inhibition zones less than or equal to 7mm were considered to have no antimicrobial effect. Positive controls were also set up using ampicillin and ciprofloxacin disc which was placed onto the surface of the agar and incubated for 48hours at 35degrees. Media controls were also set up by incubating a plate of MH agar for 48hours at 35degrees. Agar well diffusion followed the same principle as disc diffusion the only difference been well were bored in the agar already inoculated with the test microbes and different concentrations of spice extracts were placed into the wells which was incubated for 48hours and 35degrees. Biochemical characterisation The following tables show the biochemical characterisation of the two isolates used. Simmon citrate agar and MR-VR broth were used for E.coli, inoculated and were incubated for 48hours at 35degrees, while the coagulase test was used for S.aureus and was also incubated for 48hours at 35degrees. The catalase test was also carried out on S.aureus (Table 1). Citrate utilization test showed E.coli does not utilize citrate due to the media remaining unchanged this is a typical E.coli reaction. When the MR reagent was added a bright red color appeared this is regarded as a positive result while when the VP reaction was added there was no change, this is regarded as a negative result which is typical to E.coli (Table 2). Coagulase test was positive due to clot which showed S.aureus produces coagulase and the presence of gas bubbles also indicated that the catalase test was positive. Morphological Characterisation The following table shows the morphological characterisation and gram reaction of E.coli and S.aureus (Table 3). Morphological characterisation of E.coli was characterised by the presence of red rod shaped cells in random order and purple cocci shaped cells in clusters for S.aureus with indicates a typical morphological characterisation for both cells. Minimum inhibitory concentration The following tables show the result for the Minimum Inhibitory Concentration of both the raw and boiled extract of both species which was diluted in Mueller Hinton broth to get specific concentrations, inoculated with overnight culture of E.coli and S.aureus and incubated for 48hours at 35degrees (Table 4). Due to the nature of the extracts the negative controls were used to determine the presence and absence of growth by comparing it with the inoculated tubes. The Negative controls showed no growth in them ( Table 5). The presence of growth was identified by the turbidity of the tubes and the presence of a whitish pellicle on the surface of the tubes. Also the presence of no growth was identified by the similarity of the tubes to the negative controls ( Table 6). The presence of growth was identified by the turbidity of the tubes and the presence of a whitish pellicle on the surface of the tubes. Also the presence of no growth was identified by the similarity of the tubes to the negative controls (Table 7). Due to the nature of the extracts the negative control were used to determine the presence and absence of growth by comparing it with the inoculated tubes. The Negative controls showed no growth in them ( Table 8). The presence of growth was identified by the turbidity of the tubes and the presence of a whitish pellicle on the surface of the tubes. Also the presence of no growth was identified by the similarity of the tubes to the negative controls ( Table 9). The presence of growth was identified by the turbidity of the tubes and the presence of a whitish pellicle on the surface of the tubes. Also the presence of no growth was identified by the similarity of the tubes to the negative controls. Minimum bactericidal concentration The following tables show the result for the minimum bactericidal concentration of all tubes inoculated, a loopful was transferred unto partitioned sections on a plate of Mueller Hilton agar and incubated for 35degrees at 48hours (Table 10). MBC results showed that 80% concentration of raw onion was capable of killing the E.coli cells. The ginger extract on the other hand was not capable of killing the cells (Table 11). MBC results showed that 70% concentration of raw onion was capable of killing the S.aureus cells. The ginger extract on the other hand was not capable of killing the cells. MBC for boiled onion and ginger extract (Table 12). MBC results showed both the onion and ginger extract was not capable of killing the cells (Table 13). Disc diffusion The following tables shows the results for the antimicrobial activity of the spice extract against E.coli and S.aureus measured as the diameter of growth inhibition using impregnated discs with different concentration of the spice extracts which were diluted in different volumes of distilled sterile water. Positive controls were also carried out using ampicillin and ciprofloxacin disc on inoculated plates of E.coli and S.aureus incubated for 48 hours at 35 degrees (Table 14) & (Table 15). The result showed onion to have good antimicrobial properties S.aureus but also showed ginger having no antimicrobial properties against S.aureus (Table 16). The result showed boiled spice extract to have no antimicrobial properties against both organisms (Table 17). Agar well diffusion The following tables shows the results for the antimicrobial activity of the spice extract against E.coli and S.aureus measured as the diameter of growth inhibition from the wells bored in the agar, each well contained a different concentrations of spice extracts. The plates were incubated for 48hours at 35degrees. The diameter of the wells were 7mm (Table 18). The zones of inhibition gotten above showed both onion and ginger to have no antimicrobial activity on E.coli (Table 19). The result showed onion to have fairly good antimicrobial properties S.aureus but also showed ginger having no antimicrobial properties against S.aureus. Table 4 Negative controls of raw extracts from onion and ginger which was incubated at 35degrees for 48hours Controls Onion Ginger Description Slightly clear with particles settled at the bottom Yellowish slightly clear solution with particles settled at the bottom. Observation of Tubes Whitish pellicle on the surface of the tubes. Whitish pellicle on the surface of the tubes. Whitish pellicle on the surface of the tubes. Whitish pellicle on the surface of the tubes. Whitish pellicle on the surface of the tubes. Whitish pellicle on the surface of the tubes. Whitish pellicle on the surface of the tubes. Whitish pellicle on the surface of the tubes. Whitish pellicle on the surface of the tubes Whitish pellicle on the surface of the tubes. Table 7 Negative controls of boiled extracts from onion and ginger which was incubated at 35degrees for 48hours Controls Onion Ginger Description Slightly clear with particles settled at the bottom Yellowish slightly clear solution with particles settled at the bottom. The diameter of the disc was 7mm. The zones of inhibition gotten above showed both onion and ginger to have no antimicrobial activity on E. coli. Discussion Natural products including spices have been used for years because of their therapeutic and antimicrobial properties. The purpose of this project was to investigate the antimicrobial properties of two of such spices, onion and ginger. The experiment was conducted in two stages, the preliminary and final stage, the result for the former were not recorded in the previous chapter due to irregularities and numerous errors resulting from improper aseptic technique during the extraction of the spices which lead to cross contamination and unreasonable results. The result of this experiment showed Staphylococcus aureus to be more susceptible to Escherichia coli, which makes E.coli more resistant to the spice extracts, thus confirming the first hypothesis stated earlier. Results were obtained using three techniques (MIC,MBC, Agar diffusion) based on this ginger showed little to no antimicrobial activity against either organism particularly E.coli, this was similar to the results gotten by Serthi et al. 1 Based on the results, the raw onion extract showed a greater antimicrobial activity against S.aureus with an MBC concentration of 70% v/v and an inhibition zone of 28mm been the largest. The activity on E.coli was different as no zones of inhibition were gotten which was similar to results gotten by Onyeaba et al. 15 90% v/v concentration was shown to kill E.coli, this was unusual as the same concentration showed no zone of inhibition when re-evaluated using disc diffusion. This could be as a result of two possible lab errors, either the bacterial lawn was too thick which rendered the extract inactive or no cells were transferred from the MIC tube to the MBC plates. In addition, the boiled spice extract showed no antimicrobial activity against either organism thus confirming the second hypothesis. This could be as a result of the high temperature which must have deactivated the active agents rendering them ineffective against both bacteria. Due to the nature of the spice extracts, the broth dilution technique was not ideal in evaluating MIC as it was difficult to identify the presence and absence of growth, this was shown extensively during the preliminary stage. As such other techniques such as agar dilution and Epislometer test should be used when dealing with the same or similar spices. It is also important to note that the experiment was affected by various factors which include concentration of spice extracts, volume of agar, and concentration of culture and incubation times. Future studies should be conducted in a more controlled environment were these factors are constant. Also future research should observe if the phytochemicals present in spices showed synergetic or antagonistic properties against each other. In conclusion, each particularly phytochemical should be extracted, purified and tested individually for its antimicrobial properties.

v3-fos

2017-03-31T01:47:46.655Z

2015-08-01T00:00:00.000Z

89874

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Single Nucleotide Polymorphism in the Coding Region of Bovine Chemerin Gene and Their Associations with Carcass Traits in Japanese Black Cattle Chemerin, highly expressed in adipose and liver tissues, regulates glucose and lipid metabolism and immunity in these tissues in ruminants and mice. Our previous reports showed that chemerin is involved in adipogenesis and lipid metabolism in adipose tissue as an adipokine. The aim of the present study was to identify single nucleotide polymorphisms (SNPs) in the coding region of the chemerin gene and to analyze their effects on carcass traits and intramuscular fatty acid compositions in Japanese Black cattle. The SNPs in the bovine chemerin gene were detected in 232 Japanese Black steers (n = 161) and heifers (n = 71) using DNA sequencing. The results revealed five novel silent mutations: NM_001046020: c.12A>G (4aa), c.165GT (92aa), c.321 A>G (107aa), and c.396C>T (132aa). There was no association between 4 of the SNPs (c.12A>G [4aa], c.165GG [107aa], and c.396C>T) and carcass traits or intramuscular fatty acid compositions. Regarding the remaining SNP, c.276C>T, we found that cattle with genotype CC had a higher beef marbling score than that of cattle with genotype CT, whereas cattle with genotype CT had a higher body condition score (p<0.10). Further, cattle with genotype CC had significantly higher C18:0 content in their intramuscular fat tissue than that of cattle with genotype CT (p<0.05). On the other hand, cattle with genotype CT had significantly higher C14:0 and C16:0 content in their intramuscular fat tissue (p<0.05). Moreover, the number of individuals carrying the minor allele of c.276C>T SNP is small. It is suggested that the c.276C>T SNP of the chemerin gene has potential in cattle breeding using modern methods, such as marker assisted selection. So, further functional and physiological research elucidating the impact of the chemerin gene on bovine lipid metabolism including fatty acid synthesis will help in understanding these results. INTRODUCTION Chemerin, also known as retinoic acid receptor responder protein 2 or tazarotene-induced gene 2, plays a regulatory role not only in the immune system but also in energy metabolism. Chemerin is a secreted protein whose gene expression is observed in several tissues, especially in adipose tissue and the liver (Roh et al., 2007), which are essential organs for energy metabolism. Recent studies in mice and humans indicate that chemerin regulates glucose and lipid metabolism (Goralski et al., 2007;Sell et al., 2010) and is involved in metabolic disorders such as obesity, insulin resistance in skeletal muscle tissue, and nonalcoholic fatty liver disease (Bozaoglu et al., 2007;Sell et al., 2009). In our previous studies, chemerin was upregulated during bovine adipocyte differentiation, and chemerin analog regulated insulin secretion related to glucose metabolism and the release of triglycerides in sheep in vivo (Roh et al., 2006;Song et al., 2010;. In addition, in fully differentiated adipocytes, the treatment of tumor necrosis factor (TNF)-α and adiponectin up-regulated both chemerin and chemokine-like receptor 1 (CMKLR1) gene expression levels, although leptin did not show such effects . These results suggest that the expression of chemerin in bovine adipocytes might be regulated by chemerin itself and other adipokines, which indicates the possible role of chemerin in modulating the adipokine secretions in adipose tissues. Japanese Black cattle are well known for their highly marbled beef and greater content of unsaturated fatty acids and they seem well adapted to accumulate oleic acid in their adipose tissue (Smith et al., 2006). Marbling abundance is an important component of the beef carcass quality grade, and a high concentration of oleic acid contributes to palatability and softness of beef (Yang et al., 1999). These traits are influenced by a large number of genes, and many genetic factors related to these traits have been investigated. The bovine chemerin gene is located on chromosome 4 (NCBI reference sequence: NM_001046020.2) and consists of six exons, with exons 2, 3, 4, and 5 coding a protein with 162 amino acids. Single nucleotide polymorphism (SNP) A868G (c.12 A>G), located in exon 2 of the Bos taurus chemerin gene, was detected in six Chinese cattle breeds (Tian et al., 2011). A868G was a synonymous mutation, and it affected some carcass traits in Qinchuan heifers. Chemerin expression in subcutaneous adipose tissue from cross-bred heifers was correlated with body condition score (BCS) and yearling weight (Lindholm-Perry et al., 2012). In this paper we report the discovery of new SNPs in the coding region of the bovine chemerin gene and attempt to show their significance for carcass traits and intramuscular fatty acids composition in Japanese Black cattle. Animals and data collection Japanese Black cattle (161 steers and 71 heifers) were raised and slaughtered in Miyagi Prefecture, Japan, and used to analyze the chemerin allelic frequencies and for the association study. The average slaughtering age and carcass weight (CW) of the cattle were 30.7±2.2 months and 459.9±67.3 kg, respectively. A cross-sectional portion of the intramuscular adipose tissue was collected from longissimus thoracis muscle at the 6th to 7th thoracic vertebrae, and a portion of skeletal muscle tissue was sampled from Japanese Black carcasses. The adipose and muscle tissues were stored at -20°C until analyses of fatty acid composition and genomic DNA extraction, respectively. DNA extraction Genomic DNA was extracted from approximately 50 mg of muscle tissue using DNA extraction apparatus QG-800 (Fujifilm, Tokyo, Japan) and QuickGene DNA tissue kit S (Fujifilm, Japan). The DNA concentration was measured using a NanoDrop ND-1000 Spectrophotometer V3.7.1 (Thermo Fisher Scientific, Wilmington, MA, USA). Single nucleotide polymorphism identification and genotyping According to the sequence of the bovine chemerin gene (GenBank accession no. NM_001046020), two pairs of primers were designed to amplify a coding region of the chemerin gene that included exons 2 to 4. The gene primer sequences were: E1-forward: E2-forward: 5'-CTGCAGGATAGTTCTGACTTTTG-3', E2-reverse: 5'-GCTTTATTAGCTCAGGGGTCA-3'. Polymerase chain reaction (PCR) amplifications were performed in 30-μL reaction mixtures, each containing 10 to 30 ng DNA template, 5 μM of each primer, 3 μL 10×PCR Buffer for KOD-Plus-, 0.20 mM dNTPs, 1.67 mM MgSO 4 , and 0.5 U KOD-Plus-Polymerase (Toyobo, Osaka, Japan). The PCR protocol was 94°C for 9 min followed by 35 cycles of 30 s at 94°C, 30 s at 63°C or 60°C, and 1 min at 72°C and a final extension at 72°C for 5 min. Next, the PCR products were electrophoresed through 1.5% agarose gel and single band was confirmed. Then, the products were purified using Fast Gel/PCR Extraction Kit (Nippon Genetics Co, Ltd, Tokyo, Japan). Finally, the PCR products were sequenced to detect the presence of SNPs. Bi-directional DNA sequencing of PCR amplicons was carried out using an ABI PRISM 3730 sequencer (Applied Biosystems, Foster, CA, USA). Sequencing variants were detected by visual examination of the sequencing map followed by alignment using CLUSTAL (Higgins and Sharp, 1988). Fatty acids composition To analyze fatty acid compositions, we performed gas chromatography analysis as described in a previous report (Yokota et al., 2012). Briefly, total lipid was extracted by adding 1 mL of n-hexane to approximately 10 mg of intramuscular adipose tissue of longissimus thoracis muscle. The mixture was vortexed and incubated at room temperature for 30 min. Two hundred microliters of 2 N sodium hydrate/methanol solution (1:1 v/v) and 600 μL of hydrogen chloride-methanol reagent (5% to 10%) were added to saponify and methylate the mixture (Tokyo Chemical Industry, Tokyo, Japan). The methylated lipid was concentrated by flowing in N 2 gas. The concentrated lipid was then diluted with 500 μL of hexane before proceeding to GC analysis. The fatty acid was analyzed by gas chromatography (6850 Network GC System; Agilent Technologies, Palo Alto, CA, USA). The inlet temperature was 230°C, and the oven temperature was programmed to gradually increase from 100°C to 170°C at 10°C/min and from 170°C to 224°C at 3°C/min. The detector sensor temperature was set at 230°C. We used helium as the carrier gas and a capillary column (DB-23, 0.25 mm ID×30 m, df = 0.25 μm; Agilent Technologies, USA) and a flame ionization detector for detection. The data for fatty acid composition comprised percentages of C14:0 (myristic acid), C16:0 (palmitic acid), C16:1 (palmitoleic acid), C18:0 (stearic acid), C18:1 (oleic acid), C18:2 (linoleic acid), saturated fatty acid (SFA) (C14:0+C16:0+C18:0), and monounsatureated fatty acid (MUFA) (C16:1 + C18:1). Statistical analysis Gene frequencies were determined by direct counting. The effects of chemerin genotypes on carcass traits and intramuscular fatty acid compositions in Japanese Black cattle were analyzed by analysis of variance using the general linear model procedure of SAS (SAS Institute, Cary, NC, USA). The statistical model included fixed effects (sex, sampling year, and genetic information of the chemerin gene) and the covariate effect of slaughter age. Differences were considered significant at p<0.05, and differences at p<0.10 were considered to indicate a tendency. The statistical model of the genotype of c.276 of the chemerin gene was as follows: y ijk = μ+sex j +year k +bx ijk +Chemerin12+e ijk or y ijk = μ+sex j +year k +bx ijk +Chemerin276+e ijk or y ijk = μ+sex j +year k +bx ijk +Chemerin321+e ijk y ijk : the trait measured on the individual i μ: the overall mean for the trait sex j : the fixed effect of sex j (2 classes: male and female) year k : the fixed effect of sampling year k (3 classes: years 2009, 2010, and 2011) b: the covariate coefficient with x ijk x ijk : the random effect of slaughter age ijk Chemerin276: the fixed effect of the c.276 genotype in the chemerin gene (2 classes: CC and CT) e ijk : the residual effect Single nucleotide polymorphisms identified and genotypes Eight hundred fifteen and 823 bp fragments of the chemerin gene were amplified and sequenced with two different primers, respectively. As a result, five silent mutations, c.12A>G (4aa), c.165G>A (55aa), c.276C>T (92aa), c.321A>G (107aa), and c.396C>T (132aa), were detected within the coding region of the chemerin gene ( Figure 1). Table 1 shows the genotype distribution for each SNP site. The highest frequencies at c.12A>G, c.165G>A, c.276C>T, c.321A>G, and c.396C>T were for genotypes AA, GG, CC, AA, and CC, respectively. Most of the alleles at c.276 were allele C, and frequency of allele T was low. Because the frequency of genotypes with the minor allele T was very low (<0.03), we analyzed the effects of individual SNPs only. Single nucleotide polymorphism and carcass trait associations In Table 2, the genotypes of 232 individuals were compared with the phenotypic data for 7 carcass traits and 6 intramuscular fatty acid compositions. The carcasses were evaluated by official graders according to the guidelines of the Japanese Meat Grading Association. The recorded traits, including carcass weight, rib eye area, rib thickness, subcutaneous fat thickness, and yield estimate, were measured or calculated. There were no associations between 4 of the SNPs (c.12A>G [4aa], c.165G<A [55aa], c.321 A>G [107aa], and c.396C>T [132aa]) and carcass traits or intramuscular fatty acid compositions. In the case of the SNP marker of c.276C>T in the exon 3 region, however, cattle with the genotype CC tended to have a higher beef marbling score (BMS), and cattle with genotype CT tended to have a higher BCS (p<0.10) ( Table 2). In addition, cattle with genotype CC had a significantly higher percentage of C18:1 (p<0.05) and tended to have a higher percentage of MUFA (p<0.10) ( Table 2). In contrast, cattle with genotype CT had significantly higher percentages of C14:0 and C16:0 (p<0.05) ( Table 2). These findings indicate that a larger proportion of these two saturated fatty acids led to the high proportion of SFA in cattle with genotype CT (p<0.10). These results suggested that the c.276C>T SNP of the chemerin gene may affect carcass and meat quality traits in Japanese Black cattle. DISCUSSION Our study is the first to demonstrate that the polymorphic loci identified within the bovine chemerin gene might be associated with marbling score and intramuscular fatty acid compositions in Japanese Black cattle. Previously, several genetic variations of the fatty acid synthase, stearoyl-CoA desaturase, and GH (growth hormone) genes associated with bovine carcass traits had been reported (Taniguchi et al., 2004;Abe et al., 2009;Matsuhashi et al., 2011;Yokota et al., 2012). Our previous studies showed that SNPs in the GH gene of Japanese Black cattle were associated with some carcass traits, fatty acid composition, the expression of lipogenic genes, and the regulation of plasma insulin level in Japanese Black heifers (Ardiyanti et al., 2009;Ardiyanti et al., 2012). The c.12 A>G SNP that we detected in the coding region of the chemerin gene in Japanese Black cattle was identical to the A868G SNP detected in six Chinese cattle breeds (Tian et al., 2011). In agreement with their report, genotype AG of this SNP was the dominant genotype in Japanese Black cattle. However, there was no association between carcass traits and AA/AG/GG genotypes in the Japanese Black cattle used in our study. Thus, these results suggest that the effect of the c.12A>G SNP might differ among breeds. A human study revealed that the rs17173608 SNP located on intron 3 of the chemerin gene was associated with visceral adipose tissue mass (Mussig et al., 2009). Another report showed that this SNP increased the risk of metabolic syndrome in an Iranian population (Hashemi et al., 2012). These results suggest that chemerin gene polymorphisms located in noncoding regions could affect the regional fat deposition in humans. Intramuscular fat deposition (marbling) measured as BMS is one of the economically important traits in Japanese Black cattle, with a high level of BMS associated with higher intramuscular fat deposition. To date, there are no reports on the direct mechanism underlying chemerin's regulation of the intramuscular fat deposition of cattle, to the best of our knowledge. However, increasing lipid content induced higher chemerin gene expression in bovine differentiated adipocytes (Song et al., 2010). The difference in fatty acid composition between genotypes CC and CT of the c.276C>T SNP suggests that this SNP might affect the differentiation of adipocytes and intramuscular fatty acid compositions by changing the expression of chemerin. The 'silent' polymorphism could change the substrate specificity by altering its conformations (Kimchi-Sarfaty et al., 2007). Moreover, exonic cis-elements are important for correct splice-site identification and are distinct from the classical splicing signals (Cartegni et al., 2002). Chemerin regulates the differentiation of adipocytes and myoblast cells (Yang et al., 2012). Therefore, we need to evaluate the effects of c.276C>T SNP on chemerin gene expression and reveal the mechanisms that affect intramuscular fatty acid compositions. In this study, we found 5 SNPs in the coding region of the bovine chemerin gene in Japanese Black cattle, and the c.276C>T SNP was associated with marbling and fatty acids compositions. However, further studies are required to elucidate the biological relevance and mechanism by which the chemerin gene polymorphism affects these traits in Japanese Black cattle. ACKNOWLEDGMENTS This study was partly supported by a grant-in-aid from JSPS.

