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      Community terminal restriction fragment length polymorphisms reveal insights into the diversity and dynamics of leaf endophytic bacteria

      research-article
      1 , 2 , 1 ,
      BMC Microbiology
      BioMed Central
      Leaf bacterial endophytes, Ecology, T-RFLP, Biodiversity

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          Abstract

          Background

          Plant endophytic bacteria play an important role benefiting plant growth or being pathogenic to plants or organisms that consume those plants. Multiple species of bacteria have been found co-inhabiting plants, both cultivated and wild, with viruses and fungi. For these reasons, a general understanding of plant endophytic microbial communities and their diversity is necessary. A key issue is how the distributions of these bacteria vary with location, with plant species, with individual plants and with plant growing season.

          Results

          Five common plant species were collected monthly for four months in the summer of 2010, with replicates from four different sampling sites in the Tallgrass Prairie Preserve in Osage County, Oklahoma, USA. Metagenomic DNA was extracted from ground, washed plant leaf samples, and fragments of the bacterial 16S rDNA genes were amplified for analysis of terminal restriction fragment length polymorphism (T-RFLP). We performed mono-digestion T-RFLP with restriction endonuclease DdeI, to reveal the structures of leaf endophytic bacterial communities, to identify the differences between plant-associated bacterial communities in different plant species or environments, and to explore factors affecting the bacterial distribution. We tested the impacts of three major factors on the leaf endophytic bacterial communities, including host plant species, sampling dates and sampling locations.

          Conclusions

          Results indicated that all of the three factors were significantly related (α = 0.05) to the distribution of leaf endophytic bacteria, with host species being the most important, followed by sampling dates and sampling locations.

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          Most cited references29

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          T-REX: software for the processing and analysis of T-RFLP data

          Background Despite increasing popularity and improvements in terminal restriction fragment length polymorphism (T-RFLP) and other microbial community fingerprinting techniques, there are still numerous obstacles that hamper the analysis of these datasets. Many steps are required to process raw data into a format ready for analysis and interpretation. These steps can be time-intensive, error-prone, and can introduce unwanted variability into the analysis. Accordingly, we developed T-REX, free, online software for the processing and analysis of T-RFLP data. Results Analysis of T-RFLP data generated from a multiple-factorial study was performed with T-REX. With this software, we were able to i) label raw data with attributes related to the experimental design of the samples, ii) determine a baseline threshold for identification of true peaks over noise, iii) align terminal restriction fragments (T-RFs) in all samples (i.e., bin T-RFs), iv) construct a two-way data matrix from labeled data and process the matrix in a variety of ways, v) produce several measures of data matrix complexity, including the distribution of variance between main and interaction effects and sample heterogeneity, and vi) analyze a data matrix with the additive main effects and multiplicative interaction (AMMI) model. Conclusion T-REX provides a free, platform-independent tool to the research community that allows for an integrated, rapid, and more robust analysis of T-RFLP data.
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            Effects of plant genotype and growth stage on the betaproteobacterial communities associated with different potato cultivars in two fields.

            Bacterial communities in the rhizosphere are dynamic and susceptible to changes in plant conditions. Among the bacteria, the betaproteobacteria play key roles in nutrient cycling and plant growth promotion, and hence the dynamics of their community structures in the rhizosphere should be investigated. Here, the effects of plant cultivar, growth stage, and soil type on the communities associated with potato cultivars Aveka, Aventra, Karnico, Modena, Premiere, and Désirée were assessed for two different fields containing sandy soil with either a high or low organic compound content. Thus, bacterial and betaproteobacterial PCR-denaturing gradient gel electrophoresis analyses were performed to analyze the effects of plant cultivar and growth on the rhizosphere community structure. The analyses showed that in both fields all cultivars had a rhizosphere effect on the total bacterial and betaproteobacterial communities. In addition, the plant growth stage strongly affected the betaproteobacterial communities in both fields. Moreover, the community structures were affected by cultivar, and cultivars differed in physiology, as reflected in their growth rates, root development, and estimated tuber starch contents. Analyses of betaproteobacterial clone libraries constructed for two selected cultivars (one cultivar that produced low-starch-content tubers and one cultivar that produced high-starch-content tubers), as well as bulk soil, revealed that the rhizospheres of the two cultivars selected for specific bacteria, including plant-growth-promoting bacteria, such as Variovorax and Achromobacter spp. In addition, quantitative PCR-based quantification of the Variovorax paradoxus-specific functional gene asfA (involved in desulfonation) indicated that there were clear potato rhizosphere effects on the abundance of this gene. Interestingly, both cultivar type and plant growth stage affected the community