v3-fos

2022-11-19T15:24:00.793Z

2015-10-30T00:00:00.000Z

253640654

s2

Genetic variation and inheritance of phytosterol and oil content in a doubled haploid population derived from the winter oilseed rape Sansibar × Oase cross Identification of QTL for phytosterol content, oil content, fatty acids content, protein content of defatted meal, and seed weight by multiple interval mapping in aBrassica napusDH population. Phytosterols are minor seed constituents in oilseed rape which have recently drawn wide-interest from the food and nutrition industry due to their health benefit in lowering LDL cholesterol in humans. To understand the genetic basis of phytosterol content and its relationship with other seed quality traits in oilseed rape, QTL mapping was performed in a segregating DH population derived from the cross of two winter oilseed rape varieties, Sansibar and Oase, termed SODH population. Both parental lines are of canola quality which differ in phytosterol and oil content in seed. A genetic map was constructed for SODH population based on a total of 1638 markers organized in 23 linkage groups and covering a map length of 2350 cM with a mean marker interval of 2.0 cM. The SODH population and the parental lines were cultivated at six environments in Europe and were phenotyped for phytosterol content, oil content, fatty acids content, protein content of the defatted meal, and seed weight. Multiple interval mapping identified between one and six QTL for nine phytosterol traits, between two and six QTL for four fatty acids, five QTL for oil content, four QTL for protein content of defatted meal, and three QTL for seed weight. Colocalizations of QTL for different traits were more frequently observed than individual isolated QTL. Major QTL (R2 ≥ 25 %) were all located in the A genome, and the possible candidate genes were investigated by physical localization of the QTL to the reference genome sequence of Brassica rapa. Introduction Oilseed rape (Brassica napus L.; genome AACC, 2n = 38) is the world's third-leading source of vegetable oil for human nutrition and industrial products. Almost all of the oilseed rape cultivation is "double low" or "canola" quality" with low content of erucic acid in the oil and glucosinolates in the seeds (Friedt and Snowdon 2010). While oilseed rape breeding has achieved remarkable success over the past few decades, there is still much to learn about the genes regulating seed oil content and quality traits. Increasing concerns about rapid population growth, demands for improved nutritional oil, and expansion of biofuel production have also led to the call for further enhancement in quantity and quality of seed oil. In the case of a complex trait like seed oil content, the number of QTL as reported by numerous studies varied between 3 and 27 QTL and were found distributed among 17 of the 19 chromosomes in B. napus (Rahman et al. 2013). In addition, these QTL individually explained between 2 and 10 % of the phenotypic variance while the additive effect ranged from 0.2 % to more than 1.0 % (Rahman et al. 2013). Therefore, increasing oil content through breeding would have to rely on progressive stacking of positive alleles. Besides considering the environmental influence on the QTL, identifying the underlying candidate genes as well as recognizing the pleiotropic effect or correlation between traits would greatly increase the efficiency of breeding. Several studies have shown that oil content is influenced by the fatty acid composition or vice versa (Ecke et al. 1995;Möllers and Schierholt 2002;Hobbs et al. 2004;Zheng et al. 2008). Since triacylglycerols constitute about 90 % of the oil, the fatty acid composition which represents the overall composition of the triacylglycerols is an important quality parameter determining the value and suitability of the oil for nutritional or industrial applications. Although the canola quality oilseed rape possesses a nearly ideal fatty acid profile, there is still room for improvement on the thermal stability of oil by further increasing oleic acid and reducing the polyunsaturated fatty acids content. Recently, some minor salutary oil constituents such as carotenoids (Shewmaker et al. 1999;Yu et al. 2008;Wei et al. 2010), phytosterols (Amar et al. 2008b), and tocopherols (Marwede et al. 2005;Fritsche et al. 2012;Wang et al. 2012b) have also drawn the attention among plant breeders and researchers to study and improve the content and composition due to their conferred health-benefiting properties. Phytosterols are widely known for their cholesterol lowering properties since 1950s (Peterson 1951;Pollak 1953). An effective dose of 1-3 g day −1 leads to reduction between 8 and 15 % in LDL cholesterol (Quilez et al. 2003). Other promising effects include anti-cancer (Woyengo et al. 2009), anti-atherosclerosis (Moghadasian et al. 1997), anti-inflammation (Bouic 2001), and anti-oxidation (Van Rensburg et al. 2000). These health-promoting properties have led to the development of functional foods enriched with phytosterols as bioactive ingredients. A variety of foods fortified with phytosterols, including margarines, mayonnaises, vegetable oils, salad dressings, milk, dairy products, beverages, and snack bars, are now widely available in the market (Berger et al. 2004). The most common sources of phytosterol added to foods are tall oil-a byproduct of the pulping industry that is rich in sitosterol and sitostanol (Jones et al. 1998)-and distillate fraction from vegetable oil refining. While most crude vegetable oils contain about 1-5 g kg −1 of phytosterol, corn oil contains about 8-16 g kg −1 and oilseed rape oil contains about 5-10 g kg −1 (Piironen et al. 2000). The high amount of phytosterol in oilseed rape means that it may serve as valuable base stock for the health and nutrition industry. Phytosterols include a wide variety of molecules that are structurally similar to cholesterol. The structural variations of phytosterols arise from different number of carbon atoms on C-24 in the side chain as well as the number and position of double bonds in the tetracyclic skeleton ( Fig. 1). In oilseed rape, the phytosterol profile consists mainly of sitosterol, campesterol, brassicasterol, and avenasterol, while cholesterol and stigmasterol occur only in trace amounts (Appelqvist et al. 1981). Brassicasterol is a characteristic sterol of Brassicaceae species and in oilseed rape, it amounts to about 13 % of total phytosterol content. Among the adapted winter oilseed rape populations, modern cultivars with canola quality contain higher amount of total phytosterols than the genetically diverse or resynthesized lines that are of non-canola quality. This observation is due to the close negative correlation between total phytosterol content and erucic acid content (Amar et al. 2008b). In a winter oilseed rape DH population segregating for erucic acid, QTL mapping showed that two of the three QTL identified for total phytosterol content colocalized with two erucic acid genes (Amar et al. 2008a). Based on the fact that cytoplasmic acetyl-CoA is required in the synthesis of both erucic acid and phytosterols, colocalizations of QTL are most likely attributed to pleiotropic effect exerted by the erucic acid genes. To further investigate the inheritance of phytosterols and their relations to other important seed quality traits, a DH population constructed from the two canola quality winter oilseed rape cultivars, Sansibar and Oase, was used in this study. The parental lines were shown to differ with respect to phytosterol and oil content based on a previous screening (Amar et al. 2009). It was anticipated that this DH population, which does not segregate for erucic acid, may have greater power for detection of QTL with novel alleles for phytosterol content than previous studies. The main objectives of this study were (1) to identify QTL for phytosterol content, fatty acids, oil content, protein content of defatted meal, and seed weight; (2) to investigate the correlation between the analyzed traits; and (3) to inspect for possible candidate genes underlying the major QTL. Plant material The experimental population consisted of 226 F1 microspore-derived DH lines derived from the Sansibar × Oase cross. The two parental lines were among the 27 canola quality winter oilseed rape cultivars analyzed by Amar et al. (2009) and were chosen due to their contrasting total phytosterol content and oil content in seed; Sansibar had the highest total phytosterol content (~480 mg 100 g −1 seed) and lowest oil content (43 %), while Oase had the lowest total phytosterol content (~360 mg 100 g −1 seed) and highest oil content (46 %). The DH population was developed in the Division of Plant Breeding at Georg-August-Universität Göttingen and was named as SODH population. Field experiments The SODH population and the parental lines were cultivated in six environments: two environments at Göttingen, Germany during growing seasons 2009/2011 and 2010/2011; one environment at Einbeck, Germany during growing season 2010/2011 by KWS Saat AG; one environment at Asendorf, Germany during growing season 2011/2012 by Deutsche Saatveredelung (DSV) AG; and two environments at Svalöv, Sweden during growing seasons 2010/2011 and 2011/2012 by Lantmännen SW Seed. The field trials were carried out in small plots in a complete randomized design without replication. Seeds of ten open pollinated plants from each line were harvested and bulked for analyses. Molecular markers Genomic DNA of the SODH population and their parental lines were isolated from young leaves of 4-5 week-old greenhouse-grown seedlings using Nucleon PhytoPure plant extraction kits (GE Healthcare, Illustra™) according to manufacturer's instructions. DNA was quantified using Bio-Rad Fluorescent DNA Quantification Kit (Bio-Rad Laboratories CA, USA). Simple sequence repeats (SSR) and amplified fragment length polymorphism (AFLP) markers SSR analysis was carried out following the M13-tailing PCR technique (Schuelke 2000). PCR reactions were performed in 96-well PCR plates with a volume of 20 μl per reaction, containing 25 ng of genomic DNA, 0.05 μM of forward primer with a M13 tail of 19 bp at the 5′ end, 0.05 μM of reverse primer, 0.05 μM of M-13 primer, 2.5 mM MgCl 2 , 0.2 mM of each dNTP, 1× PCR buffer, and 1 U of Taq DNA polymerase. A two-step touchdown PCR program was performed in a Biometra T1 Thermocycler (Biometra GmbH, Göttingen, Germany): 95 °C for 2 min; 5 cycles of 95 °C for 45 s; 68 °C (−2 °C/cycle) for 5 min, 72 °C for 1 min; 5 cycles of 95 °C for 45 s, 58 °C (−2 °C/ cycle) for 1 min, 72 °C for 1 min; 27 cycles of 95 °C for 45 s, 47 °C for 30 s and 72 °C for 1 min; and 72 °C for 10 min. A total of 350 primer pairs obtained from various sources were screened for polymorphisms between the parents. The SSR primer pairs prefixed with "BRA" and "CB" were developed by Celera AgGen consortium, and prefixed with "MR" and "MD" were developed by Division of Plant Breeding at Georg-August-Universität Göttingen. AFLP analysis was performed by adapting the method described by Vos et al. (1995). A total of 16 primer combinations made up from 8 EcoRI fluorescence-labeled primers and 4 MseI primers were used: E32M48, E37M50, E36M51, E36M59, E39M48, E38M50, E37M51, E37M59, E44M48, E40M50, E38M51, E38M59, E45M48, E44M50, E44M51, and E44M59. The PCR products of AFLP and SSR were separated on the ABI PRISM 3100 genetic analyzer (Applied Biosystems) with GeneScan-500 ROX size standard (Applied Biosystems) using 36 cm capillary arrays. The results were analyzed with GeneScan software version 3.7 (Applied Biosystems) and scored using Genotyper software version 3.7 NT (Applied Biosystems). Single nucleotide polymorphism (SNP) markers A total of 125 polymorphic SNP, designated with prefix "ra" were genotyped by the breeding company KWS Saat AG and were kindly provided to us for map construction. Diversity arrays technology (DArT) and Silico-DArT markers The SODH population was genotyped with the B. napus v1.0 DArT microarray comprising 3072 markers, designated with the prefix "brPb." A subset of 183 lines from the SODH population was genotyped with 4787 Silico-DArT markers (www.diversityarrays.com/dart-application-dartseq-data-types), designated with the suffix "| F |0." Genotyping with DArT and Silico-DArT markers was performed by Diversity Array Technology Pty Ltd, Yarralumla, Australia. The sequences for DArT markers were retrieved from http://www.diversityarrays.com/dart-map-sequences while the sequences for Silico-DArT clones were provided by Diversity Array Technology Pty Ltd, Yarralumla, Australia. Candidate gene-based markers Five candidate genes involved in the regulation of phytosterol synthesis and one candidate gene involved in the triacylglycerol synthesis were selected to develop candidate gene-based markers. The details of the candidate genes are described in Supplementary Table 1 with the aid of the phytosterol biosynthetic pathway depicted in Fig. 1. The approach involved first designing a locus-specific marker to differentiate between homologs based on locus-specific SNP, followed by sequencing of the amplicons to screen for allelic SNP between the parental lines. If an allelic SNP was found, an allele-specific marker was developed for the pertaining homolog. Due to the limited number of locus-specific SNP and a lack of polymorphisms between the parental lines, only four candidate gene-based markers were developed: HMG1A07-O1 for 3-hydroxy-3-methylglutaryl-CoA reductase 1 (HMG1) and HMG2A10-2 for hydroxy-3-methylglutaryl-CoA reductase 2 (HMG2), and D120E-3 and Dx-3 for diacylglycerol acyltransferase 1 (DGAT1). The DGAT1 primer pairs were kindly provided by Dr. Renate Schmidt from IPK Gatersleben. Primer sequence of candidate gene-based markers are listed in Supplementary Table 2. Linkage map of SODH population Linkage map was constructed using MAPMAKER/EXP 3.0 (Lincoln et al. 1992) with the aid of a purpose-built Perl script (unpublished; Wolfgang Ecke, personal communication) that automates the mapping process. Segregation of each marker was tested by χ 2 analysis (P = 0.05) to assess the goodness-of-fit for the expected segregation ratio (1:1). Markers which were significantly deviating from 1:1 segregation ratio were regarded as skewed segregated markers while markers which were not significantly different from 3:1 or beyond were defined as strongly skewed segregated markers. Markers with strongly skewed segregation were initially excluded for map construction and were attempted for mapping after the initial map was built. Markers were assigned to linkage groups to construct a core map by the "group" command with the minimum LOD score parameter set to 4 and the maximum distance parameter set to 35 cM. The most probable marker order within each group was determined by the command "order" and the resulting high-fidelity map was built upon by adding markers using the command "try." Markers that showed more than the predetermined numbers of crossovers were excluded in the high-fidelity map. Markers that were not supported by a LOD score of 3 in the high-fidelity map were placed at their most likely position in the linkage group. Following this, the "ripple" command was used to find the optimal marker order in the linkage groups. Genetic distances between loci were calculated using the Kosambi mapping function (Kosambi 1944). The resulting map consisted of high-fidelity markers which are supported by a LOD score of at least three and placed markers which are supported with LOD score of less than three. The map was further optimized by constructing each linkage group 200 times with a random subset of five highly informative markers according to MAPMAKER/ EXP3.0 command order to obtain the possible variant of a high-fidelity map. The optimal variant was selected to have as many markers as possible, as few double crossover as possible, and that the markers were as evenly distributed as possible. The map was aligned with common marker loci on established genetic maps based on SSR (Piquemal et al. 2005;Radoev et al. 2008; Sharpe and Lydiate, unpublished data), DArT (Raman et al. 2013), and SNP (KWS Saat AG, unpublished data). Linkage groups were named according to the nomenclature of Parkin et al. (1995) as A01-A10 and C01-C09. For QTL mapping purpose, a subset of markers were selected from the high-fidelity markers on the basis that the distance between adjacent markers was about 5-10 cM. The term framework map was used to refer to the map used for QTL mapping. Phytosterols Phytosterol content was analyzed by adapting the protocol of Amar et al. (2008b) and Fernández-Cuesta et al. (2012), following a direct alkaline hydrolysis method which involves three major steps: alkaline hydrolysis (saponification), extraction of the non-saponifiable matter, and derivatization of the sterols to trimethylsilyl (TMS)-ether derivatives. The main advantage of using this method is that it bypasses the lipid extraction step, facilitating large number of seed samples to be analyzed more economically. The downside of this method is that alkaline hydrolysis could only quantify free sterols and steryl esters, but not steryl glycosides. The hydrolysis of acetal bond between phytosterol and the carbohydrate moiety requires acidic condition which may be destructive to the compound and laborious for routine analysis. Hence, it is possible that the present analysis would underestimate the total phytosterol concentration in the seed sample. For each sample analysis, 200 mg of seed was weighed and placed in a polypropylene tube. Two milliliter of 2 % potassium hydroxide (Carl Roth, Germany) in ethanol (w/v) was added for alkaline hydrolysis, followed by 200 μl of 2 % cholesterol (99 % purity, Sigma-Aldrich, Germany) in hexane-ethanol (3:2) solution, used as an internal standard to quantify phytosterol content. By placing one stainless steel rod (1.1 cm length; 0.4 cm diameter) in each tube, seeds were crushed and homogenized using a custom-built vertical homogenizer (Institute of Applied Plant Nutrition, Georg-August-Universität Göttingen) for 3 min at a speed deemed sufficient to homogenize the seeds. The tubes were subsequently incubated for 15 min at 80 °C in a water bath and cooled at room temperature for 30 min. To extract the phytosterols, 1.0 ml of hexane and 1.5 ml of distilled water were added, briefly vortexed, and centrifuged for 10 min at 4000 rpm. The upper hexane layer was transferred to a new tube and left over night on a hot plate at 37.5 °C to evaporate. The residue obtained after evaporation was dissolved with 80 μl hexane and derivatized with 20 μl of silylating agent, composed of hexamethyldisilazane (Fluka analyti cal):trimethylchlorosilane (Sigma-Aldrich purum >98 %; GC grade) 3:1. The solution was pipetted into a GC vial, capped, and incubated at room temperature for 20 min. To settle the precipitate, the derivatized samples were centrifuged for 10 min at 3000 rpm prior to GC analysis. Analysis of derivatized sterols was performed using capillary gas-liquid chromatograph (Chrompack CP-9003), equipped with autosampler, split injector (320 °C; injection volume of 3 μl with a split ratio of 100:1), and flame ionization detector 320 °C, with fused silica capillary column of medium polarity (SE-54, 50 m long, 0.1 μm film thickness, 0.25 mm i.d. coated with 5 %-phenyl-1 %-vinylmethylpolysiloxane) (IVA Analysentechnik, Meerbusch, Germany). Hydrogen (carrier gas) pressure was set at 150 kPa. Initial oven temperature was set at 240 °C with an increment of 5 °C per min to final oven temperature at 275 °C and held for 20 min. Total analytical time was 25 min. Phytosterol content was expressed as mg 100 g −1 seed The phytosterol traits evaluated in this study include contents of brassicasterol, campesterol, sitosterol, avenasterol, total phytosterol, 24-methyl sterol, 24-ethyl sterol and campesterol to sitosterol ratio, and 24-methyl to 24-ethyl sterol ratio. Total phytosterol content was calculated as the sum of brassicasterol, campesterol, sitosterol, and avenasterol contents. 24-Methyl sterol was calculated as the sum of brassicasterol and campesterol contents. 24-Ethyl sterol was calculated as the sum of sitosterol and avenasterol contents. Fatty acids Fatty acid composition was analyzed by gas chromatography using a method adapted from Thies (1971). Approximately 200 mg of seed, 1 ml of Na-methylatemethanol (0.5 mol l −1 ), and one stainless steel rod (1.1 cm length; 0.4 cm diameter) were added in a propylene tube. The seeds were then homogenized using a custom-built vertical homogenizer (Institute of Applied Plant Nutrition, Georg-August-Universität Göttingen) for 3 min. Following incubation for 20 min at room temperature, 300 μl isooctane and 100 μl 5 % NaHSO 4 in water were added, briefly vortexed, and centrifuged for 3 min at 4000 rpm. About 200 μl of the upper phase was pipetted into a GC vial and 3 μl was injected into a gas chromatograph (Thermo Trace GC Ultra), equipped with autosampler, split injector (split ratio 70:1), flame ionization detector (320 °C), and capillary FFAP-phase (0.25 mm × 25 m; Macherey & Nagel). Hydrogen (carrier gas) pressure was set at 100 kPa. Oven temperature was set at 210 °C. Total analytical time was 6 min. Oil and protein content of defatted meal Oil and protein content in seeds were estimated by NIRS using calibration raps2012.eqa provided by VDLUFA Qualitätssicherung NIRS GmbH (Teichstr. 35, D-34130 Kassel, http://h1976726.stratoserver.net/cms, accessed September 24, 2015). Oil content and protein content of defatted meal were expressed as a percentage of seed dry matter content at 9 % moisture. Protein content of defatted meal was calculated by using the estimated seed oil content and seed protein content obtained from the NIRS prediction as follows: Seed weight Thousand seed weight was obtained from weight conversion of 500 seeds. The seeds were counted using a seed counter (Model:Contador, Pfeuffer GmbH, D-97318 Kitzingen, http://www.pfeuffer.com). Statistical analysis Variance components, heritability, and means were estimated using PLABSTAT software version 3A (Utz 2011). The model implemented in ANOVA analysis was as follows: % Protein of defatted meal = % Seed protein 100 − % Seed oil × 100 %. where Y ij is the trait value of the ith genotype in the jth environment, µ is the general mean, g i is the effect of ith genotype, e j is the effect of jth environment, and ε ij is the random error mean of the ith genotype in the jth environment confounded with residual error and genotype × environment interaction. The genotype was treated as fixed effect, whereas environment was treated as random effect. Broad-sense heritability ĥ 2 was estimated as follows: where σ 2 G and σ 2 E are variance components for genotype and random error; n e refers to number of environment. Mean values across all environments were used to calculate Spearman's rank correlation coefficients between traits. QTL mapping QTL detection was performed with WinQTL Cartographer software ver. 2.5 ) using means of phenotypic data obtained from 6 environments and a framework map consisting of 273 markers. QTL were initially detected with composite interval mapping (CIM) using the default model (model 6) that selects certain markers as control markers by using additional parameters. For each trait, the LOD significance threshold (α = −0.05) were estimated by 1000 permutation tests. Five markers selected by a forward and backward regression method were used as cofactors. CIM tests were performed at 1-cM steps with a 10-cM window size. Peaks were treated as separate QTL when the distance is more than 5 cM and the minimum LOD value exceeds one between any two adjacent peaks. Subsequently, multiple interval mapping (MIM) was performed to refine the QTL positions, to search for more QTL, and to investigate epistatic effects among the detected QTL (Kao et al. 1999). The MIM model was built upon a priori model from CIM analysis and progressively refined using the BIC-M2 = 2ln(n) criterion. QTL positions that did not remain significant when fitted with others were then dropped from the model. QTL effects and their percentage of phenotypic variance explained by individual and all the QTL were estimated with the final model fitted in MIM. A one-LOD drop from the peak position was used as a confidence interval for each QTL. In silico mapping of sequence-informative markers on the B. napus genome Sequences of DArT, silico-DArT, SSR, and KASP markers were used to search in the B. napus Darmor-bzh genome sequence assembly (Chalhoub et al. 2014) using the nucleotide MEGABLAST algorithm. The word size was set at 28 and the cutoff e-value was set at 1e-10. When multiple hits were obtained, the physical marker position was predicted based on the alignment with the genetic map. Identification of possible candidate genes for major QTL Based on known key regulatory genes from the literature, an attempt was made to investigate whether the predicted genes were in fact underlying the major QTL. Since all the major QTL were located in the A genome, sequences of predicted Arabidopsis genes were searched in both B. rapa Chiifu (Wang et al. 2011) andB. napus Darmor-bzh (Chalhoub et al. 2014) genome sequence assemblies. Likewise, markers within the major QTL region were searched against both B. rapa and B. napus genome sequence assemblies using the nucleotide MEGABLAST algorithm with word size of 28 and cutoff e-value at 1e-10. Polymorphism of molecular markers and linkage map development of SODH population Different types of molecular markers were used in the construction of the genetic map for the SODH population: AFLP, SSR, DArT, Silicor-DArT, SNP, KASP, and candidate gene-based markers. With 16 AFLP primer combinations, a total of 75 polymorphic markers could be scored in the SODH population. Of the 350 SSR primer pairs screened, 23 (0.07 %) were found polymorphic between the parents and exhibited clear and unambiguous amplification. Seven of the 23 SSR primer pairs amplified more than one polymorphic locus, resulting in 32 SSR loci. Approximately 13 % (407/3072) of DArT and 42 % (2005/4787) of silico-DArT markers were polymorphic between the parents. After removal of markers with a minor allele frequency of less than 10 %, a total of 2555 marker loci were available for map construction. The resulting linkage map for SODH population has 1638 markers mapped onto 23 linkage groups and covered 2350.2 cM with a mean interval distance of 2.0 cM between markers. The unmapped markers were either ambiguously linked to various linkage groups, unlinked, or formed small linkage groups that were excluded for estimation of the linkage map length. About 50 % (457/913) of the unmapped markers showed skewed segregation of which 47 % (217/457) showed strongly skewed segregation. The number of markers, map size, marker density, and mean distance between markers are summarized in Table 1 and the genetic map is shown in Supplementary Fig. 1 and Supplementary Table 3. All linkage groups could be assigned with chromosome names according to the nomenclature of Parkin et al. (1995) as A01-A10 and C01-C09. The 23 linkage groups represented 19 chromosomes in B. napus, additional four linkage groups (A08-II, C02-II, C03-II, and C04-II) were formed due to loose or no linkage to their main linkage groups. The map has an average density of 0.70 marker per cM with distribution of markers varying from 0.20 to 1.37 cM across the linkage groups (Table 1). The A genome comprised more markers (987) as compared to the C genome (655), with a mean interval distance between markers of 1.6 cM in the A genome and 2.4 cM in the C genome. The number of markers mapped in an individual linkage group ranged from 7 (A08-II) to 164 (A07). A total of 910 sequence-informative markers were physically mapped to the B. napus genome sequence. The alignment of the SODH genetic map and the physical map of B. napus genome sequence was generally in agreement although some regions showed disruption of colinearity which may suggest chromosomal rearrangements, error in genome sequence assembly, or inaccuracies of the map ( Supplementary Fig. 2). The genomic locations of sequence-informative markers and their homology are provided in Supplementary Table 4. Phenotypic analysis Highly significant effects for the genotype and the environment were found for all traits in the SODH population (Table 2). Broad-sense heritability (ĥ 2 ) estimates were high, ranging from 0.80 to 0.90, indicating that much of the phenotypic variance were genetically determined. The total phytosterol content ranged from 311.2 to 486.9 mg 100 g −1 seed, with a mean of 401.9 mg 100 g −1 seed (Table 3). Among the four quantified end-products of the sterol pathway, sitosterol was the most prominent sterol, followed by campesterol, brassicasterol, and avenasterol. The 24-ethyl sterol content, which includes sitosterol and avenasterol, was higher than the 24-methyl sterol content, which comprises campesterol and brassicasterol. Between the parents, Sansibar consistently showed a higher phytosterol content than Oase while Oase had a higher 24-methyl to 24-ethyl sterol ratio than Sansibar and only a small difference was observed for the campesterol to sitosterol ratio. The oil content was high in this population, ranging from 41.2 to 48.6 %, with a mean of 46.3 %. Between the parents, Oase had a higher oil content than Sansibar. Highly significant correlations (P = 0.01) were observed between total phytosterol and the four individual sterols (Table 4). All nine phytosterol traits were positively correlated to C16:0 while brassicasterol in particular was correlated to all the major fatty acids. Oil was positively correlated with total phytosterol and oleic acid and negatively correlated with linoleic and linolenic acids. Except for brassicasterol, no significant correlation was observed between phytosterols and protein content of the defatted meal. QTL mapping Multiple interval mapping identified between one and six QTL for nine phytosterol traits, between two and six QTL for four fatty acids, five QTL for oil content, four QTL for protein content of defatted meal and three QTL for seed weight (Table 5). These QTL were distributed on 13 linkage groups as shown in Fig. 2. Colocalizations of QTL for different traits were more frequently observed than individual isolated QTL. Fatty acids, oil content, and protein content of the defatted meal Between two and six QTL were identified for fatty acids, five QTL for oil content, and four QTL for protein content of the defatted meal ( DE18:3.6, and DE-Oil.5 on C08; the directions of the additive effects were identical to the QTL at the first QTL hotspot region. All of the five QTL for oil content had negative additive effects, indicating that the alleles increasing the oil content were derived from Oase. Interestingly, the largest QTL DE-Oil.3 on A07 was located within the confidence interval of QTL DE-Bra.5 for brassicasterol with the same direction of the additive effect. Seed weight The three QTL detected for seed weight were found on linkage groups A02, A07, and C03-II (Table 5; Fig. 2). Individual QTL explained between 6.1 and 10.7 % of the phenotypic variance, which collectively accounted for 27.1 % of the total phenotypic variance. Additive effects were positive for QTL located on A02 and C03-II and negative for QTL located on A07. The QTL DE-SW.1 on LG linkage group a CI 1-LOD confidence interval b Additive effect is the substitution effect of one Oase allele by one Sansibar allele ; the direction of the additive effects indicate that the Oase allele led to an increase in seed weight, oleic acid, and protein content of the defatted meal and to a decrease in phytosterols. The QTL DE-SW.3 on C03-II overlapped with QTL ; the directions of the additive effects indicate that the Oase allele reduced seed weight and Brassicasterol content but increased protein content of the defatted meal. Identification of possible candidate genes for major QTL Sequences of markers associated with the major QTL and the predicted genes were searched in genome sequences of both B. rapa Chiifu (Wang et al. 2011) and B. napus Darmor-bzh (Chalhoub et al. 2014). The congruency of marker orders was better with B. rapa Chiifu genome sequence assembly while some markers and most of the predicted genes were found matching on random, non-anchored scaffolds in B. napus Darmor-bzh genome assembly. Therefore, the B. rapa Chiifu genome sequence assembly was used as a reference for the investigation of the underlying candidate genes. The alignments of the major QTL on A01, A04, A06, and A09 with B. rapa are provided in Supplementary Figs. 3-6. Within the genomic region of 64.8-92.2 cM on A01, major QTL for C18:1 and C18:3 were found colocalized with FAD2 (not annotated in B. rapa) while the minor QTL for C16:0, C18:2, and oil content were colocalized with LPAAT (Bra037553). The major QTL for brassicasterol on A04 colocalized with two orthologs of CYP710A2 which are annotated as CYP710A1 in B. rapa (Bra021916 and Bra021917). On A06, major QTL for campesterol to sitoterol ratio and 24-methyl to 24-ethyl sterol ratio colocalized with SMT2. On A09, the major QTL for C16:0 colocalized with the homolog of FATB (Bra031631). Molecular markers and linkage map The two parental lines, Sansibar and Oase, revealed a narrow genetic background based on their low level of polymorphisms for most marker types. The numbers of polymorphic markers were greatly increased with array-based high-throughput DArT and silico-DArT markers which at the same time were also sequence informative. As a 10-cM interval between marker loci is commonly used for QTL analysis, the SODH map can be considered suitable for performing QTL analysis. High level of segregation distortions observed in this study have also been reported in other B. napus maps (Kaur et al. 2009;Zhang et al. 2011;Delourme et al. 2013;Raman et al. 2013). Such phenomenon appears to be common in maps of microspore-derived DH populations, which may be due to the differential responsiveness between the two parental lines to microspore culture during in vitro androgenesis and plantlet regeneration (for a review see Ferrie and Möllers 2011). The SODH map has a higher number of markers mapped on the A genome than on the C genome, similar to a few reported studies (Bancroft et al. 2011;Delourme et al. 2013;Raman et al. 2013). Phenotypic analysis Results from the phenotypic analysis revealed a relatively large and significant phenotypic variation for all the traits. Total phytosterol content which ranged from 311.2 to 486.9 mg 100 g −1 seed was comparable to the range from 356.6 to 480.0 mg 100 g −1 seed reported in 27 modern rapeseed cultivars (Amar et al. 2009) and higher than the range from 257 to 410 mg 100 g −1 seed reported in a DH population segregating for erucic acid content (Amar et al. 2008b). By taking oil content into consideration, the theoretical phytosterol content in oil ranged from 718 to 1123 mg 100 g −1 oil in the SODH population, which was lower than the range from 766 to 1402 mg 100 g −1 oil in 12 different spring canola varieties (Abidi et al. 1999) but higher than the range from 464 to 807 mg 100 g −1 oil in nine canola lines (Vlahakis and Hazebroek 2000) and the range from 448 to 928 mg 100 g −1 oil in three different DH populations of winter oilseed rape (Amar et al. 2008b). The high total phytosterol content found in the SODH population may be attributed to the low erucic acid content of the seed oil as a negative correlation between the two traits has been reported by Amar et al. (2008b). Among the individual phytosterols, sitosterol was the most prominent sterol, followed by campesterol, brassicasterol, and avenasterol, which is in accord with the relative contents reported from literatures (Vlahakis and Hazebroek 2000;Verleyen et al. 2002;Amar et al. 2008aAmar et al. , b, 2009. The transgressive segregation for individual and total phytosterol content of the DH population can be explained by the fact that both parents contributed positive alleles. The range of seed oil content from 41.2 to 48.6 % was within the range of commercial cultivars which usually contain about 40-50 % of oil. Significant genotypic variation and high heritability observed in all traits suggest that SODH population is suitable for QTL analysis. From a nutritional point of view, negative correlations between oil content and polyunsaturated fatty acids such as linoleic and linolenic acids and positive correlations between oil content and both oleic acid and total phytosterol content are desirable as reduced levels of polyunsaturated fatty acids and increased level of oleic acid will increase oxidative stability of oil while phytosterols can lower LDL cholesterols. QTL mapping Results from multiple interval mapping (MIM) indicate that additive effects were the main factors contributing to variation in all traits as no significant epistatic interaction was detected in any case. For total phytosterol content, the present study identified only two minor QTL located on A07 and C08 while Amar et al. (2008a) detected two major QTL on A08 and C03 and a minor QTL on C08. The disappearance of two major QTL in the present study corroborate the findings of Amar et al. (2008a) who found that the two major QTL for total phytosterol content were most likely due to pleiotropic effects exerted by the erucic acid genes. As a matter of fact, the present study did not detect any QTL on A08 and C03 for all the nine phytosterol traits except for the one minor QTL for brassicasterol identified on C03-II (DE-Bra.6). By disregarding the QTL on A08 and C03 from the study of Amar et al. (2008a, b), the number of QTL was almost the same as detected in the present study except for avenasterol in which four additional QTL were detected in the study of Amar et al. (2008a). Similarly, the present study shows that more QTL were detected for individual phytosterol content than for total phytosterol content. Of the eight linkage groups that harbored QTL for phytosterols in this study, only two linkage groups (A02 and A07) were not found to have QTL in the study of Amar et al. (2008a). Major QTL and possible underlying candidate genes Of the 16 traits analyzed, QTL with major effects were found for C18:1 and C18:3 on A01, brassicasterol on A04, campesterol to sitosterol ratio and 24-methyl to 24-ethyl sterol ratio on A06, and C16:0 on A09. A good colinearity observed between the genetic and the B. rapa physical map positions enabled the investigation of the possible underlying candidate genes. On A01, major QTL for C18:1 and C18:3 colocalized with FAD2 which encodes the enzyme endoplasmic Δ-12 oleate desaturase that desaturates C18:1 to C18:2. In B. napus, four loci located on A01, A05, C01, and C05 have previously been reported for FAD2 (Schierholt et al. 2000). However, in the reference genome of B. napus, homologs of FAD2 were only found on A05, C05, and scaffold chromosome. Because there were many cases like this, where the position of the candidate gene could not be determined, the reference genome of B. rapa was used for the investigation of candidate genes. About 2-3 cM above the major QTL for C18:1 and C18:3, there were minor QTL for C16:0, C18:2, and oil content which were found colocalized with the LPAAT gene, which encodes the second enzyme of the Kennedy pathway that acylates the sn-2 hydroxyl group of lysophosphatidic acid to form phosphatidic acid. In Arabidopsis, expression of the oilseed rape microsomal LPAAT isozyme has shown enhancement of seed oil content and seed mass (Maisonneuve et al. 2010). Moreover, studies have shown that LPAAT is a substratespecific acyltransferase and appears to discriminate against saturated acyl CoA in most oilseeds (Norton and Harris 1983;Sun et al. 1988;Lassner et al. 1995), which somehow explains the opposite direction of additive effects observed between QTL for oil content (DE-Oil.1) and QTL for palmitic acid and oleic acid ). In addition, the negative correlation between palmitic acid and oleic acid (r s = −0.53**) in SODH population may suggest an enhanced flux in de novo fatty acid synthesis pathway. As reported by Möllers and Schierholt (2002), an enhanced C16/C18-fatty acid ratio of the seed oil may indicate an improved seed oil synthesis by a top-down control mechanism. In maize, simultaneous enhancement of oil and oleic acid contents in seed are linked to a gene encoding diacylglycerol acyltransferase (DGAT) that catalyzes the final committed step in the Kennedy pathway leading to triacylglycerol production (Zheng et al. 2008). Similarly, expression of DGAT in wild-type Arabidopsis thaliana and the high linoleic acid fad3fae1 mutant have both shown a striking increase in seed oleic acid content (Zhang et al. 2013). One explanation for the influence of DGAT on fatty acid composition, or more specifically the increased oleic acid content, is that the enhanced triacylglycerol synthesis mediated by DGAT limits the flux through the phosphatidylcholine-based desaturation reactions (Aznar-Moreno et al. 2015). In our study, we show that the positive correlation between oil and oleic acid content could also be due to close linkage between FAD2 and LPAAT ( Supplementary Fig. 3). Because the nature of metabolic control is such that a single gene (or enzyme) rarely leads to a huge effect for a complex trait like oil content, recognizing the pleiotropic effect and close linkage of the involved genes may facilitate gene stacking for improving seed oil content as well as the quality traits. Therefore, it would also be of interest to identify the functional polymorphisms of LPAAT and FAD2 between Sansibar and Oase as well as to investigate the allelic diversity using a broader germplasm to develop functional marker for marker-assisted selection. The major QTL DE-Bra.3 on A04 collocated with two orthologs of CYP710A2, annotated as CYP710A1 in B. rapa. The CYP710A genes have been known to encode cytochrome P450 enzyme that catalyzes the C-22 desaturation reaction, converting both 24-epi-campesterol and sitosterol to brassicasterol and stigmasterol, respectively (Morikawa et al. 2006). In Arabidopsis, three C-22 sterol desaturases encoded by CYP710A1, CYP710A2, and CYP710A4 (Morikawa et al. 2006;Arnqvist et al. 2008) are able to catalyze the synthesis of stigmasterol while only one C22-desaturase encoded by CYP710A2 is able to produce brassicasterol (Morikawa et al. 2006 3) with opposite effects also suggests that CYP710A1 gene exerts a pleiotropic effect on the composition of phytosterols. Given that brassicasterol is synthesized via two enzymatic steps from 24-methylene cholesterol, and campesterol is synthesized directly from 24-methylene cholesterol, a trade-off between campesterol and brassicasterol is usual in the case of parallel biosynthetic pathways (Fig. 1). Overlapping QTL between brassicasterol and campesterol on A04 as well as the opposite additive effects were similarly observed in the study of Amar et al. (2008a), indicating that they may be the same loci in both populations. On A06, two major QTL for campesterol to sitosterol ratio and 24-methyl to 24-ethyl sterol ratio colocalized with two other minor QTL for campesterol and 24-methyl sterol (Table 5; Fig. 2). About 30 cM below this genomic region, there was a colocation of two minor QTL for sitosterol and 24-ethyl sterol with positive additive effects as opposed to the upper genomic region. Alignment between the genomic region and physical map of B. rapa revealed that the SMT2 gene was within the genomic region of major QTL (Supplementary Fig. 5). The SMT2 gene encodes the enzyme sterol methyltransferase 2 which regulates the ratio between campesterol and sitosterol or between 24-methyl sterol and 24-ethyl sterol. Campesterol to sitosterol ratio is of interest because it is important in plant growth and development (Schaeffer et al. 2001) and in humans, it determines the efficacy of cholesterol lowering ability (Miettinen 2001). The ability of plants to synthesize sterols with branched ethyl groups (as in sitosterol and stigmasterol) has also been proposed to be part of an evolutionary adaptation process to cope with wider temperature fluctuations, and to maintain the essential membrane associated metabolic processes, as compared to animals (Dufourc 2008). Considering the fact that no unfavorable pleiotropic effect or close linkage with other quality traits were observed, independent effect of this major QTL or SMT2 on A06 could be of interest for modifying phytosterol composition. On A09, the major QTL DE-16:0.2 for palmitic acid was found colocalized with the FATB gene (Table 5; Supplementary Fig. 6), which encodes the enzyme that hydrolyzes the thioester bond of C16:0-ACP and releases C16:0 from acyl-ACP (Bonaventure et al. 2003). Acyl-ACP thioesterases are known to be responsible for regulating the chain termination during de novo fatty acid synthesis and in channeling carbon flux between the plastid and cytosol in plants. The FATB gene belongs to one of the two isoforms of acyl-ACP thioesterase which primarily hydrolyzes C8-C16-saturated acyl-ACPs (Jones et al. 1995). Given that no other QTL were found overlapping with DE-16:0.2, this further supports the hypothesis that FATB is the underlying candidate gene. Similar assumption can also be made for QTL DE-16:0.5 on C09 but the homologs of FATB in B. napus C subgenome were located on C05 and C08. Minor QTL In contrast to the large effects of QTL identified for phytosterols and fatty acids, five minor QTL distributed on five linkage groups were identified for oil content. The alleles increasing oil content were all derived from Oase, the parent with a high oil content, which explains why only slight transgressive segregation was observed in the SODH population. The SODH population is similar to the RNSL population used in the study of Delourme et al. (2006) as the parents were also chosen from the elite winter oilseed rape germplasm which differs in oil content. The study reported a total of 10 genomic regions associated with oil content which were distributed on 10 linkage groups. A comparison between the two populations showed that QTL were similarly detected on five linkage groups (A01, A07, A08, C03, and C08) but it could not be confirmed if they were the same loci in both populations as the genetic maps do not share any common markers. In this study, the QTL with the largest effect was located on A07 (DE-Oil.3) and was found colocalized with QTL for brassicasterol (DE-Bra.5) and the candidate gene-based marker of HMG1 (HMG1A07O) at 120 cM. Given that HMG1 gene is responsible for regulating the carbon flux into the isoprenoid pathway and both de novo fatty acid synthesis and phytosterol synthesis share the same precursor (acetyl-CoA) (Fig. 1), the collocation of both QTL with HMG1 may be caused by a downstream effect of HMG1 gene or alternatively, it might be due to close linkage between the causative genes and HMG1. Besides the HMG1, two other candidate genes, HMG2 and DGAT1, were also mapped on A07 but were not found colocated with any QTL. Above the overlapping QTL for oil and brassicasterol on A07 lies a genomic region (38-54 cM) which harbored six QTL associated with different traits (brassicasterol, campesterol, 24-methyl sterol, oleic acid, protein of defatted meal, and seed weight). All of the six QTL showed minor effects; however, QTL for protein content of defatted meal and seed weight were the individual QTL which have the largest effect in their respective trait. Particularly for seed weight, numerous studies have consistently detected QTL on A07 in different populations with diverse genetic backgrounds Udall et al. 2006;Shi et al. 2009;Basunanda et al. 2010;Cai et al. 2012) while in the latest study, 12 candidate genes underlying 8 QTL for seed weight were identified through comparative mapping among Arabidopsis and Brassica species but no candidate genes could be inferred for the two major QTL detected on A07 (Cai et al. 2012). In conclusion, the results show that phytosterol composition and content can be improved without hampering genetic progress in improving seed oil content. Major QTL were found exclusively on the A genome and the identified candidate genes would need to be confirmed in future studies for implementing marker-assisted selection. Notably, the colocation of QTL for oil content and fatty acids with LPAAT and FAD2 on A01 could either be due to independent or combined effects of the genes.

v3-fos

2018-04-03T02:00:37.678Z

2015-10-01T00:00:00.000Z

5173594

s2

Cholesterol esterase inhibitory activity of bioactives from leaves of Mangifera indica L Background: In the earlier studies, methanolic extract of Mangifera indica L leaf was exhibited hypocholesterol activity. However, the bioactive compounds responsible for the same are not reported so far. Objective: To isolate the bioactive compounds with hypocholesterol activity from the leaf extract using cholesterol esterase inhibition assay which can be used for the standardization of extract. Materials and Methods: The leaf methanolic extract of M. indica (Sindoora variety) was partitioned with ethyl acetate and chromatographed on silica gel to yield twelve fractions and the activity was monitored by using cholesterol esterase inhibition assay. Active fractions were re-chromatographed to yield individual compounds. Results and Discussion: A major compound mangiferin present in the extract was screened along with other varieties of mango leaves for cholesterol esterase inhibition assay. However, the result indicates that compounds other than mangiferin may be active in the extract. Invitro pancreatic cholesterol esterase inhibition assay was used for bioactivity guided fractionation (BAGF) to yield bioactive compound for standardization of extract. Bioactivity guided fractionation afford the active fraction containing 3 β -taraxerol with an IC50 value of 0.86µg/ml.Conclusion: This study demonstrates that M.indica methanol extract of leaf have significant hypocholesterol activity which is standardized with 3 β -taraxerol, a standardized extract for hypocholesterol activity resulted in development of dietary supplement from leaves of Mangifera indica . INTRODUCTION H y p e r ch o l e s t e r o l e m i a , i s a p r e r e q u i s i t e f o r atherogenesis which leads to myocardial ischemia and other cardiac complications. People with elevated low-density-lipoprotein (LDL) cholesterol are prone to the development of coronary heart disease through multiple stages of the process. Lowering of serum LDL cholesterol is the primary target of anti-hyperlipidemic therapy. A large number of clinical trials on cholesterol-lowering therapy resulted in using 3-hydroxy-3-methylglutaryl coenzyme-A reductase inhibitors (statins [STs]). [1] Bile acid sequestrants acted on by interrupting enterohepatic recycling of bile acids, they have shown adverse effects like constipation and bloating hemorrhoidal bleeding. Fibric acid derivatives are another class of drugs reduce cholesterol by increased lipolysis of triglyceride via lipoprotein lipase. Ezetimibe reduces cholesterol by blocking the uptake of cholesterol into jejunal enterocytes. [2] STs are drugs of first choice for the patients with hypercholesterolemia, especially in those at high cardiovascular risk, however some of these patients are intolerant to STs. [3] Dietary ingredients include vitamins, minerals, amino acids, and herbs or botanicals, as well as other substances that can be used to supplement the diet. Several plant based nutraceuticals have been suggested to improve plasma lipid profile. [4] Red yeast rice, sugar cane-derived policosanols, artichoke leaf extracts are currently used as dietary supplements for their potential LDL-cholesterol-lowering effects. [5] Extensive literature survey supports Mangifera indica L. as one of the ingredients which could be utilized for controlling cholesterol level. Mangifera indica commonly known as mango is a large avenue tree which seen throughout India. It belongs to the family Anacardiaceae. M. indica is one of the most popular of all tropical trees. There are more than 1000 varieties of mango trees all over the world. Most parts of the tree (fruit, seeds, pulp, stem bark, roots and leaves) have medicinal properties. [6] It is native to tropical Asia and has been cultivated in the Indian subcontinent for over 4000 years and is now found naturalized in most tropical countries. [7] Phytochemical studies on various parts of M. indica L. revealed that it contains phenolic acids, phenolic esters, flavonoids, xanthanoid-mangiferin etc. In vitro studies revealed that these compounds exhibit many biological activities. Mangiferin, a major compound of M. indica have studied for many pharmacological activities like anti-diabetic, rheumatid arthritis, anti-inflammatory, hypolipidemic, antioxidant activities. [8][9][10][11][12] Flavonoid rich fraction of kernels of M. indica, leaf and bark extract have shown anti-atherogenic activity and excretion of cholesterol through faeces. [13,14] The ethanolic extract of immature leaf have shown favorable hypolipidemic and hepatoprotective activities. [15] Thus, this study was focused to isolate bioactive compounds through bioactivity guided fractionation for isolation of bioactives from the extract using cholesterol esterase inhibition assay along with their purification and structural elucidation. The active fraction can be used to develop a dietary supplement of M. indica for hypocholesterol activity. An effort is also made to develop a high performance liquid chromatography (HPLC) method to determine the amount of bioactive compounds in different varieties of M. indica screened for activity. Experimental details Plant materials Fresh leaves of different varieties of M. indica like Badami, Totapuri, Sindoora, Sannabeejada Kayi and Valaja were collected from Krishnagiri District, Tamil Nadu, India and identified by Dr. Santhan, Taxonomist, Natural Remedies Pvt. Ltd, Bangalore. Voucher specimens for each variety have been deposited in Agronomy Department of Natural Remedies Pvt. Ltd. General The hydrogen nuclear magnetic resonance ( 1 H NMR) and carbon NMR ( 13 C NMR) spectra were recorded using a Bruker AMX-400 (400 MHz) instrument. Deuterated chloroform, dimethyl sulphoxide are used for NMR analysis. HPLC analysis is carried by using Shimadzu LC 2010AHT which uses ultraviolet (UV) and photodiode array detector with LC solution software. The mass spectra were taken on a LC-MSD-Trap-XCT-plus instrument. Fractions were monitored by thin layer chromatography and the spots were visualized under UV (254 nm) light and further developed by spraying with anisaldehyde sulphuric acid and heated at 105°C. TLC was carried out on precoated silica gel 60F 254 (0.25 mm thick, Merck, Darmstadt, Germany) and column chromatography was performed on silica gel (120 mesh, Swambe chemicals). Standards-gallic acid, mangiferin (phytocompounds scale up Lab, Natural Remedies, Pvt., Ltd.). Bioassay guided fractionation The dried leaves of M. indica of different varieties were used for extraction by reflux method. Sindoora variety (5 kg), and others 200 g each of Badami, Totapuri, Sannabeejada Kayi and Valaja were extracted three times with methanol. The methanol extracts of all variety were filtered, combined and concentrated at 60°C under vacuum using a rotary evaporator. Final powder form of methanol extract was used for bioassay studies. Sindoora variety extract was used for bioactivity guided fractionation studies due to easy and commercial availability of leaf. The crude methanol extract of Sindoora leaf was subjected to liquid/liquid partition using water and ethyl acetate. The ethyl acetate layer (250 g) subjected to column chromatography (mesh size #60-120). Hence, it is sprayed with anisaldehyde sulphuric acid reagent followed by heating at 105°C for 5 min). Fractions collected from 20% chloroform to 30% chloroform in petroleum ether was enriched with compound 1, followed by repeated crystallisation by dissolving in hexane to get pure 3 β-taraxerol [ Figure 2] characterized based on spectroscopic data [Figures S1-4] matched with literature values. [16] Compound 2 The 100% methanol fraction (fraction no XII) from silica column was rechromatographed on silica gel, eluted with increasing polarity using petroleum ether and ethyl acetate followed by increasing percentage of methanol. Each individual collected fraction was concentrated in rotary evaporator to distil the solvents. The 20% methanol in ethyl acetate fraction obtained from above was applied on diaion HP-20 resin column for further purification of compound 2, which was eluted with decreasing polarity using water and acetone. The 20% acetone in water fraction was applied on sephadex LH-20 for further purification of compound eluted using water as solvent. Different fractions were collected and combined based on TLC observation. Final purification was carried out by using the prep HPLC (Kromasil C18 preparative HPLC column [250 mm × 21.2 mm, 5 µm]) was used. The preparative HPLC method was developed using isocratic method containing 15% methanol in water to yield compound 2 (360 mg) which was identified as Iriflophenone-3-β-C-glucoside [ Figure 3] based on the spectroscopic data [ Figures S5-7]. [17] 3 β-taraxerol (compound 1) Infrared: 3600 (strong broad, OH), 2900 (CH stretching), 1540 (C = C). The HPLC instrument employed for the analysis of the amount bioactives consists of Shimadzu SIL-10A auto injector, sample cooler, two CTO-10A pumps, CTO-10A column oven, SPD-M10AVP diode array detector and SCL 10 AVP central unit (Shimadzu Ltd, Kyoto, Japan). A Kromasil C 8 column (250 mm × 4.6 mm, particle size 5 µ) was used. The isocratic mobile phase consists of a water (solvent A) and methanol (solvent B) in proportion of 5:95. Flow rate of the effluent was 1.5 ml/min, with run time of 30 min, the detector wavelength of 205 nm and the volume of injection was 20 µL. Sample concentration used in the range of 5-10 mg/ml solutions. Cholesterol esterase inhibition assay Hypercholesterolemia is the source for major health issues like coronary heart disease and atherosclerosis. One of the major therapeutic strategies to effectively control these diseases by regulating plasma cholesterol level which is produced by the biosynthesis and dietary intake. Inhibition of human cholesterol esterase is very important target in the treatment of hypercholesterolemia, which is involved in the regulation of plasma cholesterol level. [18] The pancreatic cholesterol esterase inhibition assay was performed in triplicates according to Pietsch and Gutschow. [19] Various concentrations of each compound/ fractions were incubated with mixtures containing 100 µl of 5.16 mM taurocholic acid, 90 µl of 0.2 mM p-nitrophenylbutyrate in 100 mM sodium phosphate buffer diluted with100 mM NaCl, pH 7.0. The reaction was initiated by adding 5 µl of porcine pancreatic cholesterol esterase (1 µg/mL). After the incubation of 5 min at 25°C, absorbance of the mixtures was measured at 405 nm. Simvastatin was used as a positive control for this study. RESULTS AND DISCUSSION Cholesterol and free fatty acids are liberated within the intestinal lumen after hydrolysis of long chain fatty acid esters of cholesterol by pancreatic cholesterol esterase (EC 3.1.1.13), also known as a bile salt-dependent lipase. The process of hydrolysis of cholesterol ester into cholesterol by cholesterol esterase in the intestinal lumen is required for absorption because cholesterol ester is not directly absorbed by the intestinal epithelial cells. WAY-121,898, a synthetic novel inhibitor of cholesterol esterase was shown the lipid lowering effect upon oral and parenteral administrations. [20] The efficient decrease in the absorption of dietary cholesterol esters in animal [21,22] was supported that the hydrolysis of dietary cholesterol ester into cholesterol by cholesterol esterase in the intestinal lumen is an essential process for absorption. Therefore, inhibition of cholesterol esterase enzyme activity could inhibit absorption of dietary cholesterol esters. The leaf methanol extract of M. indica is known to have hypocholesterol activity. [21] Scientific literature search has indicated that cholesterol lowering constituents are not identified so far. Polyphenols like gallic acid, catechin and epicatechin from the grape seed are exhibiting hypocholesterol activity. [23] Mangiferin has shown reduced cholesterol, anthihyperlipidemic and antiatherogenic properties in diabetic rats. [24][25][26] These evidences motivated us to test these compounds for the cholesterol lowering activity. However, the preliminary result revealed that the mangiferin, a polyphenol is not active in cholesterol esterase inhibition assay. The effects of mangiferin on targets other than cholesterol esterase in in vivo may results in nonresponding to cholesterol esterase inhibition assay. Hence, bio-active guided studies study is conducted to identify hypocholesterol responsible constituents in the extract. The methanol extract was partitioned between ethyl acetate and water to afford polar (water) and nonpolar (ethyl acetate) fractions. The nonpolar fraction from the methanol extract has shown highest Figure 1]. The scheme of isolation of marker compounds indicates that the 3 β-taraxerol (compound 1) enriched fraction has shown IC 50 value of 0.86 µg/ml [ Figure 4]. However, 3 β-taraxerol isolated from the fraction was not soluble in buffer assay; hence, it was not able to carry out IC 50 value of the same. Methanol extract of different mango are tested in cholesterol esterase inhibition assay to understand the variability of the activity and constituents [ Table 1]. Valaja and totapuri variety have shown best activity when compared to other mangoes. Maximum yield of 16% from sindoor variety but the activity is very less. The sterols appeared to have same Rf values in TLC/ HPTLC. Hence, there is requirement of developing HPLC based analysis for 3 β-taraxerol. It was found to be in the range of 0.4-0.9% w/w in different variety mango leaves [ Figure 5]. The developed method is useful to quantify the 3 β-taraxerol without any interference 3 β-taraxerol is a sterol, having basic structure containing oleanan-3-ol lacking the methyl group at position 14, with α-methyl substituent at position 13 and a double bond between positions 14 and 15. Phytosterols are naturally found in various oils from plants. These compete with cholesterol in the mixed micelles, needed for cholesterol absorption by the small intestine. As a result, cholesterol absorption, either from food or from bile salts is lowered by about 50%, leading to a lowering of about 10% of blood cholesterol level, even though an increase in hepatic cholesterol synthesis. This reduction is achieved when phytosterols are given as monotherapy, and also in a combination therapy along with ST. [27] Phytosterols and dietary fibre act as low density lipoprotein cholesterol lowering agents. [28] Dietary supplements can help to get an adequate dietary intake of essential nutrients; others may help in reducing the risk of disease. The standardized leaf methanol extract of M. indica for anti-hypercholesterol could be used as dietary supplement to prevent hypercholesterolemia and the developed HPLC method may be used as a chromatographic fingerprint for this plant extracts or its formulations. CONCLUSION The present study demonstrated the hypocholesterol activity of methanolic extract of M. indica leaves in cholesterol esterase inhibition assay along with the isolation of bio-actives from their fractions. HPLC analysis of 3 β-taraxerol is developed to quantify in the methanol extract. 3 β-taraxerol is found to be in the range of 0.4-0.9% w/w in the mango leaves. The developed method is useful to quantify the 3 β-taraxerol without any interference of the other compounds (specific). However, the method should be validated for quality control purpose. Iriflophenone-3-C-β-glucoside was isolated from fraction XII have shown IC 50 value of 8.70 µg/ml. However, the compound isolated from this fraction didn't show the cholesterol esterase inhibition activity. But, iriflophenone-3-C-β-glucoside is an alpha glucosidase inhibitor. [29] This compound is also need to be tested in other responding in vitro assays for better understanding of mechanism of the action of hypocholesterol activity of the fraction generated during fractionation. The leaf methanol extract of M. indica is standardized with 3 β-taraxerol for hypocholesterol activity using cholesterol esterase inhibition assay. Figure S1: Carbon Nuclear magnetic resonance spectrum in CDC l3

v3-fos

2016-05-04T20:20:58.661Z

2014-01-14T00:00:00.000Z

17345534

s2

Simple Indirect Enzyme-Linked Immunosorbent Assay to Detect Antibodies Against Bovine Viral Diarrhea Virus, Based on Prokaryotically Expressed Recombinant MBP-NS3 Protein Background: Bovine viral diarrhea (BVD) is an economically important disease of cattle distributed worldwide. Diagnosis of BVD relies on laboratory-based detection of its viral causing agent or virus specific antibodies and the most common laboratory method for this purpose is Enzyme-Linked Immunosorbent Assay (ELISA). Objectives: The current study was aimed to develop a simple indirect ELISA to detect antibodies against Bovine Viral Diarrhea Virus (BVDV) in the sera of infected cattle. Materials and Methods: A new simple indirect ELISA method was developed to detect BVDV infection by prokaryotically (Escherichia coli, BL21 strain) expressed recombinant whole nonstructural protein 3 (NS3) of BVDV (NADL strain). Four hundred bovine serum samples were evaluated by the newly developed NS3-ELISA and virus neutralization test (VNT) as the gold standard method to diagnose BVD. Among these samples, 289 sera had been previously tested by a commercial ELISA kit. Results: Statistical analyses showed a very high correlation between the results of the developed NS3-ELISA and VNT (kappa coefficient = 0.935, P < 0.001), with the relative sensitivity and specificity of 94% and 98.8%, respectively. There was also a high correlation between the results of NS3-ELISA and the commercial ELISA kit (kappa coefficient = 0.802, P < 0.001) with the relative sensitivity and specificity of 90.72% and 91.15%, respectively. Conclusions: The newly developed simple indirect ELISA showed high sensitivity and specificity with respect to VNT. Developing such a simple, sensitive, and specific ELISA which is much less expensive than the available commercial ELISA kits can improve the detection of BVDV infections, help to eliminate the disease from herds, and decrease economic losses caused by this disease. Background Bovine Viral Diarrhea (BVD) is one of the most important cattle diseases characterized by diarrhea, abortion, stillbirth, return to estrus, and milk drop. Economic impact of BVD has led a number of countries in Europe to start eradication or control programs (1,2). BVD is caused by Bovine Viral Diarrhea Virus (BVDV), which belongs to Pestivirus genus, Flaviviridae family. Diagnosis of BVD relies on laboratory-based detection of its viral causing agent or virus specific antibodies. Although virus neutralization test (VNT) is the gold standard method to detect the virus specific antibodies (3), Enzyme-Linked Immunosorbent Assay (ELISA) is a popular laboratory method for this purpose (4). The most immunogenic proteins of BVDV (NADL strain) are Erns, E2, and nonstructural protein 3 (NS3) (5). NS3 molecule is an 80 kDa protein which is highly conserved among Pestiviruses. This protein, containing a serine protease domain at N-terminal and a domain with RNA helicase activity at C-terminal (6)(7)(8), is essential for pestivirus RNA replication (9). NS3 is strongly recognized by polyclonal convalescent sera (10). Animals vaccinated with modified live vaccines also have a strong antibody response to this protein (11). Antibodies to NS3 do not neutralize the virus infectivity; however, these antibodies can be readily detected by other serological tests. Therefore, NS3 is an important antigen in BVDV serology (4) and is suggested as a proper candidate antigen to detect antibodies against the virus. The current study used a prokaryotically expressed (Escherichia coli, BL strain) NS3 fusion protein as an ELISA antigen to develop a simple indirect ELISA for the detection of antibodies against BVDV in the sera of infected cattle. Objectives The current study aimed to develop a simple indirect ELISA to detect antibodies against BVDV in the sera of infected cattle. Preparation of ELISA Antigen Full-length cDNA coding region of the whole NS3 molecule (nucleotide 5423 to 7471, 2049 base pair, 683 residues) was cloned into pMAL-c2X expression vector at the XbaI/PstI sites. The recombinant maltose binding protein (MBP)-NS3 fusion protein was expressed in E. coli (BL21 strain) followed by purification of the protein with an amylose-resin column chromatography as previously described (12). Bovine Serum Samples Four hundred bovine serum samples were randomly collected from slaughtered cattle of both sexes and different ages at the abattoir. All serum samples were heat inactivated at 56°C for 30 minutes. Among these samples, 289 sera had been previously examined by a commercial ELISA kit (IDEXX, USA) to detect the anti-BVDV antibodies. Virus Neutralization Test All bovine sera were duplicately tested for the presence of virus neutralizing antibodies against BVDV according to the standard microtitration procedure described in the OIE (World Organization for Animal Health) manual (13). Briefly, 50 μL of 1:5 dilution of each serum sample was added into two wells of a 96-wells cell culture microplate; thereafter, 50 μL (400 TCID50) of BVDV-1 (cytopathic NADL strain) was added into each well and the plate was incubated at 37°C for one hour in a humidified CO 2 incubator. Finally, 1.5 × 104 BT cells were added to each well and the micro plate was incubated at 37°C for five days. The plate was observed daily for the presence of virus cytopathic effects. Four wells were allocated to each of the cell and virus controls and the results were documented compared to these control. Indirect ELISA Procedure The optimum concentration of ELISA antigen, serum dilution, and conjugated anti-antibody dilution were determined using checkerboard titration method. The amylose-resin purified recombinant MBP-NS3 fusion protein, at 300 ng/well, was applied into the 96-well polystyrene microtiter plates (Karizmehr Co., Iran) as ELISA antigen and incubated for 16 hours at 4°C. All the subsequent incubations were at room temperature and the plates were washed four times with PBST (PBS containing 0.05% Tween 20) after each step. Blocking of the unreacted sites was carried out for three hours using PBS containing 0.2% Tween 20. After washing the plate, a 1/400 dilution of the bovine serum samples, in PBST containing 10% chicken serum, were added to the wells and incubated for 30 minutes. The serum diluent buffer also contained 5% of soluble proteins of IPTG (Isopropyl β-D-1-thiogalactopyranoside) induced E. coli cells containing pMAL-c2X plasmid and purified MBP at a final concentration of 1.2 mg/L. After a washing step, a 1/1300 dilution of rabbit anti-bovine IgG-HRP (Sigma, USA) in PBST containing 1% rabbit serum was added to the wells and incubated for one hour. The ELISA plate was washed again with PBST and tetramethylbenzidine substrate solution was added to the wells and incubated for 10 minutes. The reactions were then stopped by addition of 0.1 M HCl and finally, optical density (OD) values were measured at 450 nm with a plate reader (Pishtazteb Co., Iran). Statistical Analyses Receiver Operating Characteristic Curve (ROC) was used to determine the cut-off value of the MBP-NS3-ELISA, considering the maximum correlation between the results of VNT and MBP-NS3-ELISA. Thereafter, relative sensitivity and specificity of MBP-NS3-ELISA compared to VNT and the commercial ELISA kit were calculated and analyzed statistically. Preparation of ELISA Antigen Recombinant MBP-NS3 protein was purified by amyloseresin column chromatography as shown in the Figure 1. This recombinant protein was expressed as a fusion protein of about 117 kDa with MBP (42.5 kDa) at the N-terminal part. Virus Neutralization Test Each bovine serum sample was tested in duplicate. The results of VNT for all sera were summarized in Table 1. ELISA and Statistical Analyses Considering the maximum correlation between the results of MBP-NS3-ELISA and VNT as the gold standard method for serodiagnosis of BVDV infection, the calculated cut-off value by ROC was 0.303. While 150 serum samples were positive with VNT, and 144 serum samples were positive by MBP-NS3-ELISA. On the other hand, 250 serum samples negative for VNT, whereas 256 of all samples were negative for MBP-NS3-ELISA. A visual comparison of the results of these two methods is shown in Figure 2. McNemar's test indicated no significant difference between the results of the two methods (P > 0.05). Kappa coefficient showed a very high correlation between these two methods (Kappa coefficient = 0.935, P < 0.001). The relative sensitivity and specificity of MBP-NS3-ELISA with respect to VNT were 94% and 98.8%, respectively and the accuracy of the developed MBP-NS3-ELISA was 97%. Quantitative comparison of the VNT and MBP-NS3-ELISA results is summarized in Table 2. The results of 289 serum samples previously tested with commercial ELISA kit were compared to the results of MBP-NS3-ELISA. While 97 serum samples were positive with commercial ELISA kit, 105 serum samples were positive by MBP-NS3-ELISA. On the other hand, 192 serum samples were negative with commercial ELISA kit, whereas 184 of all samples were negative for MBP-NS3-ELISA. A visual comparison of the results of these two methods is shown in Figure 3. McNemar's test indicated no significant difference between the results of the two methods (P > 0.05). Kappa coefficient showed a high correlation between these two methods (Kappa coefficient = 0.802, P < 0.001). The relative sensitivity and specificity of MBP-NS3-ELISA with respect to commercial ELISA kit were 90.72% and 91.15%, respectively; the accuracy of the developed MBP-NS3-ELISA was 91%. Quantitative comparison of the commercial ELISA kit and MBP-NS3-ELISA results is summarized in Table 3. Discussion The first step to control eradicate BVD is to eliminate persistently infected (PI) animals and determine the prevalence of antibodies against the viral causal agent in order to monitor the virus circulation. The first generations of diagnostic ELISA kits utilized the extracts of virus infected cell cultures as ELISA detector antigens (14,15). Several ELISAs have been recently developed to detect BVDV infections using recombinant NS3 protein (16)(17)(18)(19). Eukaryotic or prokaryotic recombinant NS3 is used for this purpose and this protein has shown high sensitivity and specificity to detect BVDV infection in comparison with whole virus antigen (16,20). Thus, production of a recombinant form of NS3 in large amounts is economically very important and can be very useful to manufacture BVDV antibody ELISA kits. Although it is shown that eukaryotic expression of NS3 increases sensitivity and specificity of ELISA kits (18), prokaryotic expression of NS3 is still considered, since it is simple and less expensive than eukaryotic expression of the protein. So far, several ELISAs are developed using prokaryotically expressed NS3 to detect anti-BVDV antibodies. Reddy et al. (17) cloned a 917-bp segment of NS3 (p80) into pGex-2T plasmid vector containing the glutathione-S-transferase (GST) gene and the recombinant protein was expressed in E. coli and used as an ELISA antigen to detect anti-BVDV antibodies. A 1152-bp cDNA fragment of NS3 (2/3rd of NS3 gene from C-terminal) was cloned into pGEMT Easy Vector and expressed in E. coli by Bhatia et al. (21) and used as a detector ELISA antigen in their developed competitive inhibition ELISA (CI-ELISA) using a monoclonal antibody. Lecomte et al. (16) used a recombinant 80 kDa antigen of the BVDV/Osloss virus strain as a fusion protein with β-galactosidase to detect BVDV specific antibodies by ELISA. They also developed a competitive ELISA which was more specific than the direct assay. Vanderheijden et al. (18) inserted a 2183-nucleotide fragment containing encoding sequence of p80 (Osloss strain) into the pARHS3 plasmid and analyzed the expressed protein by a competitive ELISA. Although the prokaryotic expression of recombinant NS3 is a simple and inexpensive method, compared to the eukaryotic expression, researchers tried to develop competitive ELISAs by monoclonal antibodies and/or eukaryotically expressed recombinant NS3 molecule to increase sensitivity and specificity of the assay. In fact, one of the major problems of prokaryotic expression of NS3 molecule, particularly whole NS3 molecule, is that it is a large insoluble protein which aggregates as inclusion bodies in the bacterial host cells. Thus, researchers had to use high concentration of urea to solubilize the recombinant NS3 protein (inclusion bodies) and then renature it by gradient dialysis of the solution and it is obviously a time-consuming and expensive process. This problem was solved by another plasmid vector, pMAL-c2X, to clone and express the recombinant NS3 molecule used in this study. This vector encodes a maltose binding protein at the N-terminal part of the molecule which is a soluble protein and can even solubilize the recombinant protein expressed with it as a fusion protein (22,23). MBP tag makes it possible to purify the recombinant protein by amylose-resin. In addition, with the enhanced solubility of the expressed recombinant NS3, there was no need to treat the recombinant protein with urea for the purification. The current study developed an indirect ELISA using the recombinant MBP-NS3 molecule as an ELISA detector antigen. The protein was expressed in E. coli in a simple and inexpensive manner and it was sufficiently soluble to be purified without any treatment. MBP-NS3 based ELISA showed a high degree of sensitivity and specificity in comparison with the results of VNT; kappa coefficients indicated a very high correlation between MBP-NS3-ELISA with VNT (kappa = 0.935, P < 0.001) and the commercial ELISA kit (kappa = 0.802, P < 0.001), although there were some differences. Nine out of 150 positive samples by VNT were negative by MBP-NS3-ELISA. This could be due to 1) a recent infection which results in the production of IgM; therefore, the OD values of such serum samples may decrease significantly causing the samples to be determined as negative by MBP-NS3-ELISA, 2) presence of non-specific inhibitor factors in the tested serum samples that prevented virus propagation in the cell culture, and 3) vaccination of animals with an inactive vaccine that induces the production of neutralizing antibodies but not antibodies against BVDV NS3 protein. On the other hand, 3 VNT negative samples were positive by MBP-NS3-ELISA. This could also be due to the strain of the employed virus in VNT and/ or the amount (TCID50) of the employed virus in VNT. In this study, maximum recommended TCID50 of the virus (400 TCID50) was utilized in VNT. So, it was more stringent and this could decrease the sensitivity and increase the specificity of the assay. The differences between the results of MBP-NS3-ELISA and the commercial ELISA kit probably resulted from the type of employed ELISA detector antigen(s) or standardization of the procedures. Meanwhile, the developed ELISA is a simple ELISA which has no need to monoclonal antibodies to increase sensitivity or specificity of the assay. Statistical analyses showed that this developed ELISA is highly sensitive and specific in comparison with the viral neutralization test, which is the reference test for the serological diagnosis of BVDV. Developing such a simple, sensitive, and specific ELISA which is much less expensive than the available commercial ELISA kits can improve the detection of BVDV infections, help to eliminate the disease from herds, and decrease the economic losses caused by this disease. The current study developed a new simple indirect ELISA with a prokaryotically expressed recombinant whole NS3 molecule as ELISA antigen to detect anti-BVDV antibodies in sera of the infected cattle. The developed ELISA showed high sensitivity and specificity with respect to VNT.

v3-fos

2019-04-21T13:08:28.689Z

2015-01-01T00:00:00.000Z

124447184

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Ascertainment of K Nutrient Availability Class for Maize by Several Methods Research was conducted in Gowa, South Sulawesi at dry land farmer during two years. The aims of the research was to get the best method in ascertainment of availability class of potassium (K) for maize in dry land. The research used a single location approach, which made some of K nutrient artificial. Result of this research indicated that K nutrients class which reached by several methods are: (1) by Cate-Nelson method : two class, ie low and high class, (2) by curve continue method: two until three classes, ie very low to moderate class, low and moderate, and low to high class; and (3) by analysis of variance modified method: three classes, ie low to high class. Ascertainment of K nutrient availability classes by modified analysis of variance method was the best methods compared to other methods. Critical level of K nutrient for maize according to modified analysis of variance method by several extractant is: 0.40 me K 100 g-1 for NH4OAc pH 4.8 extractant; 0.40 to 0.60 me K 100 g-1 for NH4OAc pH 7 extractant; 200-300 ppm K2O for Bray-1 extractant, and 215-250 ppm K2O for Olsen extractant. [How to Cite: Marthen PS and P Tandisau. 2015. Ascertainment of K Nutrient Availability Class for Maize by Several Methods. J Trop Soils 19: 21-27. Doi: 10.5400/jts.2015.20.1.21][Permalink/DOI: www.dx.doi.org/10.5400/jts.2015.20.1.21] INTRODUCTION Potassium fertilizer plays an important role in improving crop production because of its role in physiological processes, such as cell division, photosynthesis, nitrate reduction and protein synthesis, and enzyme activity (Leiwakabessy and Sutandi 1996). Therefore, potassium is often referred to as a catalyst in the process of ensuring the life because the reaction in the plant life. Potassium is one of the macro nutrients that plants need quite a lot of early corn growth before flowering plants even 30% of the potassium they need is absorbed by plants (Laddong 1988). However, the use of K fertilizer on crops are generally not rational and balanced because fertilization has not been based on the status and dynamics of nutrients in the soil as well as the needs of the plant optimum. Therefore, nutrient status and plant nutrient dynamics are needed to be known through soil testing approach (Sabiham 1996). Estimate of nutrient availability in the soil can be done through soil testing, chemical analysis is an activity that is simple, fast, inexpensive, accurate, and reproduceable (Leiwakabessy and Sutandi 1996;Leiwakabessy and Koswara 1985). Ascertainment the status of nutrients in the soil through a soil test can be used as a guide the land's ability to provide nutrients for plants. However, the ability of plants to absorb nutrients from the soil is determined by the nutrient status of the soil, and it is also influenced by the availability of water, infiltration rate, drainage, salinity, exchangeable cations, and the presence of compounds that are toxic as well as the plant it self (Soepartini et al. 1994). Thus, the status of nutrients in the soil can not be used as a probe response to fertilization and crop fertilizer requirements for crops in question before calibrated with experimental results fertilization (Widjaja-Adhi 1993;Voss 1998). Several methods can be used to ascertainment the class of the availability of nutrients such as Cate-Nelson graphical method (Widjaja-Adhi 1996). In this method, the critical threshold values obtained indicate that the nutrient status of the soil nutrients that have a soil test value is lower than the critical threshold value considered low class, whereas greater than the critical threshold value including high class. Cate-Nelson graphical method only provides two classes (categories) soil test, which is a low class (response to fertilizers) and high class (no response to fertilizer). Another method that gives the class of nutrient availability more than two classes is a continuous curve method (Leiwakabessy 1996;Dahnke and Olson 1990) or analysis of variance modified method (Widajaja-Adhi 1986). Ascertainment of K nutrient availability class with some methods are very important in terms of getting the best method in the preparation of fertilizer recommendations for maize. K soil test calibration studies conducted with a single location to remember the way the approach is relatively inexpensive, easy and fast, but to obtain more accurate data needs to be done through a multi-site approach. The aims the research was to get the best method in determining of K nutrient class for corn in dry land. MATERIALS AND METHODS Nutrient soil test calibration study K for corn conducted on dry land by using the approach of a single location, that is by making artificial soil nutrient status of very low to very high and subsequently implement fertilization experiment in each nutrient status. Type of soil in the experiment at the level of orders include Alfisols and at the level classified as Typic Rhodustalfs family. Average rainfall at the site is 291 mm/month with 18 days the number of rainy days. The highest rainfall occurs in February which reached 1,008 mm while the lowest rainfall occurs in August. The event was doing in Gowa, South Sulawesi during two years. The experiment consisted of two stage, namely the first stage of making artificial soil nutrient status K, and the second stage K fertilization experiments were placed in locations that have been conditioned of K nutrient status. Stage I: Preparation of Artificial K Nutrient Status K fertilizer was given at a dose of 0X (K status is very low), 1/4X (low), 1/2X (medium), 3/4X (high), and X (very high), where X is the number of K fertilizer given that K in the soil solution reached 0.02 mg K/l (Widjaja-Adhi et al. 1990), defined by the absorption curve of K according to Fox and Kamprath method (1970). K dose used in the first stage is based on K maximum absorption, ie 200 kg ha -1 of Potassium Clorida (KCl). Potassium clorida is given to the respective main plots in accordance with the treatment before planting. Evenly spread fertilizer and soil stirred until completely homogeneous. Urea and ZA, respectively at a dose of 300 kg (3 times application) and 50 kg ha -1 given bolt spread on crops, while the SP-36 at a dose of 250 kg ha -1 provided with Potassium Clorida. Experimental plots were 30 m × 20 m. The experimental design was in a randomized completely block design with three replications. The experiment consisted of five treatments, namely: (1) very low K nutrient, (2) low K nutrient, (3) moderate K nutrient, (4) high K nutrient, and (5) very high K nutrient status. Indicator plants are maize with a spacing of 75 cm × 20 cm. In the first phase of the experiment (incubation experiments), in addition to soil is expected to reach reaction equilibrium constant (steady stage) or K nutrients from manure turned into the soil nutrient K, is also the basis for fertilization experiment in the second stage at various K nutrient status of the soil. StageII : K Fertilization on Corn in Dry Land After the completion of the first stage of the experiment, soil samples were taken from each plot treatment by composite about 1 kg for ascertainment of soil K levels with 25% HCl, NH 4 OAc pH 7, pH 4.8 NH 4 OAc, Bray-1 and Olsen method. Further experimental plots in stage I, split into plots measuring 6 m × 4 m. The experimental design used in the second stage is a split plot with three replications. In the main plot is K nutrient status of the soil (which acquired the activities of stage I), namely: very low K nutrient (A), low K nutrient (B), moderate K nutrient (C), high K nutrient (D), and very high K nutrient (E ), while the subplot is K fertilizers, namely: 0 kg K ha -1 (K0), 20 kg K ha -1 (K1), 40 kg K ha -1 (K2), 80 kg K ha -1 (K3), and 160 kg K ha -1 (K4). Total number of treatment is 60 treatments. Plant indicators which using are corn of Lamuru variety. Potassium clorida is given prior to planting at a dose of 0, 20, 40, 80, and 160 kg K ha -1 for the treatment consecutive K0, K1, K2, K3, and K4. Whereas urea, ZA and SP-36, respectively at a dose at 300 kg urea (3 times applications), ZA 50 kg, and 250 kg SP-36 ha -1 given the same as in the first stage. The parameters measured in the experiment is the result of the second stage of the maize plant. Ascertainment of Nutrient Availability Several methods can be used in the ascertainment of K nutrient availability, including by Cate-Nelson graphical method (Widjaja-Adhi 1996), continuous curve (Leiwakabessy 1996;Dahnke and Olson 1990), and analysis of variance were modified (Nelson and Anderson 1977). Cate-Nelson Graphical Method Ascertainment procedures of nutrient availability class with this method are as follows: (1) make the distribution diagram of the relative percentage yield on the Y axis and K soil test values on the X axis, (2) the distribution diagram put the two lines intersect (cross axis) which divides quadrant into four parts, the lower left quadrant and right upper quadrant as positive, and the left upper quadrant and lower right quadrant as a negative, (3) the cross slide axis in a fixed position until the number of points in the positive quadrant as much as possible, while at the quadrant negative as little as possible, and (4) the intersection of the cross axis with X axis is a critical level nutrient value of K. Areas that are to the left of the critical threshold is low class and the area to the right of the critical threshold is high class (Figure 1). Continuous Curve Method Ascertainment of availability class K nutrient by this method are as follows: (1) pair of points mapped relative to the value of the results of the soil test K, (2) create a curve through the points, (3) for the curve into several classes according to the criteria of Cope and Rouse (1973), namely: (a) very low: <50%, (b) low: 50-75%, (c) are: 75-100%, (d) high and very high:> 100% relative yield, as in Figure 2. (3) Groupping the data into several groups DY max with basic considerations in drawing boundaries subgroups as follows: (a) there must be a considerable reduction of DY max between values either side of the separation barrier and the average DY max should up, (b) dividing line is not drawn between the two soil test values are the same or nearly the same, and (c) members of at least two. (4) Calculated data pairs (ni), deviation standard (Si), and the average DY maxi of the group to-1 and pooled S of all groups. (5) Test the difference between two D Y max median of consecutive groups with Student's t-test one-way by the formula: t = (DY max i -D Y max i+ 1 ) / S (1/n i + 1/n i + 1 ) 0.5 When the difference D Y max on average between two successive groups are not real, then the two groups merged into one. Based on the number of new groups, the procedure returns to step 4 and continue to step 5. This is repeated until the value of the average difference between the two groups were sequentially real (Nelson and Anderson 1977). Statuss of Soil K Nutrient Data experiment of K nutrient which obtained through experiments in the first step on the field is very irregular (erratic). One of the suspected causes for the land has not reached equilibrium constant (steady stage) or in other words K fertilizer nutrients has not changed completely into nutrient soil K during incubation in the field in a state of drought. Other causes are factors beyond the ability of management experiments, both in the field and in the laboratory. Cate-Nelson Graphical Method Availability class of K nutrient which obtained by Cate-Nelson graphical method with various extractans consist of two classes, namely low class and high class (Table 1). Low K class is obtained in the area to the left of critical level values, while the high K class areas that are on the right of the critical level value. Boundary between low K class and high K class referred to as the "critical level". Ascertainment of availability class of K nutrient with Cate-Nelson graphical method (Figure 3) is very weak because it only consists of two classes, low and high, or response and no response to fertilizer K. But according to Dahnke and Olson (1990), the class categories are located in the critical level value. Cate-Nelson graphical method has disadvantages as well, also has the advantage, that it can know the critical value of a nutrient in relation to whether or not to do an act of fertilization. Critical threshold values obtained with the K nutrient Cate-Nelson graphical method can be used as a reference in the act of fertilization on a soil type. Continuous Curve Method Ascertainment of availability class of K nutrient with continuous curve method at various extractors K are presented in Figure 4. Based on the continuous curve method, extracting NH 4 OAc pH 7 provides two classes of nutrient availability, ie low and medium K class, while the other extractors generate three classes, namely the class K is very low to moderate (NH 4 OAc pH 4.8 and Olsen), and low K class to high class (Bray-1), as in Table 2. Only extractors Bray-1 that provides high-class categories K, while the other extractors obtained through the class K is very low to moderate. Analysis of Variance Modified Method Ascertainment of availability class of K nutrient by using analysis of variance modified methods (Nelson and Anderson 1977) showed that a class of nutrients K obtained by extracting NH 4 OAc pH 4.8, NH 4 OAc pH 7, Bray-1 and Olsen, each producing three groups, namely K low, moderate, and high class. The third methods of determining the availability class of K nutrient to corn in dryland is known that the analysis of variance modified method gives three Table 2. Availability class of K nutrient for maize based on continuous curve method. Effect of Fertilization for Maize Grain yield drying of maize obtained in the second stage of the experiment. From this data will be seen that in general, the average of the yield highest maiz obtained on fertilization at 80 kg K ha -1 for K nutrient very low class to moderate class, and the fertilization at 20-40 kg K ha -1 for high K nutrient class. K fertilization until dose 80 kg K ha -1 at different K nutrient classes increased yield, but if the dose increased until to 160 kg K ha -1 the decreasing of yield. Thus, K fertilization for Lamuru variety on Alfisol type is 80 kg K ha -1 is sufficient. To determine the optimum dose of the preparation needs to be done recommended dose using response curve fertilization (Widjaja-Adhi 1993;. Based on this curve, optimum K fertilizer is determined by following the laws of economics. Recommended dose of fertilizer is fertilizer dose to achieve optimum results. Optimum fertilizer usually occur when crop reaches 90% maximum yield (Affandi et al. 2001) CONCLUSIONS Ascertainment of K nutrient class for maize by some of the methods are as follows: (a) Cate-Nelson graphical method provides two classes of nutrient K, the low-and high-K classes, (b) continuous curve method gives two to three classes, namely the class K very low and moderate, and low-to high-K class, and (c) a modified method of analysis of variance gives three classes, namely low-to high-K class. The average highest yield of maize obtained at fertilization rate 80 kg K ha -1 for very low K class through moderate class, and fertilization rate 20 kg -40 kg K ha -1 for high K nutrient class. Analysis of variance modified method give the best yield for determination of availability class K soil nutrient for corn in dry land. To obtain the nutrient availability of class K is more accurate for a certain soil-plant system is recommended with multi-location approach.

v3-fos

2016-03-14T22:51:50.573Z

2015-02-01T00:00:00.000Z

7139364

s2

Transcriptome Profiling and Identification of Transcription Factors in Ramie (Boehmeria nivea L. Gaud) in Response to PEG Treatment, Using Illumina Paired-End Sequencing Technology Ramie (Boehmeria nivea L. Gaud), commonly known as China grass, is a perennial bast fiber plant of the Urticaceae. In China, ramie farming, industry, and trade provide income for about five million people. Drought stress severely affects ramie stem growth and causes a dramatic decrease in ramie fiber production. There is a need to enhance ramie’s tolerance to drought stress. However, the drought stress regulatory mechanism in ramie remains unknown. Water stress imposed by polyethylene glycol (PEG) is a common and convenient method to evaluate plant drought tolerance. In this study, transcriptome analysis of cDNA collections from ramie subjected to PEG treatment was conducted using Illumina paired-end sequencing, which generated 170 million raw sequence reads. Between leaves and roots subjected to 24 (L2 and R2) and 72 (L3 and R3) h of PEG treatment, 16,798 genes were differentially expressed (9281 in leaves and 8627 in roots). Among these, 25 transcription factors (TFs) from the AP2 (3), MYB (6), NAC (9), zinc finger (5), and bZIP (2) families were considered to be associated with drought stress. The identified TFs could be used to further investigate drought adaptation in ramie. Introduction Abiotic stresses, such as drought and high salt, are becoming increasingly common because of global climate change, and severely inhibit plant growth and development [1,2]. Among all the abiotic stresses, drought has probably the most significant effect on plant distribution, growth, and productivity in natural and agricultural systems. Studies on the molecular responses to drought are vital, and will ultimately lead to enhanced stress tolerance in crops [3]. However, the responses and adaptations of plants subjected to drought conditions are complex. During the process of domestication, plants have developed numerous physiological and biochemical strategies to cope with adverse conditions [1,4]. For example, when the leaf-to-air vapor pressure or relative humidity changes, a plant's leaves close their stomata to reduce water loss through transpiration; when suffering from progressive water loss, the leaves begin to curl to protect the photosynthetic machinery [5]. Meanwhile, the water content of cells is maintained by synthesizing and accumulating various small molecule compounds, such as soluble sugars [6] and proline [7]. Nevertheless, the production of reactive oxygen species (ROS), which are caused by severe drought stress [8], could have detrimental effects on plant growth and development [9]. To cope with oxidative damage, several enzymes are activated in plant cells, including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), and non-enzymatic antioxidants, such as ascorbic acid and glutathione [8]. Studies on the molecular mechanisms of plant responses to these stresses and damages are increasing. Recently, analyses of a large number of transcriptome sequence data sets have revealed many new stress-responsive genes in Arabidopsis [10] and rice [11]. However, there have been few reports on ramie [12,13]. Ramie is an important natural fiber crop, widely planted in China, India, and other Southeast Asian and Pacific Rim countries [12]. It is the second major fiber crop after cotton in China and plays an important role in Chinese economy. Ramie fibers possess high tensile strength, antibacterial properties, and good moisture absorption characteristics. In China, ramie is mainly planted by the Yangtze River and can be harvested three times per year. However, the per capita arable land area is shrinking as urbanization and industrialization accelerate. Therefore, it is a good practice to plant ramie on sloping land so that more land can be used to grow grain. However, ramie yields have shown dramatic decreases recently because of reduced rainfall in the summer or autumn. Thus, drought tolerance research in ramie (especially research into breeding drought-resistant varieties) has become particularly important. However, to date, only a few reports have focused on ramie physiological traits [12,13]. In a previous study, research into the ramie universal transcriptome has identified 24 genes that could be transcription factors (TFs), but only 12 of them have been shown to be authentically involved in drought stress responses [13]. In this study, hydroponic ramie seedlings, propagated from stem cuttings of cultivar Huazhu No. 5 and transplanted into half-strength Hoagland's solution, were generated and subjected to simulated drought stress (PEG treatments). Four physiological traits were observed (the relative water content, RWC; the peroxidase activity, POD; the malondialdehyde content, MDA; and the proline content) from the leaves of the seedlings to distinguish the stress severity and for collecting samples for transcriptome sequencing. A total of 16,798 (9281 in leaves and 8627 in roots, respectively) unigenes were differentially expressed in the transcriptome data. Among these, 25 TFs from the AP2 (3), MYB (6), NAC (9), zinc finger (5), and bZIP (2) families were considered to be associated with drought stress because of their expression patterns (coincided with the propensities of the observed physiological traits) in leaves or roots. This study increased our understanding of the molecular responses to drought in ramie, which will improve drought resistance breeding in ramie. Physiological Responses of Ramie to Drought Stress Leaves harvested at 0, 12, 24, 48, and 72 h after being treated with 15% PEG6000 (w/v) were prepared for measuring the four physiological traits (the RWC, the POD activity, the MDA content, and the proline content). RWC seemed to decline consistently (Figure 1a), most significantly after 24 h. The highest RWC was at 0 h and the lowest RWC was at 72 h under drought stress. The critical time-points were observed as 0, 24, and 72 h after drought stress. The POD activity first increased but then decreased ( Figure 1b). The highest POD activity was at 24 h and the lowest POD activity was at 0 h. The POD activity increased most significantly after 12 h and declined most significantly after 48 h. The POD activity remained stable after 72 h under drought stress. The critical time-point was 24 h after drought stress. The leaves were harvested at 0, 12, 24, 48, and 72 h after treatment with 15% (w/v) PEG6000. The error bars mean standard error. a, b, c, d, and e indicated significant differences (p < 0.05) among different time-points. By contrast, the MDA and proline content increased throughout the entire drought stress periods (Figure 1c,d). The highest MDA and proline contents were at 72 h and the lowest were at 0 h under drought stress, the critical time-points were determined as 0 and 72 h after drought stress. Based on the critical time-points of the change of the RWC, the POD activity, the MDA content, and the proline content under drought stress, we chose three time-points (0, 24, and 72 h after PEG treatment), to investigate the transcriptome. Illumina Paired-End Sequencing, Reads Assembly, and Annotation Illumina paired-end sequencing technology yields 2 × 300 bp independent reads. After stringent quality checking and data cleaning, approximately 33,976,322,460 bp (30G) of high-quality data (94.02% of the raw data) were generated under the Q20 standard. The sequence data generated in this study have been deposited at the NCBI in the Short Read Archive database under accession SRP041143. Assembly of the high-quality sequencing reads yielded 138,381 unigenes, with an average length of 730.6 bp and a range from 201 to 20,860 bp. The lengths distribution of the assembled contigs is shown in Figure 2. The lengths distribution of the unigenes is given in Figure 3. To provide putative annotations for the assembled unigenes, Blastp similarity searches were performed against the non-redundant protein (Nr) and the Swiss-Prot protein databases. The paired-end reads were realigned to contigs and the contigs in one transcript were assembled by Trinity and were defined as unigenes. The unigene sequences were compared to the non-redundant (nr) protein database with a cut-off E-value of 1 × 10 −5 . As a result, 47,565 (Supplementary file 1) unigenes (34%) were annotated. All sequences of the unigene are shown in Supplementary file 2. Functional Classification and Metabolic Pathway Assignment To provide putative functional classifications for the transcriptome assembly, all the assembled unigenes were evaluated using Blastp similarity searches against the Gene Ontology (GO) database, which classified 22,058 (Supplementary file 3) matched unigenes into the three functional categories ( Figure 4). In molecular function, those that matched unique sequences were clustered into 25 subcategories. The largest subcategory in the molecular function class was "catalytic activity" (11,849; 21.54%) and the second was "binding" (11,620; 21.12%) ( Figure 4). For the cellular component category, the sequences were divided into 11 subcategories. The most represented cellular components were "cell" (18,176; 30.58%) and "intracellular" (16,053; 27.01%) ( Figure 4). For the biological process category, the sequences were classified into 21 subcategories. The most represented biological processes were "cellular process" (16,499; 27.67%) and "macromolecule metabolism" (10,676; 17.90%) ( Figure 4). The Kyoto Encyclopedia of Genes and Genomes (KEGG) [14] database can be used to analyze the gene products of metabolic processes. A total of 6502 (Supplementary file 4) assembled sequences were observed to be associated with 2755 predicted KEGG metabolic pathways, and were grouped into five KEGG biochemical pathways: Genetic information processing, organismal systems, cellular processes, environmental information processing and metabolism ( Figure 5). The metabolic pathways were well represented by carbohydrate metabolism, amino acid metabolism, energy metabolism, lipid metabolism, nucleotide metabolism, metabolism of cofactors and vitamins, and the biodegradation and metabolism of xenobiotics. Those pathways related to genetic information processing included genes involved in translation, transcription, replication and repair, and folding, sorting and degradation. Pathways related to cellular processes and environmental information processing were also well represented by unigenes from ramie. These results represented a valuable resource for investigating metabolic pathways in ramie. Analysis of Differential Gene Expression Gene expression was calculated according to the reads per kilo base of transcript per million reads mapped (RPKM) method [15], using the MA-plot-based method with Random Sampling (MARS) model from the DEGseq [16] program package. 16,798 (Supplementary file 5) genes (12.14% of all genes) were identified as differentially expressed genes (DEGs) in roots and leaves. These amounted to 9842 genes (accounting for 7.11% of all genes) in the leaves of ramie and 9748 genes (accounting for 7.04%) in the roots. The numbers of unigenes that had differential expression patterns are shown in Table 1. Seven altered pathways were significantly enriched (corrected p-value ≤ 0.05), with genes involved in amino acid metabolism, carbohydrate metabolism, lipid metabolism, signal transduction, translation, energy metabolism, and folding, sorting, and degradation. These seven altered pathways were the most significantly enriched in the ramie leaves (Supplementary file 6); the Y-axis denotes the numbers of unigenes that were annotated to the enrichment between the two sample combinations in each KEGG pathway). For roots, the altered pathways contained xenobiotic biodegradation and metabolism genes, in addition to the seven altered pathways previously mentioned (Supplementary file 6). Identification of Drought-Responsive TFs Given that TFs appear to have a major effect on drought-responsive genes, one of the objectives of this study was to identify drought-inducible TFs. Among the differentially expressed unigenes, those with expression patterns that coincided with the patterns of the physiological traits (as displayed in Figure 1) were considered important. Generally, more unigenes were generated as "all-up" (unigenes up-regulated in L2 compared with L1, and simultaneously up-regulated in L3 compared with L2) or "all-down" patterns than "up-down" (unigenes were up-regulated in L2 compared with L1, while down-regulated in L3 compared with L2) and "down-up" patterns in leaves. While more TFs showed up-down or down-up patterns than all-up and all-down patterns in roots (Table 2). Accordingly, unigenes from the five main TF families (AP2, MYB, NAC, zinc finger, and bZIP) that shared all-up or all-down patterns in leaves and up-down or down-up patterns in roots were considered to have consistent physiological trait trends and were selected for further quantification. Altogether, 25 genes were found to encode known or putative TFs. Of the 25 TFs selected, three belonged to the AP2, six to the MYB, nine to the NAC, five to the zinc finger, and two to the bZIP families (Supplementary file 7). To further evaluate the role of these TFs, we analyzed their expression levels using quantitative real-time reverse transcription PCR (qRT-PCR). As shown in Figure 6, these TFs were all-up-regulated, all-down-regulated, up-down-regulated, or down-up-regulated by drought stress. Furthermore, when the transcriptome sequencing was performed, only one cDNA library was constructed for each sample; therefore, the effectiveness of the transcriptome data was validated (due to lack of duplication when preparing the transcriptome sequencing). Specifically, 11 unigenes (comp41385, C2; comp79146, E1; comp77912, I1; comp75203, K2; comp80737, M2; comp86676, N1; comp84340, O2; comp83977, T1; comp56037, U2; comp83361, V2 and comp58004, Y1 in Figure 6) from the 25 TFs were randomly chosen, and their expression profiles in leaves or roots (where they were picked out originally, as listed in Supplementary file 7) were tested (Supplementary file 8) using similarly treated plant samples. The results showed that they had the same expression trends (Supplementary file 9) with the qRT-PCR output from the residual RNA ( Figure 6), and simultaneously correlated with the transcriptome data (Supplementary file 7). These data validated our transcriptome result. Comp56509, comp79146, comp80892, comp72628, comp73235, comp77912, comp78350, comp75203 and comp80372 (D, E, F, G, H, I, J, K and L, respectively) were from the NAC TF family. Comp80737, comp86676, comp84340, comp51469, comp54867 and comp33749 (M, N, O, P, Q and R, respectively) were from the MYB TF family. Comp95985, comp83977, comp56037, comp83361 and comp52860 (S, T, U, V and W, respectively) were from the zinc finger TF family. Comp28477 and comp58004 (X and Y, respectively) were from the bZIP TF family. All of them were chosen for qRT-PCR quantification, using GAPDH as an internal control (Supplementary file 8). The line charts A1-Y1 were carried out in leaves, while A2-Y2 were carried out in roots. Each line chart (A1-Y2) was formed from the order of samples subjected to minor, moderate, and severe drought stress (X-axis) for their quantitative results (Y-axis) in three biological replicates. The significance analyses were performed for each unigene among three samples using the ANOVA method of Sigmaplot software with a cut-off p-value of 0.05. * indicate that the corresponding unigenes shared regular expression patterns from leaves (all-up or all-down) or roots (up-down or down-up) where they were picked out (Supplementary file 7). The error bars indicate the standard error. Characterization of the Ramie Transcriptome The Illumina sequencing method, with the largest output and lowest reagent cost, has been widely used for deep sequencing of model and non-model organisms [12,17,18]. In the previous studies, the Illumina paired-end sequencing platform was used for high-throughput sequencing of the ramie transcriptome [12,13]. There were some evident advantages to this study. First, after stringent quality checking and data cleaning, approximately 33,976,322,460 bp (30G) of high-quality data (94.02% of the raw data) were generated under the Q20 standard, which was more than previous studies in ramie [12,13]. Second, based on the high-quality reads, the sequencing assembly yielded 138,381 (Supplementary file 1) unigenes, which were more than that created by other transcriptome sequences. Third, 16,798 (Supplementary file 5) genes (12.14% of all genes) were identified as DEGs in roots or leaves, which comprised 9842 genes (accounting for 7.11% of all genes) in the leaves and 9748 genes (accounting for 7.04%) in the roots of ramie. The number of DEGs was higher than that found in previous studies [12,13]. Finally, the six samples, specifically the leaves (L1-L3) and roots (R1-R3) under increasing drought stress, were determined by the physiological traits (RWC etc., as displayed in Figure 1) and phenotypes (data not shown). Thus, understanding on molecular regulation of ramie under drought stress was specifically ascertained by the results conducted from the ramie seedlings submitted to mild (L2 and R2) and severe (L3 and R3) drought stress compared with the control (L1 and R1). The transcriptome sequence generated in this study will be valuable for further ramie research on drought stress. Physiological Traits Changed under PEG Treatment The PEG-simulated drought approach has many advantages: The water potential (ψW) can be controlled precisely and a large number of treatments can be performed quickly [19]. In this study, 15% (w/v) PEG6000 was used to induce drought stress. Many abiotic stresses trigger the production of ROS, which disrupt normal metabolism by oxidative damage of membrane lipids, proteins, and nucleic acids [20,21]. SOD and POD (among other so-called scavengers) are able to eliminate these harmful molecules [22]. In this study, more unigenes were generated as all-up or all-down patterns than up-down and down-up patterns in leaves. However, more up-down or down-up patterns than all-up and all-down patterns were observed for the root genes (Table 2), which coincided with the trends of the physiological traits (RWC, MDA, and proline content shared all-up or all-down trends, while POD activity was decreased after increasing, as indicated in Figure 1). The Five Main Families of TFs Responding to Drought Stress in Ramie Proteins that are characterized as being involved in the protection of plant cells from dehydration stress damage include molecule chaperons, osmotic adjustment proteins [23], ion channels [24], transporters [25] and antioxidation or detoxification proteins [26]. Transcriptome analyses using microarray technology, together with conventional approaches, have revealed that dozens of TFs are involved in the plant response to drought stress [27][28][29]. The expressions of these stress-related functional proteins are largely regulated by specific TFs [30,31]. More than 30 families of TFs have been predicted to be associated with drought stress from Arabidopsis [23]. Members of the NAC, AP2, MYB, bZIP, and zinc-finger families have been shown to have roles in the regulation of plant defense and stress responses [23][24][25][26][27]. Unigenes that shared all-up or all-down patterns in leaves and up-down or down-up patterns in roots were chosen for further quantitative analyses. Specifically, 25 TFs from the AP2 (3), MYB (6), NAC (9), zinc finger (5) and bZIP (2) families were putatively identified as directly associated with drought stress. Further expression analysis of these genes ( Figure 6) and validation of randomly chosen unigenes (Supplementary file 9) by qRT-PCR confirmed their involvement in ramie's response to drought stress and were consistent with the transcriptome data. NAM (no apical meristem), ATAF1-2, and CUC2 (cup-shaped cotyledon) [32] constitute one of the largest TF families (NAC), and this family is plant-specific. NAC proteins play vital roles in many plant developmental processes [33] and also in biotic and abiotic stress tolerance [34,35]. The involvement of NAC TFs in the regulation of drought responses was first reported in Arabidopsis [35]; and NAC TFs have subsequently been shown to improve drought tolerance in rice [36] and cotton [7]. NAC TFs (encoding comp79664, comp77101, and comp41385) showed up-down or down-up expression patterns only in roots. Overexpression of the rice NAC gene SNAC1 improved drought and salt tolerance by enhancing root development [7]. The proline content was enhanced. The MDA content was decreased in the transgenic cotton seedlings under drought and salt treatments compared with the wild-type [7]. In this study, the up-down or down-up expression of three genes (comp79664, comp77101, and comp41385) might be related with the increase of MDA and proline contents throughout the entire drought stress periods. In a previous report, 18 NAC domain factors were identified from expression profiling of stress-treated rice [37]. OsNAC10 and SNAC1/OsNAC9 were believed to have similar stress response functions because of their sequence similarity [37]. OsNAC10-overexpression in rice resulted in enlarged roots, and enhanced drought tolerance and grain yield under field drought conditions [37]. These results suggested the NAC TFs might confer drought resistance through altered root architecture. The MYB TFs were previously reported to be regulatory genes in drought stress. They altered the expression levels of some drought stress-responsive genes and affected several physiological traits to overcome adverse conditions [38]. For example, AtMYB2 plays a role in ABA-dependent drought stress responses [39]. The expression of AtMYB41 is up-regulated when under drought, ABA or salt stress [40,41]. In a previous study, AtMYB60 (an R2R3-MYB gene of Arabidopsis) was shown to be involved in the regulation of stomatal movements [42]. AtMYB60 is a transcriptional modulator of physiological responses in guard cells, and its expression is negatively modulated during drought [42]. The water loss rate was lower in detached leaves from transgenic Arabidopsis compared with the WT leaves [43]. Overexpression of AtMYB44 enhanced stomatal closure to confer abiotic stress tolerance in transgenic Arabidopsis [44]. Arabidopsis, AtMYB88 and AtMYB124 are also involved in the regulation of stoma aperture development [45,46]. In this study, a MYB TF (encoding comp80737) was up-regulated, while comp86676, comp84340, and comp51469 were down-regulated in leaves under drought stress. Comp54867 was first up-regulated and then down-regulated, whereas comp33749 was first down-regulated and then up-regulated in ramie roots under drought stress (Supplementary file 5). MYB TFs have different expression patterns in the leaves and roots of ramie. One MYB gene (TaMYBsdu1) was markedly up-regulated in the leaf and root of wheat under long-term drought stress [47]. These data suggested that MYB TFs might have different expression patterns in different crops. The MYB TFs might play an important role in the regulation of stomatal movements and improving water retention. This hypothesis will require further experimental testing. The zinc finger TFs were previously reported to be positive regulators of plant tolerance to drought stress [48,49]. The transgenic lines that showed DgZFP3-overexpression (a drought stress-responsive Cys2/His2-type zinc finger protein gene) in tobacco plants did not accumulate as much H2O2 under drought stress. They accumulated more proline and showed greater POD and superoxide dismutase activities than in the WT plants under both the control and drought stress conditions [50]. DST (drought and salt tolerance) is a zinc finger transcription factor that negatively regulates stomatal closure via direct modulation of genes related to H2O2 homeostasis [51]. In this study, two zinc finger TFs (comp95985 and comp83977) were down-regulated in leaves under progressive drought stress (Supplementary file 5). They may be positive regulators in the increased accumulation of proline. Otherwise, two of them (comp83361 and comp52860) were first up-regulated and then down-regulated, whereas one (comp56037) was first down-regulated and then up-regulated in ramie roots under progressive drought stress (Supplementary file 5). We propose that the zinc finger TFs may play a key role in the increased accumulation of proline ( Figure 1d) and in the improved peroxidase (POD) (Figure 1b) activities, which would enhance ramie tolerance to drought stress. bZIP TFs were previously reported to be negative regulators of the drought stress response in rice [52] and tobacco [53], probably by deploying a better ROS-scavenging system to be an integral part of the defense against drought in the transgenic plants expressing PtrABF (a bZIP transcription factor) [53]. Our results ( Figure 6X1,Y1) were consistent with previously reported results [52,53]. We inferred that the bZIP TFs might play an important role in reducing the accumulation of ROS and increasing the activities and expression levels of antioxidant enzymes (POD, Figure 1b) during drought stress. The AP2/ERF family is a large family of plant-specific transcription factors that share a well-conserved DNA-binding domain [54]. This transcription factor family includes DRE-binding proteins (DREBs), which activate the expression of abiotic stress-responsive genes via specific binding of the dehydration-responsive element/C-repeat (DRE/CRT) cis-acting element in their promoters [54]. Transgenic alfalfa plants overexpressing WXP1 (a putative Medicago truncatula AP2 domain-containing TF gene) showed improved drought tolerance compared with the wild-type [55]. Transgenic Arabidopsis plants indicated an AP2/EREBF TF (AtERF7) reduced the sensitivity of guard cells to ABA and increased transpirational water loss [56]. In this study, two AP2 TFs (comp79664 and comp77101) were first up-regulated and then down-regulated, whereas comp41385 was first down-regulated and then up-regulated in roots under progressive drought stress (Supplementary file 5). We hypothesize that the AP2 TFs might have important roles in ABA responses during drought stress. However, more evidence is needed to support this conclusion. The putative drought stress-related TFs that were identified in this study will further increase our understanding of gene expression, transcriptional regulation, and signal transduction during plant responses to drought. Our data will also help future investigations into drought adaptation in ramie. We have also developed an efficient regeneration and transformation protocol for ramie via Agrobacterium-mediated genetic transformation [57]. Furthermore, ongoing research in our laboratory using genetic engineering and several functional or regulatory genes has demonstrated activation or repression of both specific and broad pathways related to drought tolerance in ramie. Plant Materials Preparation, RNA Extraction, and cDNA Library Construction In this study, the elite ramie variety, "Huazhu No. 5", was used as the plant material and was obtained from the Ramie Germplasm Resources Garden, located at Huazhong Agricultural University, Wuhan, China. Two weeks after planting, the "Huazhu No. 5" seedlings were propagated from stem cuttings, which were transplanted into half-strength Hoagland's solution for 20 days [7]. The period of seedling was when the plant height reached about 10 cm [7]. Physiological traits (the RWC, POD, the MDA, and the proline content) of ramie leaves at 0, 12, 24, 48, and 72 h after being subjected to drought stress (15% PEG6000) were measured to evaluate the severity of the stress. Every experiment was repeated at least three times. All stress experiments were conducted in a greenhouse at 28 ± 2 °C/23 ± 2 °C (day/night) with a relative humidity of 50%-70% under a 16/8-h (light/dark) photoperiod and a photon flux density of 350 µmol·m −2 ·s −1 . Based on the changing trends of these parameters, we chose three time points, which were 0, 24, and 72 h after being treated with PEG, to investigate the transcriptome. Total RNA was isolated from leaves and roots harvested at 0 (L1 and R1), 24 (L2 and R2), and 72 (L3 and R3) h, using an RNAprep Pure Plant Kit (Tiangen Biotech, Beijing, China) with three replicates. The RNA integrity and quality were confirmed by gel electrophoresis and using a NanoDrop 2000 spectrophotometer (Thermo, Waltham, MA, USA). Twenty micrograms of RNA were separately pooled from the six samples for cDNA library preparation. The residual RNA was used for qRT-PCR. After digestion by DNase I (Takara, Japan) at 37 °C for 30 min, poly(A) RNA was isolated from 30 μg of total RNA using Dynabeads ® Oligo (dT) 25 (Life, Lake Success, NY, USA). The volume of purified mRNA was adjusted to 100 μL before adding 100 μL Binding Buffer, incubating at 65 °C for 2 min and immediately cooling on ice. Pre-rinsed magnetic beads (100 μL) were then added and the samples were then mixed well for 5 min at RT, and placed in the magnetic shelf (Life, Lake Success, NY, USA) for 1-2 min; the supernatant was then discarded. The magnetic beads were washed twice using 200 μL of Washing Buffer B, resuspended by 15 μL of Tris-HCl (10 mM, pre-cooled), and then heated at 75-80 °C for 2 min. The supernatant containing the purified mRNA was then generated using the magnetic shelf. The cDNA library was constructed from 100 ng of mRNA using NEBNext ® UltraTM RNA Library Prep Kit from Illumina (NEB, Ipswich, MA, USA), following the manufacturer's instructions. The ligated cDNAs, ranging from 200 to 500 bp, were subjected to PCR amplification for 12-15 rounds using the Universal PCR Primer and the Index (X) Primer under the following procedures: 98 °C for 15 s, 65 °C for 30 s, and 72 °C for 30 s. Finally, the library was subjected to high-throughput sequencing after purification using an isometric volume of AMPure XP Beads (Agencourt, Webster, TX, USA). Sequence Assembly, Annotation, and GO Terms/KEGG Pathways Construction Transcriptome sequencing was performed for six equally pooled cDNA libraries, which were separately conducted from leaves (L1-L3) and roots (R1-R3) subjected to progressive drought stresses. Sequencing was performed on an Illumina HiSeq2500 genome analyzer. The raw reads were cleaned using FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/) to remove the adaptor sequences, empty reads and low-quality sequences (reads with unknown "N" sequences or those that were less than 20 bp long). The clean reads were assembled into non-redundant transcripts using Trinity [58]. The resulting sequences were used for Blastp searches and annotation against the Nr protein, the GO, the KEGG and the Swiss-Prot databases using an E-value cut-off of 1 × 10 −5 . The predicted protein coding sequences were annotated to the non-redundant protein databases in GenBank, Swiss-Prot, and TrEMBL using Blastp. GO mapping was carried out by GoPipe [59]. The KEGG pathway annotations were performed using Blast against the KEGG [14] databases. Differential Expression Redundancy and Enrichment Analyses After assembly and annotation, the universal reads from the six separately pooled samples (L1-L3, and R1-R3) were mapped to unigenes to calculate the RPKM value [15] for each assembled unigene. The differences in expression redundancy among the three samples from the leaves (L1-L3) or the roots (R1-R3) were determined using the RPKM value [15] for each unigene using the MARS model from the DEGseq program package [16]. The p value threshold was <0.001. The differentially expressed genes were used for GO terms/KEGG pathway enrichment analyses using the hyper geometric test to measure significantly enriched terms: where N is the number of genes with GO/KEGG annotations and n represents the number of differentially expressed genes in N. The variables, M and m, represent the total number of genes and the number of differentially expressed genes, respectively, in each GO/KEGG term. The threshold used to determine significant enrichment of the gene sets was corrected to a p value ≤0.05 and a false discovery rate (FDR) <0.01. Real-Time Quantitative RT-PCR The residual RNA from transcriptome sequencing was used for qRT-PCR. The RNA was reverse transcribed into cDNA using the GoScript Reverse Transcription System (Promega, Fitchburg, WI, USA), following the manufacturer's protocol. QRT-PCR was performed in a 20-μL reaction vessel containing 10 μL iTaq Universal SYBR Green supermix (Bio-RAD; Hercules, CA, USA), 10 pmoL of forward and reverse gene-specific primers, and 1 μL cDNA. Forward and reverse primers (Supplementary file 8) were designed for each gene of interest using Primer 3 online software version 4.0.0 [60] and adjusted by oligo software version 7.56 [61]. Additionally, primers specific to GAPDH were designed to serve as an endogenous control to normalize the data for differences in input RNA. PCR amplification was performed in an IQ5 (Bio-Rad, Hercules, CA, USA), according to the manufacturer's instructions. Following a denaturation step at 95 °C for 5 min, the amplification step comprised 40 cycles at 95 °C for 15 s and 60 °C for 30 s. A melting curve was constructed to determine the specificity of each PCR primer by maintaining the reaction at 95 °C for 1 min, cooling the sample to 55 °C for 1 min and finally heating to 95 °C at a rate of 0.5 °C per 6 s. The reactions were carried out in triplicate to ensure reproducibility. Estimations of the RWC, the MDA, the Proline Content, and POD The RWC was calculated according to the following method [48]: RWC (%) = (FW − DW)/(TW − DW) × 100 (2) FW, fresh leaf weights; TW, turgid leaf weights; DW, dry leaf weights. The MDA levels (a measure of lipid peroxidation) were assessed as previously described [7] by measuring the amount of thiobarbituric acid reactive substances present in the samples. MDA content (μmoL·L −1 ) was calculated using the formula: The extraction and colorimetric determination of proline from leaves were carried out according to a previously described method [39]. POD was determined using the guaiacol oxidation method as previously described [48]. Conclusions In this study, we used Illumina paired-end sequencing technology to characterize the transcriptome of ramie under progressive drought stress. After stringent quality checking and data cleaning, approximately 33,976,322,460 bp (30G) of high-quality data (94.02% of raw data) were generated from 170 million raw sequence reads. Differential gene expression analysis revealed 9281 putative genes in leaves and 8627 in roots that might be associated with drought tolerance. Among these, 25 TFs shared consistent expression patterns with physiological traits associated with drought stress, and thus they were picked out and validated by qRT-PCR. This study represents a fully characterized transcriptome, and provides a valuable resource for genetic and genomic studies in ramie plants, especially under drought stress. Our work is useful for breeding drought-resistant ramie varieties.

v3-fos

2016-05-04T20:20:58.661Z

2015-06-01T00:00:00.000Z

13943894

s2

Copper Tolerance and Biosorption of Saccharomyces cerevisiae during Alcoholic Fermentation At high levels, copper in grape mash can inhibit yeast activity and cause stuck fermentations. Wine yeast has limited tolerance of copper and can reduce copper levels in wine during fermentation. This study aimed to understand copper tolerance of wine yeast and establish the mechanism by which yeast decreases copper in the must during fermentation. Three strains of Saccharomyces cerevisiae (lab selected strain BH8 and industrial strains AWRI R2 and Freddo) and a simple model fermentation system containing 0 to 1.50 mM Cu2+ were used. ICP-AES determined Cu ion concentration in the must decreasing differently by strains and initial copper levels during fermentation. Fermentation performance was heavily inhibited under copper stress, paralleled a decrease in viable cell numbers. Strain BH8 showed higher copper-tolerance than strain AWRI R2 and higher adsorption than Freddo. Yeast cell surface depression and intracellular structure deformation after copper treatment were observed by scanning electron microscopy and transmission electron microscopy; electronic differential system detected higher surface Cu and no intracellular Cu on 1.50 mM copper treated yeast cells. It is most probably that surface adsorption dominated the biosorption process of Cu2+ for strain BH8, with saturation being accomplished in 24 h. This study demonstrated that Saccharomyces cerevisiae strain BH8 has good tolerance and adsorption of Cu, and reduces Cu2+ concentrations during fermentation in simple model system mainly through surface adsorption. The results indicate that the strain selected from China’s stress-tolerant wine grape is copper tolerant and can reduce copper in must when fermenting in a copper rich simple model system, and provided information for studies on mechanisms of heavy metal stress. Introduction Copper (Cu) is unavoidable in winemaking: long-term use of copper fungicide [1][2] may increase the copper level in soil [3][4] and grape berry; winemaking equipment [5] and copper selecting wine yeast strains with high copper-adsorption and resistance to high level copper in grape must during alcoholic fermentation, and could give a better understanding on the adsorption mechanism of Saccharomyces cerevisiae to copper. Yeast strains Three Saccharomyces cerevisiae strains were used; one laboratory strain, BH8 (B), separated (from BeiHong grape must) and stored at the laboratory (China Agricultural University, Beijing), identified as S. cerevisiae by Institute of Microbiology, Chinese Academy of Sciences [29]; two industrial strains, AWRI R2 (A; Maurivin Co., Australia) and Freddo (F; Erbslöh Co., Germany), commonly used by Chinese winemakers for their good fermentation performances. Fermentation experiments CuSO 4 5H 2 O was added into MSM in a graded Cu 2+ series of 0 (control), 0.50 mM (32 mg/L), 1.00 mM (64 mg/L) and 1.50 mM (96 mg/L) [12]. 4 mL yeast precultures were inoculated in 500 mL flasks containing 400 mL MSM to obtain a density of 10 6 cells /mL [29]. Flasks were sealed with glass capillary stoppers filled with concentrated H 2 SO 4 to prevent weight loss caused by water evaporation . Cultures were constantly shaken at 28°C, 120 r/m in thermostatic shaker (SKY-2102C, Shsukun Co. Ltd., Shanghai) [29]. Mass loss caused by CO 2 evolution was monitored by weighing the fermentation flasks every 24 h [26]. Fermentation was considered to have stopped when mass loss was less than 0.02 g for 3 days. Samples of fermentation must were taken before and every 24 h after inoculation. Fermentation experiments were separated into two groups: one group for weighing, another group for sampling, and each group was carried out in triplicate. Determination of cell growth and viability Cell growth was followed by measuring OD 600 of the fermenting MSM [29] with a UV1800 spectrophotometer (Shimadzu, Japan). MSM free of Cu 2+ was used as blank control. Viable cell level of strain B was determined by cell counting using the following procedure: 1 μL of five times diluted MSM sample was embedded on a cytometer and dyed with 1μL of 0.1% methylene blue, a dye commonly used in distinguishing viable and dead cells as it only stains dead cells. Total and viable cell were counted using optical microscope (COIC XSZ-3G) with 40× object lens. Survival rate was calculated following the equation viability % = V/T (V: viable cell amount; T: total cell amount). Analysis of fermentation performance The remaining reducing sugars and ethanol content in samples taken during alcoholic fermentation were determined by HPLC using Waters 2414 RI Detector and BIO-RAD Aminex HPX-87H resin-based column (300 Ã 7.8mm) [31], which was eluted with 5 mM H 2 SO 4 at 65°C, 0.6 mL/ min. Statistical differences for cell growth and fermentation performance of the strains were analyzed using single variable general liner model with PASW Statistics 18. Analysis of Cu biosorption Cu adsorption of wine yeast strains were determined by measuring remaining copper in their fermenting MSM. A series of sterile MSM with 0.50, 1.00 and 1.50 mM Cu 2+ were used as blank control. Samples taken during fermentation were filtrated by 0.45-μm cellulose acetate membrane filters; 4 mL of filtrate was dried at 105°C in 50 mL conical flask in dust-free drying oven, then digested with 5 mL HNO 3 -HClO 4 (4:1, GR) adding in the flask which was then covered with watch glass, and heated on hot plate at 80°C for 2 h, then 120°C for 2 h, and 190°C until no white fog visible in the flask and the remaining liquid being clear and colorless; digested samples were washed by 18.2 MO ultrapure water and filtrated to 25 mL. Glassware were soaked overnight in 20% HNO 3 and washed with ultrapure water before used. Cu concentrations of pre-treated samples were determined by ICP-AES (Perkin Elmer Optima 2000DV) at 327.393 nm. Removal ratio η and adsorption efficiency A (mg/g) of copper ion on yeast were calculated according to equations: η = (C 0 -C 1 )/C 0 , and A = (C 0 -C 1 ) ×V/M, where C 0 and C 1 are initial and final Cu concentrations of MSM ferment, respectively, and V represents volume of sample, M means dry weight of yeast separated by centrifuge from the sample Structural analysis of yeast cell Yeast cells for SEM-EDS and TEM-EDS were harvested by centrifugation of 10 mL of MSM sample at 4000 rpm (4°C, 10 min). SEM-EDS Scanning electron microscope (SEM) was used to analyze the extracellular structure; and energy dispersive spectrometer (EDS) was used for surface elemental composition analysis. Harvested yeast cells were washed in deionized water three times by centrifugation and resuspension. The cells were fixed with 2.5% glutaraldehyde-PBS overnight, washed in 30% PBS buffer (0.1 M, pH7.2) for three times (20 min each time), then post-fixed with 1% osmic acid (1 h), and washed three times with PBS. They were then dehydrated using ethanol with increasing concentrations (v/v) (30%, 50%, 70%, 80%, 90%, and 100%, each for three times, 20 min each time, followed by isoamyl acetate exchanging (three times, 20 min each time), critical point drying and gold crystal spraying. Pretreated cell samples were examined with SEM (Hitachi S-3400N) and SEM-EDS (Jeol JSM-6510A) [32]. Cell growth Growth of all three yeast strains weakened as copper concentration increased (Fig 1A, 1B and 1C). Their growth in the control MSM was fastest, reaching log-phase and the end of growth after approximately 6 h and 48 h respectively, with maximum OD 600 reaching about 2.25. For the 0.50 mM Cu 2+ medium, the growth curve of strain F mimicked control but was delayed by about 12 hours; strains A and B were slower to start and the log-growth phase tapered off sooner than their controls although OD 600 ultimately reached same levels as control. As copper concentration increased, all three strains were increasingly sluggish in growth with longer lagphases and extended log-phases with lower growth rates, reaching stable-phase after 72 h with biomass (OD 600 ratio) of approximately 80% of control. Copper was clearly inhibiting yeast growth. These results demonstrate that strain F had the highest growth and was the earliest one reached stable-phase, indicating a greater copper tolerance. Of the three strains strain B is more copper-tolerant than strain A and less than strain F. Fermentation performance For all three strains the control ferments progressed most rapidly (Fig 1D, 1E 1F and Fig 2) in the first four days and remained stable after nine days. Total CO 2 evolution was approximately 35g; residual reducing sugars were approximately 4g/L and ethanol concentration approximately 11% (v/v), with no statistical difference (CI = 0.95), indicating good fermentation performance by all strains. However in copper containing MSM, fermentations of all three strains were significantly affected, being sluggish or even becoming stuck, depending on Cu 2+ concentration. In the 0.50 mM Cu 2+ MSM, after 12 days total CO 2 evolution of the three strains were less than 50% of their controls with statistically significant differences. Fermentation in 1.50 mM Cu 2+ MSM lengthened to 14 days with total CO 2 evolution being less than 23% of controls (9.08% (A), 15.59% (B) and 22.25% (F)). Corresponding to the growth curve (Fig 1A, 1B and 1C), the curves for the reducing sugars (Fig 2A, 2B and 2C) and ethanol (Fig 2D, 2E and 2F) became stable earlier and changed less with increasing copper concentration. For the corresponding concentrations of Cu 2+ , growth activity and fermentation efficiency (Figs 1 and 2) of strain F was the highest, followed by strain B with strain A being the lowest (such as under 0.5 mM, at 24h, the OD value of strain A was 1.171, strain B 1.208, strain F 1.245; the alcohol production of strain A was 0.81, strain B 0.82, strain F 0.88) Copper biosorption It can be seen from Fig 3that Cu ion concentration of MSM ferments for all three strains went down with time during fermentation, with first four days decreasing rapidly and later period slowly (Fig 3), corresponding to the fermentation performance ( Fig 1D, 1E 1F and Fig 2), indicating relations between yeast activity and copper biosorption. Contrarily, Cu ion concentrations in control with no yeast did not reduce significantly (Fig 3). Higher initial copper concentration correlated with lower removal ratio and higher adsorption efficiency; this can been seen from a significant drop of Cu removal ratio ( Fig 4A) between groups of 0.50 mM and 1.00 mM initial Cu 2+ and a leap upward of Cu biosorption by unit yeast (Fig 4B) between groups of 1.00 mM and 1.50 mM initial Cu 2+ for all three yeast strains. The increase of adsorption efficiency as initial Cu concentration rises could be explained by yeast biomass decrease. Compared with strain A and F, strain B showed a medium removal ratio and adsorption efficiency under all three initial Cu levels (0.50, 1.00 and 1.50 mM; Fig 4). Among three yeast strains, strain A showed the strongest removal ratio (67.37%, Fig 4A) in 0.50 mM Cu and highest adsorption efficiency (15.82 mg/g, Fig 4B) in 1.50 mM Cu. Impacts of Cu on survival rate To directly reflect copper's lethal effect and the copper tolerance of strain B, living and dead yeasts were counted separately to calculate survival rates at different Cu 2+ concentrations. The survival rate of strain BH8 ( Table 1) increased with time in 24 h at each Cu 2+ level, and Impacts of Cu on surface morphology and element SEM images (Fig 5) show that toxic effects of Cu 2+ on strain B lead to increasing changes in micromorphology with increasing time and Cu 2+ concentration. EDS results (Fig 6) indicated cell surface of strain B mainly consists of carbon (C; over 60%) and oxygen (O; over 20%), with the mass fraction (Mass %) and atom% (at %) of Na decreasing with Cu increasing with time after Cu treatment. Gold (peak at 2.00 to 3.00 keV) parameters were not calculated with EDS since gold was sprayed on cell surface during the preparation for SEM testing. As the SEM images showed, yeast cells for the controls (Fig 5A and 5B) were orbicular-ovate, 4 to 6 μm long and 2 to 4 μm wide with smooth surfaces and no intercellular adhesions; besides that, there were also a little fold occurred on some individual cells, which could be a natural consequence of the sample preparation (centrifugal, deionized water washing, and ethanol dehydration) [34]; what's more, buds and bud scars were no more than three for per cell. EDS didn't detected Cu peak (Fig 6). In 0.50 mM Cu 2+ treatment level, most cells remained regular oval at 24 h ( Fig 5C), but cell deformation and pitted surface became obvious and occurred on more cells; after 48 h, there were more bud scars for per cell (Fig 5D). Meanwhile, potassium (K) was undetectable with EDS while Cu was detected with atom% less than 0.05% (Fig 6). At 1.00 mM Cu 2+ , the cells were slightly deformed and pitted after 24 h (Fig 5E) and significantly stretched with deep pits on most cells after 48 h (Fig 5F). For 1.00 mM Cu 2+ treatment, the EDS results was resembles to those of 0.50 mM with no K peak; copper was slightly higher in mass% but still low as atom% of 0.05% (Fig 6). In 1.50 mM Cu 2+ treatment, cells were mostly rough and significantly pitted on the surface with some being stretched with adhesion by 24 h (Fig 5G), and almost all yeast cells were deformed having significantly rough and uneven surfaces by 48 h (Fig 5H); nitrogen (N) was detected in increasing amounts with fermentation time and Cu was higher in the EDS results, K peak was also not detected (Fig 6). Base on these results, we deduced that the disappearance of K peak and decreasing of Na peak with increasing of Cu peak with time after Cu treatment might have certain relations. (Fig 7A) was a normal oval shape with complete cell walls and plasma membranes. The cell wall thickness was even, the organelles dispersed in plasma, and the vacuoles were small and of a similar size. In contrast the yeast taken after 48 h from the MSM ferment containing 1.50 mM Cu 2+ (Fig 7B), had rough cell walls and plasma membranes which is in agreement with its SEM image ( Fig 5H). Plasmolysis occurred with uneven plasma distribution and organelles could not be distinguished. This cytoplasm contraction could be related to Cu 2 + induced lipid peroxide activity in the plasma membrane. Fig 8 shows the intracellular element ratios determined by TEM-EDS using the same S. cerevisiae BH8 sample of Fig 7. Lead (Pb) was introduced into yeast cell from the sample preparation. Yeast mainly consists of carbon and oxygen, which is in agreement with composition of yeast surface (Fig 6). Compared to the control, yeast in 1.50 mM Cu 2+ for 48 h had no significant changes in C and O although there was some N present. No copper peak was detected, and the atomic ratio (Atomic %) of Ni decreased by 0.54 (Fig 8), indicating Ni + leakage after cell membrane deformation. That no Cu was detected inside the yeast cells suggests that strain B does not accumulate Cu 2+ in its cell and living cells of strain B reduce Cu 2+ mainly by surface adsorption. Impacts of Cu on intracellular morphology and element The mechanism of surface Cu 2+ adsorption could be further studied with atomic force microscopy and confocal laser scanning microscopy. However the sample preparation method needs to be studied since the reported methods for other microorganisms have failed on Saccharomyces cerevisiae. Discussion As there are many restrictions on natural grape juice, such as the supply of seasonal restrictions, the difference of grape juice composition caused by viticulture region and grape varieties, and the effect of solid composition of natural grape juice on separation of yeast cells, for a long time the studies on Saccharomyces cerevisiae with different researchers were hard to consistent. And MSM has the advantages of easy using, good reproducibility et al [30], in this study, we chose MSM rather than natural grape juice as fermentation medium. At a low concentration range, copper is a necessary metal elements for biological growth and metabolism and cofactors for intracellular enzymes metabolism [27]. But once grossing over the beneficial range, it will have inhibitory effect on cells, even toxicity [35]. In wine making, high copper content also affects the wine fermentation process and wine quality. In this experiment, in the MSM medium without copper, all three strains showed a good growth activity and fermentation performance, and the growth curve, CO 2 release quantity, reducing sugar utilization and alcohol production were similar between these three strains (Figs 1 and 2). Once copper was added into the MSM medium, the growth of Saccharomyces cerevisiae was delayed even stagnated, and the effect was positive correlated with the copper concentration. This was in agreement with the result of Shanmuganathan et al [36]. A possible reason for this could be that the yeast cell accumulates large amounts of reactive oxygen species (ROS) at the high concentrations, leading to protein denaturation, membrane order alteration and damage to intracellular enzyme and consequent reduced metabolism, and ultimately cell death [36]. Therefore, anaerobic fermentation was not possible with the reducing sugars not being able to be used for energy. For the copper biosorption of Saccharomyces cerevisiae, in this experiment, even if they didn't add yeast, the copper concentration would reduce slightly with the extension of time (Fig 3A). The possible reason was that copper and a small amount of sulfur ions in the solution formed precipitation [25]. After fermentation, the removal rate was 14.86~67.37% and the adsorption efficiency was 5.88~15.82 mg/g. This was in consistent with Volesky et al study [37] and Donmez et al study [38], in their study, the adsorption efficiency was 2~40 mg/g. With different yeast strains, the adsorption quantity of Saccharomyces cerevisiae was different. For these three strains, the highest removal rate and the highest adsorption efficiency were all strain A, while strain F was the lowest. In Brandolini et al study [26], the cell growth and fermentation performance of copper resistant yeast strain behaved better than normal yeast strain, and could absorb more copper ions too. By contrast, in this experiment, strain B behaved better on cell growth and fermentation performance under copper stress, but for the copper removal ratio, strain A was better. Liu [39] also reported similar results. This difference might be related with the tested strain features. Also, with different initial copper concentrations, the adsorption quantity of Saccharomyces cerevisiae was different. In this experiment, though with higher initial copper concentrations, the toxicity of copper on yeast was higher and leaded to yeast biomass decreased and the removal rate decreased, but the adsorption efficiency increased, which was in agreement with the previous research results [40]. In order to could give a better understanding on the adsorption mechanism of Saccharomyces cerevisiae to copper, SEM-OES and TEM-OES were used to observe the ultrastructure change and elemental transformation. In SEM-OES observation, yeast cell surfaces became uneven after copper adsorption together with potassium peak disappeared while copper and nitrogen appeared, but the contents of these elements were very low. It might be under coverage of the high gold peak value, or indicating a small capacity of surface copper adsorption by strain B. With the increasing of the initial copper concentration, the cell surface was more and more roughness, the copper content was getting higher and the K:Cu ratio continued decreased (Fig 8), indicated that the adsorption of copper might be associated with the release of potassium from the cell surface. When the copper concentration reached 1.50 mM, nitrogen peak appeared on cell surface, and with the extension of time, the nitrogen peak enhanced. This might because that the copper began to complex with Nitrogen groups of MSM. This was in agreement to Brady et al study [40]. They found 70% of K + was rapidly released during Cu 2+ biosorption in waste water. Hence, it might indicate that ion exchange was involved in the biosorption of Cu 2+ during fermentation. With the extension of time, though the cell surface was more and more roughness, the copper content was basically remain unchanged, which means the copper adsorption quantity on the yeast surface had reached saturation at the point of 24 h or before. In previous study in waste water, the adsorption of Saccharomyces cerevisiae on copper was divided into two stages, the first phase happened quickly on cell surface without energy consumption and the second phase was a long and slow intracellular accumulation and transformation process involving metabolism [38,40]. The SEM-OES results were fit with the first phase. Then TEM-EDS was used to observed the intracellular structure and elemental transformation. Under copper stress, the thickness of Saccharomyces cerevisiae cell wall was not uniform, the cytoplasm shrank and uneven distribution, organelles couldn't be recognized. The reason of cytoplasm shrank might be related with the lipid over oxidation of cell plasma membrane. When the copper concentration reached 1.50 mM, nitrogen peak appeared on cell surface, the intracellular Potassium content reduced, further illustrated ion exchange was involved in the biosorption of Cu 2+ during fermentation. And there was no copper detected in intracellular, indicated that Saccharomyces cerevisiae could not transport copper into internal. In combination with the results of SEM-OES, the main adsorption mechanism of Saccharomyces cerevisiae to copper during alcoholic fermentation was cell surface adsorption. As to whether intracellular accumulation exists, it still needs further studies to confirm. In conclusion, copper stress could delay even stagnate the growth of Saccharomyces cerevisiae, reduce the reducing sugar uptake and ethanol production, and the degree was related to the initial copper concentration and strains. The copper tolerance and copper adsorption ability of strains showed a negative correlation. After Saccharomyces cerevisiae adsorbed copper, the yeast surface and intracellular all changed irregularly. Ion exchange was involved in the biosorption of Cu 2+ during fermentation, and the main adsorption mechanism of Saccharomyces cerevisiae to copper during alcoholic fermentation was cell surface adsorption, reaching saturation in 24 h. Supporting Information S1

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2019-03-28T13:41:18.903Z

2015-01-10T00:00:00.000Z

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Evaluation of the Nutritive Value of Selected Browse Plant Species in the Southern Guinea Savannah of Nigeria for Feeding to Ruminant Animals One of the major problems of ruminant production in the tropics is the scarcity of quality forage all the year round. Gliricidia sepium which remains green even during drought has been extensively used for forage during forage scarcity. However, there is need to screen more browse species to broaden the feed base to improve ruminant animal production. Ten common but less utilized browse plants ( Ficus polita , Ficus cogensis, Daniella oleiveri , Prosopis africana, Parkia biglobosa , Gliricidia sepium , Ecliptaalba , Albizia odoratissima , Polyalthia longifolia and Moringaoleifera ) were evaluated for their nutrient composition. Results showed that crude protein content was highest (29.60%) in Moringa oleifera and lowest (15.84%) in Daniella oleiveri . Crude fibre compositions ranged between 13.87% - 29.33% in Albizia odoratissima and Parkia biglobosa respectively. A range of 3.25% - 6.30% and 6.68%-9.45% values were recorded for EE and ash respectively for the ten browse plants. The values reported for mineral content showed that M . oleifera had the lowest value of Ca (0.68%), P (0.15%) and Mg (0.25%). Eclipta alba was observed to be richest in mineral elements amongst the browse species with the highest concentrations of Ca, Mg, P, K, Zn, Fe, Cu and S. The fibre contents of the forages showed a range of 42.41 to 61.31, 29.51 to 49.58, 5.71 to 9.27, and 10.98 to12.90 for NDF, ADF, ADL and hemicelluloses respectively. The non-fibrous carbohydrate (NFC) contents of the browse species were observed to vary widely ranging from 6.59 ( Pakia biglobosa ) to 22.16% ( Gliricidia sepium ). The values reported for anti-nutritive factors (tannin, saponin, phytate and oxalate) were low in all species. The results of this study showed that the browse species in general contained high levels of nutrients and hence have potential as good feedstuffs for ruminant animal feeding. INTRODUCTION Ruminant animals play a very prominent role in Nigeria most especially in the southern part of the country. Ruminant animal production is a means of sustainability to people living in rural, peri-urban and urban areas. Livestock, in a mixed farming system, serves as financial reserve for the populace to face risk and uncertainty in harsh weather [1] when crops may fail. Products such as milk, meat, manure are obtained majorly from ruminants to improve people's livelihood and provide critical nutrients to support food and health. Nonetheless, poor feeding and quality of feeds available have hindered the animal productivity. Good quality forage is of importance for high productivity of animals all the year round. Studies have shown that feed intake [2] contributes to animal's productivity but milk yield and growth of ruminant animals are largely affected by the quality of forage [3]. However, due to high cost of conventional feedstuff, some workers [4,5,6] both in Nigeria and abroad have conducted research on the utilization of alternative feed ingredients-particularly browse plants which are not utilized as human foods thus not in direct competition with man [7]. Many of these browse species that may be relished by ruminants require further evaluation to determine their nutritive values for livestock. Bamikole et al. [8] in their evaluation of some Ficus species observed that the foliage of browse plants are the reservoir of valuable nutrients such as proteins, vitamins and minerals that are not found in grasses and other crop residues, that can be critically important most especially during the dry season. Sheep, a ruminant specie, are usually on popular demand during the Muslim festivals in Nigeria, especially in the parts of the country where the population is predominantly Muslims. However, inadequate nutrition has been a main factor limiting its production in the tropics, mainly attributed to the unavailability of feed all year round. Composition of feed given to animals has a very significant influence on animal performance such as fibre, milk production/quantity as well as growth performance (meat production). Various physiological stages -maintenance, growth, pregnancy, lactation, and fiber -production underlie the differing nutritional requirements of livestock. Dry season, in Nigeria signifies a period of scarcity of quality feeds to animals most especially ruminants dependent on forages as the major component of the diet. The conventional concentrate or supplemental feed resources (grains) available during this critical period are very expensive to feed ruminants because of the direct competition with human food needs. This situation has indeed necessitated the search for cheaper and readily available feed materials that can meet the nutritional requirements of farm animals for better productivity. The multipurpose trees (MPTS) used as fodders are unique in that they may also serve other purposes such as food, fruits, fibre, timber, wood, shade, live fences etc across the agro-ecological zones in Africa and in the world. In addition, they act as carbon dioxide (CO 2 ) sinks from the air and create a favourable environment during harsh weather. Most of these MPTS have hitherto not been systematically exploited for strategic year round livestock production; especially during dry the season to boost improved productivity. Nonetheless, much research had been carried out on the use of MTPS as feed resources for livestock. Luu et al. [6] studied the introduction and evaluation of Moringa oleifera and observed that the plants could be harvested seven times/ year, and the annual fresh biomass yield could be from 43 to 52 tones/ha, used as a sole feed for goats. During the early growth stage of these browse plants, arable crops can be intercropped which would serve as a source of additional income to the farmer. Ogunbosoye and Babayemi [9] reported that the daily weight gain of animals fed Albizia odoratissima was highest when compared with other existing and well recognized browse trees. In his study, Teferedegne [10] pointed to the role being played by ruminants in the livelihood of farmers in the developing world, including milk, meat, animal traction and manure for improved crop production. He further reported that local trees (legumes) have been investigated as potential supplements for ruminants because of their beneficial effect of increasing metabolizable energy, N intake and feed efficiency and thereby improving animal production. He reported that foliage of some tree plants, however, has been shown to be selectively toxic to rumen protozoa with chemical compound acting as anti -protozoa or natural defaunating agent. Khanal and Subba [11] also evaluated nutritional composition of some fodder trees in Nepal with result further revealing that they could be relevant to animal nutrition. Evaluation of more feeds will provide nutritionists with the necessary information to formulate diets from both a physiological and an economical point of view in order to optimize animal performance. Therefore, the objective of this study was to measure the nutritive characteristics of selected fodder species as feeds for ruminant animals in Nigeria. Collection of Forages The legumes (Parkia biglobosa, Gliricidia sepium, Prosopis africana, Albizia odoratissima) and non-legumes (Daniella oleiveri, Eclipta alba, Ficus polita, Ficus cogensis, Moringa oleifera, Polyalthia longifolia) browse trees and shrubs were collected at the peak of the dry season (February 2014) from Malete (Lat 8º 71ʾN and Long 4º 44ʾE), Moro Local Government, Kwara State, Nigeria. Fresh leaves were defoliated, oven dried to a constant weight at 105ºC to determine the dry matter and later ground to pass through a 1 mm sieve for subsequent analyses. Chemical Analysis The ground samples were analyzed for crude protein, crude fibre, ether extract and ash contents following standard procedures as outlined by AOAC [12]. Crude protein was determined by the micro-kjeldahl procedure (N × 6.25), ash by incineration at 500ºC for 2 hours in a closed furnace and acid detergent fibre (ADF), neutral detergent fibre (NDF), acid detergent lignin(ADL)were assessed using the method proposed by Van Soest et al. [13] Analysis for mineral levels of the sampled browse plant species was determined by wet digestion using nitric-perchloric acid and mixture. The mineral elements Calcium (Ca), Magnesium (Mg), Iron (Fe), Copper (Cu), Zinc (Zn, and Manganese (Mn) were determined by atomic absorption spectrophotometer (AAS) (Model 490 Gallen Kamp London, UK while the flame photometer (FP 419 Corning) was used to estimate the Sodium (Na) and Potassium (K) elements. Phosphorous concentration was determined colorimetrically as outlined by Parkinson and Allen [14]. The non-fibrous carbohydrate (NFC) was calculated as 100 -(%NDF + % CP + %EE + %Ash) using the equation of NRC [15] where NDF is the neutral detergent fiber, CP is the crude protein and EE is the ether extract of the tested browse plant species. RESULTS The chemical composition of the browse foliage is shown in Table 1. The browse plants were grouped in to legume (n=4) and non-legume (n=6). Although, there was no distinct different between the two groups, however, significant variations (P=0.05) were observed among the nutrients measured. Both the highest and lowest crude protein (CP) content were recorded within the non-leguminous plant species with Moringa oleifera highest (29.6%) and lowest Daniella oleiveri (15.84%). All the plant species in this study have their CP level within the acceptable range (7-14%) for ruminant diets. Variations were observed in the crude fiber contents of the browse plants. The legume (Parkia biglobosa) had the highest percentage while the least value was recorded against Daniella oleiveri (nonlegume). The highest value of ether extract was obtained in Daniella oleiveri which is an indication of high energy. The ash contents ranged from 6.68% to 9.48% in Polyalthia longifolia and Albizia odoratissima respectively. There was no significant difference in the NFC contents of the browse species. The fibre fraction of the foliage of browse plants also indicated that legumes had the higher contents of NDF, ADF, and ADL than the non-leguminous browse plant species. Table 2 shows the composition of macro and micro mineral concentrations of the browse plants. The highest concentration of all the mineral components was observed in Eclipta alba leaves which is a non-leguminous plant. This is showing the richness in minerals of Eclipta alba. Table 3 is the anti-nutritional factors of the foliage of browse plant species. The results revealed that all the browse species contain low anti-nutritional factors. Indicating that animal will be able to consume these plants to their satisfaction because none of the values was up to 1%. DISCUSSION The value of CP content in M. oleifera reported here is comparable to that reported by Fadiyimu et al. [19] for browse plants which was between 17.1 and 31.2%. All the browse species in this study have their CP level higher than the acceptable range (7-14%) for ruminants [20]. The crude protein content of all the browse in this study was however higher than the values obtained for species of Ficus and Spondias mombin [8,21]. A higher CP concentration of Moringa was observed when compared with what was obtainable elsewhere [6]. Also, Ogunbosoye [5] reported a lower percentage CP (15.99%) for Albizia odoratissima. The difference in CP content among species and between values from samples collected in different studies can be explained by inherent characteristics of each species related to the ability to extract and accumulate nutrients from soil and/or to fix atmospheric nitrogen, which is the case for leguminous plants. The other factors causing variation in the chemical composition of browse may be soil type (location), the plant part (leaf, stem, pod), age of leaf and season at which the plants were harvested. With regard to the location, some authors have reported that browse plants in the sahelian zone are higher in nitrogen compared to plants in the humid zone Rittner and Reed [22). This may likely be the case of the present study which was carried out in Southern guinea savannah area. The CP contents of these browses were higher than the minimum of 7-8% necessary to provide the minimum ammonia levels required by rumen micro-organisms to support optimum rumen activity Norton [23]. In addition, high protein in the forage should be aimed at as it will favourably enhance intake and digestibility. This indicates the browse plants under this study may be well utilized as a protein supplement to low quality feed such as grasses and crop residues due to a higher level of crude protein of all the browse plant species. The highest value of ether extract obtained in Daniella oleiveri is an indication of high energy feed. Ether extract is the lipid component and the energy derived from it is utilized by the animal for body maintenance and production. Foidl et al. [24] reported that EE in the feed is also a source of carotene and pigment, but it is observed in this work that EE was generally low and therefore may indicate a low level of carotene and pigments. Nonetheless, the EE value was higher than the report (0.31-1.08%) of Isah et al. [21] on some browse species in Edo State, Nigeria but lower than what Luu et al. [6] Ogunbosoye and Otukoya [25] reported on Moringa and some fodders in Nigeria (5.95 -18.58%). The result, nonetheless compared favourably to the report of Mbomi et al. [26] for Tephrosia species (3.39 -4.26%). The ash content of any feed indicates the mineral level and the values in this present work compared very well with the observations made in another study [26] which reported ash ranges of 9.79 to 11.85%. Non-fibrous carbohydrate (NFC) level of the fodders is higher than the values obtained by Mirzael-Aghsaghali et al. [27] suggesting these fodders to be good source of energy to ruminants. The NFC serves as sources of energy in the diets of ruminant animals but the optimal dietary of NFC in dairy diets is suggested to be between 30-40%DM [28]. The values of NFC of these browse species is moderate and may serve as good diet for all classes of ruminant animals. The fiber concentration of the tested fodders falls within the range reported by [29] but NDF and ADF contents were higher compared to the values reported by Njidda [30]. Meissner et al. [31] observed that NDF level of forage above 65% can limit feed intake. However, it is interesting to note that there is no browse species of the present study that is up to that threshold level. In general, the NDF and ADF are within the range (24 -61% and 17-61% respectively) documented for forages used in ruminant feeding [32,33]. This is suggesting that the intake of the tested browses will not be hindered. The lignin contents of browse species in question is moderate as it is known that lignin is a component of the cell wall and deposited as part of the cell wall-thickening process [34]. All the browse forages had lower Ca than the recommended requirement (g/kg -1 DM diet) for growing cattle (2.6-10.8), pregnant cows, (2.1 -3.5) and lactating cows (2.9 -5.3) [35]. Router and Robinson [36] suggested Ca requirement for maintenance of growing and lactating sheep to be 1.2-2.6 g/kg. Eclipta alba, Daniella oleiveri and some of the browse species could meet these requirements. The browse forages had lower levels of P compared to values obtained from other parts of the world. Aganga and Mesho [37] reported lower values of P for browse forages of [35] for browses of Sudan. The observation in the P contents of these browse species suggests that supplementation with phosphorus rich feed is highly necessary. The variation in the content of observed P could be due to the available soil P and soil pH, browse growth stage and proportions of leaf and stem fractions harvested for mineral analyses and sampling season. These are factors that could influence the concentration of phosphorous of any plant species. All the browse samples analyzed had sufficient Mg level in agreement with the report of Khan et al. [38]. Based on Minson's [3] recommendation of (2.0 g/kg DM Mg) in the diet of ruminants, the browse plants examined had higher levels of Mg. Shamat et al. [35] reported that Mg in tropical forage was not considered to be limiting, although Jumba et al. [39] reported exceptionally low concentrations in Kenyan forages. Na level in this study is observed adequate compared to normal levels (0.36 to 0.57% DM) reported by [35] for other browse forages with exception of Polyalthia longifolia, Grilicidia sepium and Moringa. There is general agreement that Na is deficient in most tropical grasses [40]. Sodium deficiency can be corrected by providing salt lick ad libitum which can satisfy the requirement for chloride. It has been suggested that high producing ruminants may require dietary potassium levels above 10 g/kg under stress particularly heat stress [41]. Nevertheless, the browse species had higher level of K compared to the report of Isah et al. [21]. Greene et al. [42] and Grings et al. [43] that K concentration appeared to vary as a function of absolute age of the leaf as well as the environmental conditions. The level of K is in consonance with the 70 mg/kg DM recommended daily to meet requirements for a goat weighing 50.0 kg [44,45]. The concentrations of S in the study were consistence with the wide range of data reported [33]. Regarding micro minerals, these species had higher levels of Fe than tabulated requirements of Fe for dairy and beef cattle (50 mg/kg DM) [46]. Although, its availability could vary due to the fact that Fe is absorbed according to the need and thus its absorption would depend on dietary factors, age of the animal and body Fe status. It has been suggested that 30 mg/kg Zn is a critical dietary level. Tiffany et al. [47] in north Florida reported similar values. High forage concentration of Mn in the dry season was detected and attributed to low rates of Mn translocation and accumulation of Mn in older tissue [46]. All plant species had higher levels of Mn than the normal dietary requirements of 20-40 mg/kg -1 DM [15]. Spears [48] observed that there may be decreased forage concentration of Cu with advancing maturity, climate and seasonal changes). It is believed that Cu functions as an essential component of a number of enzymes in plants. The result of this study therefore indicate that all the browse species evaluated were deficient in Cu to meet the daily requirements (18 mg/kg) of range goats [45]. The percentage components of anti-nutritional factors in this present study were in general, very low but comparable with the reports of Bamikole et al. [8]; Mbomi et al. [27]. Among the antinutritional factors, the tannin content of the browse species in the present study were considerably lower than the value of 2.05% reported for Gliricidia sepium [49]. Values obtained in F. polita, P. africana, and D. oleiveri was higher compared to value reported previously (0.13 to 6.31%) by Gidado et al. [50]. A threshold concentration of 5% tannin had been reported above which there is rejection of browse plants [51]. Min and Hart [52] also reported that the action of condensed tannins in forages markedly reduced rumen proteolytics bacterial growth and some bacteolytic populations measured in vivo. However, goats are known to tolerate a threshold level of about 9% dietary tannin [20]. The saponin content too was also low as in other leguminous browse species. Oduguwa et al. [53] reported values of 3.24% and 3.47% for Parkia biglobosa and Afzelia africana respectively. Saponins have been found to be detrimental to protozoa and have been identified as defaunating agents in the rumen [54]. The concentration of saponin in this study is still within tolerable level of 1.5 -2% [55]. The oxalate content of the browse species was not consistent with the reported values (1.49 to 5.79%) of some browse plants relished by ruminants in Nigeria [19]. It was observed that ruminants can consumed certain quantity of feeds with a high level of oxalate without any deleterious effect [56]. Hence, the concentration of the anti-nutritional factors inherent in these browse species should not pose any negative effect on the animals that may consume them. CONCLUSION In conclusion, it is generally believed that legumes are reservoirs of higher protein and other nutrients than non-legumuminous browse plants, but the results obtained in this study do not support this statement. Nonetheless, the nutritional composition of these browse plants showed that they can be utilized as sole feed or supplements to balance low quality forages for ruminants due to the high CP level, moderate levels of both macro and micro mineral elements and low anti-nutrients composition. It is suggested that Eclipta alba utilization could be encouraged due to its exceptional higher mineral concentrations. The availability of these browse species during off season periods could be an additional advantage to be used as a dry season feed.

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Chemical Composition and Bioactivities of the Essential Oil from Etlingera yunnanensis against Two Stored Product Insects The chemical composition of the essential oil of Etlingera yunnanensis rhizomes and its contact and repellent activities against Tribolium castaneum (Herbst) and Liposcelis bostrychophila (Badonnel) were investigated. The essential oil obtained from E. yunnanensis rhizomes with hydrodistillation was performed by gas chromatography-flame ionization detection and gas chromatography-mass spectrometry. The main components of the essential oil were identified to be estragole (65.2%), β-caryophyllene (6.4%), 1,8-cineole (6.4%), limonene (5.2%), and α-pinene (2.4%). It was found that the essential oil of E. yunnanensis rhizomes possessed contact toxicity against T. castaneum and L. bostrychophila (LD50 = 23.33 μg/adult and LD50 = 47.38 μg/cm2, respectively). Estragole, 1,8-cineole, and limonene exhibited stronger contact toxicity (LD50 values of 20.41, 18.86, and 13.40 μg/adult, respectively) than β-caryophyllene (LD50 = 41.72 μg/adult) against T. castaneum adults. Estragole possessed stronger contact toxicity (LD50 = 30.22 µg/cm2) than β-caryophyllene, 1,8-cineole, and limonene (LD50 values of 74.11, 321.20, and 239.62 μg/adult, respectively) against L. bostrychophila adults. Repellency of the crude oil was also evaluated. The essential oil and constituents possessed strong repellent activity against T. castaneum adults. The four individual constituents showed weaker repellent activity than the essential oil against L. bostrychophila adults. The results indicated that the essential oil of E. yunnanensis rhizomes and the individual constituents had the potential to be developed as a natural insecticide and repellent for the control of T. castaneum and L. bostrychophila. Introduction The widespread extensive use of synthetic insecticides has triggered many negative consequences (i.e., insecticide resistance, toxicity to mammals and other non-target animals, residue problems, environmental pollution) [1,2]. Risks associated with the use of synthetic insecticides have led to the growth of an environmental movement seeking sustainable alternatives in pest control [3]. Therefore, increasing attention is being given to natural products. Plants can provide potential alternatives to the currently used insecticides that seem to cause insecticide resistance and environmental and human health concerns because they constitute a rich source of bioactive chemicals, such as terpenoids, alkaloids, and avonoids, against insects, and they have evolved strategies to interact with other organisms for self-defense [4]. Many essential oils and their constituent compounds from plants have been evaluated for repellency and insecticidal activity against stored product insects and some of them are quite promising in the development of natural repellents or insecticides [5][6][7][8][9]. In ancient China, extracts of many medicinal herbs and spices were used to control grain storage insects and pests [10]. Moreover, a traditional Chinese medicinal material conservation method called antagonistic storage has also been used for medicinal materials that have special volatile odors to prevent the insects in other medicinal materials [11]. However, the gap between unsubstantiated traditional use and experimentally substantiated potential should be bridged. Thus, based on the experimental evidence, this traditional method should be developed and inherited. In order to develop this traditional method of prevention and control of storage pests, we have established a screening program and focus on the volatile substances due to their major role in the antagonistic storage process. During this screening process, the essential oil of Etlingera yunnanensis (T.L. Wu and S.J. Chen) R.M. Smith [12] was found to possess insecticidal activity against the red flour beetle, Tribolium castaneum Herbst, and the booklouse, Liposcelis bostrychophila Badonnel. The red flour beetle is one of the most widespread and destructive insect pests of stored cereals, and can be found where grains or other dried foods are stored [13]. Infestations not only cause significant losses due to the consumption of grains but also result in elevated temperature and moisture conditions, which lead to accelerated growth of molds, including toxigenic species [14]. Booklice have a worldwide distribution infesting domestic premises, raw material stores, manufacturing factories, and historical documents in museums. Additionally, new evidence indicates that psocids are perhaps the most important emerging pests in stored grains and related commodities due to their small size, and resistance to chemicals [15]. Etlingera, a genus in Zingiberaceae, has only two species, Etlingera littoralis and Etlingera yunnanensis, distributed in China [16]. E. yunnanensis is a perennial herb, and it has an attractive inflorescence-like chrysanthemum. When it is frayed, it releases an anis-like odor [12]. It is commonly used in Dai medicine and is called "Maniangbu" in a different clinical usage, i.e., for diarrhea and sunstroke [17]. A literature survey has shown that there is neither a report on the chemical composition of E. yunnanensis rhizomes essential oil nor a report on its insecticidal and repellent activity. Therefore, we decided to evaluate the contact and repellent activity of the essential oil of E. yunnanensis rhizomes and its main compounds against two stored product insects. Chemical Composition of the Essential Oil The yield of yellow essential oil from E. yunnanensis rhizomes was 0.14% (v/w) and the density of the essential oil was determined to be 0.97 g/mL. The chemical composition of the essential oil was summarized in Table 1. A total of 12 compositions of the essential oil were identified. The principal components of the essential oil were estragole (65.2%), β-caryophyllene (6.4%), 1,8-cineole (6.4%), and limonene (5.2%) ( Figure 1). * RI, retention index as determined on a HP-5MS column using the homologous series of n-alkanes (C 5 -C 36 ). The essential oil from E. yunnanensis rhizomes consisted mainly of phenylpropanoid (estragole), which accounted for 65.2%. Estragole ( Figure 1) is a phenylpropene, which consists of a benzene ring substituted with a methoxy group and a propenyl group. It is an isomer of anethole, differing with respect to the location of the double bond [18]. Its name derives from "estragon", the French and German word for tarragon (Artemesia dracunculus), a herb to which it gives its anis-like odor [19]. Furthermore, it is also the primary constituent of the essential oil of anise, fennel [20], basil [21,22], Croton zehntneri [23], Clausena anisata [24], Zanthoxylum schinifolium [25,26], Lonicera japonica [27], and so on. According to the previous paper about the essential oil from other organs of E. yunnanensis, the main components of the leaves of E. yunnanensis are comprised mainly of germacrene D (19.2%), β-pinene (11.6%), and α-amorphene (11.2%), while the stems are rich in β-pinene (23.7%), 1,8-cineole (11.0%), and α-pinene (9.6%). The major components of the root oil of E. yunnanensis were β-pinene (31.9%), α-pinene (13.7%), and 1,8-cineole (9.4%) [28]. Compared with other parts of E. yunnanensis, the rhizome oil also had some common compounds, including 1,8-cineole and α-pinene. However, some compounds like germacrene D and α-amorphene were not detected in the essential oil of its rhizomes. In addition, compared with the other two plants from Etlingera, their essential oils are also characterized by high levels of phenylpropanoids, especially in the rhizomes [29]. These differences in chemical composition and content between the essential oils of leaves, stems, roots, and rhizomes could be due to the different effects of environmental factors (such as sunlight, water, and soil) on the aerial parts and underground parts, or may result from different metabolic pathways in the plant [30]. Thus, further studies on plant cultivation and essential oil standardization are necessary. Contact Toxicity The essential oil of E. yunnanensis rhizomes showed contact toxicity against T. castaneum adults with a LD50 value of 23.33 μg/adult (Table 2). When compared with the positive control pyrethrum, the essential oil demonstrated 89.62 times less toxic activity against T. castaneum adults. E. yunnanensis essential oil also possessed contact toxicity (LD50 = 47.38 µg/cm 2 ) against the booklice. When compared with the positive control pyrethrum, the essential oil showed 2.53 times less toxic activity against T. castaneum adults (Table 2). However, compared with the other essential oils in the literature, the essential oil of E. yunnanensis possessed stronger contact toxicity against L. bostrychophila than, for example, essential oils of Lonicera japonica (LD50 = 64.04 µg/cm 2 ) [27], Litsea cubeba (LD50 = 71.56 µg/cm 2 ) [31], Foeniculum vulgare (LD50 = 90.36 µg/cm 2 ) [5], Acorus calamus (LD50 = 100.21 µg/cm 2 ) [32], Curcuma wenyujin (LD50 = 208.85 µg/cm 2 ) [33], and Artemisia rupestris (LD50 = 418.48 µg/cm 2 ) [34]. The concentration-response curves for the two toxicity assays against Tribolium castaneum and Liposcelis bostrychophila were showed in Figure 2. [35]; ** data from Yang et al. [31]. *** The mortality of the control (acetone) was 0 μg/adult for TC and 0 µg/cm 2 for LB. The main constituent compounds estragole, 1,8-cineole, and limonene exhibited stronger contact toxicity against the red flour beetles, with LD50 values of 20.41, 18.86, and 13.40 μg/adult, respectively than the essential oil (Table 2), while β-caryophyllene exhibited weaker toxicity against T. castaneum adults (LD50 = 41.72 μg/adult). However, only estragole possessed stronger contact toxicity (LD50 = 30.22 µg/cm 2 ) than the essential oil against the booklice. The previous reports using a similar bioassay method also mentioned the contact activity of the four constituents and the results were about the same [26,27,31,35,36]. Therefore, as the uppermost constituent in the essential oil of E. yunnanensis, estragole is one of the contributors to the contact activity of the essential oil. Furthermore, estragole had been shown to exhibit contact toxicity against several stored product insects such as Sitophilus zeamais [22,25], Sitophilus oryza, Callosobruchus chinensis, and Lasioderma serricorne [37]. Moreover, some modes of action of estragole were also found. From electrophysiological experiments, Huignard et al. observed that estragole specifically induces a reduction of posthyperpolarization [38]. Additionally, a prior study about acetylcholinesterase demonstrated that estragole, as well as s-carvone and camphor, produced a mixed inhibition for this enzyme, binding either to the free enzyme or to the enzyme-substrate complex but linking to a site different from the active site where the substrate binds [39]. Thus, the insecticidal activity of the essential oil of E. yunnanensis and estragole was quiet promising. On the other hand, as the uppermost constituent in the essential oil of E. yunnanensis rhizomes, estragole has been demonstrated to be genotoxic and carcinogenic. Consequently, reductions in exposure and restrictions in use levels as a flavoring substance have been recommended by the Scientific Committee on Food [40]. European Union regulations on flavorings have also established maximum levels for estragole in certain compound foods resulting from the use of food ingredients in which it naturally occurs [41]. Moreover, based on the adverse effects and risk assessments, the security doses of some main constituents must be studied further [42]. This point is best illustrated with the example of 1,8-cineole. This constituent can be used internally as a flavoring and medicinal ingredient at very low doses, but it is toxic if ingested in greater than normal doses. Although this medicinal herb is safe for human consumption because it has been used as a folk medicinal herb for hundreds of years, no experimental data about its safety is available so far. Essential oils are indeed natural, but this origin does not imply that they are safe. In terms of toxicokinetics and ecotoxicology, essential oils also have some potential problems [3]. The latter observations imply that use of essential oils can be not safe for beneficial insects as natural enemies and pollinators. Thus, to develop a practical application for the essential oil and estragole as novel insecticides, further research on the safety and effectiveness of the essential oil for humans is needed. Repellency The results of repellency assays for the essential oil against the two species of stored product insects were presented in Tables 3 and 4 and Figure 3. Data showed that at tested concentrations, the essential oil possessed strong repellency against T. castaneum adults. At the lowest assayed concentration (0.13 nL/cm 2 ), the essential oil still showed 36% repellency against the beetles at 4 h after exposure. However, the essential oil only exhibited strong repellency against L. bostrychophila at the dose of 12.63 nL/cm 2 and 2.53 nL/cm 2 . At the other concentrations, the two compounds showed some insect-attractant properties. In previous reports, estragole, limonene, 1,8-cineole, and β-caryophyllene all showed repellent activity against the red flour beetles, and Yang et al. reported the repellency of limonene against booklice [31,35,36]. Many essential oils and their constituents were evaluated for repellency against insects as well [43]. The data and literature survey has indicated that the bioactivity properties of essential oils may be related to the synergistic effects of their diverse major and minor components [35]. This paper reported that the essential oil of E. yunnanensis had contact and repellent activities to red flour beetles and booklice for the first time. These findings, considered together, suggest that the essential oil showed potential for development as a natural insecticide/repellent for stored products. Tukey's tests. PR was subjected to an arcsine square-root transformation before ANOVA and Tukey's tests. Plant Material and Extractions Rhizomes of Etlingera yunnanensis were collected in June 2013 from Yunnan Province, China. The species was identified according to the voucher specimen (BNU-CMH-Dushushan-2013-06-15-015) deposited at the Herbarium of College of Resources Science and Technology, Beijing Normal University. Rhizomes (1.4 kg) of E. yunnanensis were subjected to hydrodistillation by a modified Clevenger-type apparatus for 6 h and then extracted with n-hexane. After extraction, water in the essential oil was removed by anhydrous sodium sulphate. The essential oil was stored in an airtight container in a refrigerator at 4 °C. Insects The T. castaneum and L. bostrychophila were obtained from laboratory cultures maintained for the last two years in incubators at 29 ± 1 °C and 70%-80% relative humidity in the dark. Adult T. castaneum insects were reared in glass containers (0.5 L) containing at 12%-13% moisture content on whole wheat mixed with yeast (wheatfeed/yeast, 10:1, w/w) while booklice were reared on a 10:1:1 mixture, by mass, of flour, milk powder, and active yeast. Adults used in all the experiments were about 1-2 weeks old. All the containers housing booklice used in experiments were made escape-proof with a coating of polyterafluoroethylene (Sino-rich ® , Beijing Sino-rich Tech Co., Ltd., Xuanwu District, Beijing, China). Gas Chromatography and Mass Spectrometry (GC-MS) GC-MS analysis was performed on a Thermo Finnigan Trace DSQ instrument equipped with a flame ionization detector and an HP-5MS (30 m × 0.25 mm × 0.25 μm) capillary column. The column temperature was programmed at 50 °C for 2 min, then increased at 2 °C/min to the temperature of 150 °C and held for 2 min, and then increased at 10 °C/min until the final temperature of 250 °C was reached, where it was held for 5 min. The injector temperature was maintained at 250 °C and the volume injected was 0.1 mL of 1% solution (diluted in acetone). The carrier gas was helium at a flow rate of 1.0 mL/min. Spectra were scanned from 50 to 550 m/z. Most constituents were identified by comparison of their retention indices with those reported in the literatures. The retention indices were determined in relation to a homologous series of n-alkanes (C5-C36) under the same operating conditions. GC retention time and their mass spectra that were stored in NIST 05 and Wiley 275 libraries or from literature were used for identifying the essential oil components [44]. Relative percentages of the individual components of the essential oil were obtained by averaging the GC peak area % reports. Contact Toxicity The contact toxicity of the essential oil and the individual compounds against T. castaneum adults was measured as described [12]. The four individual compounds (estragole, β-caryophyllene, 1,8-cineole, and limonene) were obtained from Tokyo Chemical Industry (Shanghai) Development Co., Ltd., Shanghai, China. Aliquots of 0.5 μL of the essential oil (diluted with acetone at five different concentrations) were applied topically to the dorsal thorax of the insects (10 insects per replicate, five replicates per dose). Insects treated with acetone alone were used as controls. Both treated and control insects were then transferred to glass vials (10 insects per vial) and kept in incubators. Insect mortality was checked after 24 h, and the LD50 values were calculated using Probit analysis [45]. Positive control, pyrethrins (pyrethrin I and II, 37%) were purchased from Dr. Ehrenstorfer GmbH. The contact toxicity of the essential oil and the individual compounds against L. bostrychophila was tested as described [46]. A 5.5 cm diameter filter paper was treated with 300 μL of the solution of the essential oil. The filter paper after being treated with solid glue was placed in a 5.5 cm diameter Petri dish (with a coating of polyterafluoroethylene) and 10 booklice were put on the filter paper. A cover was put and all the Petri dishes were kept in incubators. Acetone was used as a negative control. Five concentrations (diluted with acetone) and five replicates of each concentration were used. Mortality of insects was observed after 24 h. The LD50 values were calculated by using Probit analysis [45]. Repellency The repellent activity of the essential oil and the individual compounds to T. castaneum and L. bostrychophila was tested using the area preference method [34]. The essential oil was diluted in acetone to different concentrations (78.63, 15.73, 3.15, 0.63, and 0.13 nL/cm 2 ) and acetone was used as the control. Filter paper (9 cm in diameter) was cut in half. 500 μL of treatment solution was placed on one half of the filter paper. The other half was treated with 500 μL of acetone. The two halves of filter are allowed to dry for 30 s. The treated side was then joined to the control side by tape and placed in glass Petri dishes (9 cm in diameter). As for the booklice, Petri dishes (with a coating of polyterafluoroethylene) and filter papers were changed to 5.5 cm in diameter and the concentrations of the essential oil used in the experiments were 63.17, 12.63, 2.53, 0.51, and 0.10 nL/cm 2 . The half filter paper was treated with 150 µL of the solution. For both tests, 20 insects were released in the center of each filter paper disk, and a cover was placed over the Petri dish. Five replicates were used. Counts of the insects present on each strip were made after 2 and 4 h. The percent repellency (PR) of essential oil was then calculated using the formula: where Nc is the number of insects present in the negative control half and Nt is the number of insects present in the treated half. The averages were then assigned to different classes (0 to V) using the following scale (percentage repellency) [12]. Class, % repellency: 0, >0.01 to <0.1; I, 0.1-20.0; II, 20.1-40.0; III, 40.1-60.0; IV, 60.1-80.0; and V, 80.1-100. Means and standard errors were conducted by Microsoft Excel 2007 for Windows XP. Analysis of variance (ANOVA) and Tukey's test were conducted by using SPSS 19.0. Percentage was subjected to arcsine square-root transformation before ANOVA and Tukey's tests. The averages were then assigned to different classes (0 to V) ( Table 4). A commercial repellent, DEET (N,N-diethyl-3-methylbenzamide), was purchased from the National Center of Pesticide Standards (Shenyang, China) and used as a positive control. Conclusions This work indicates that the essential oil of E. yunnanensis rhizomes and its four constituents have potential for being developed into natural insecticides/repellents for the control of insects in stored products. However, further studies are needed to focus on the safety of the essential oil for humans and to improve the potency and stability of these potential insecticides/repellents for practical use.

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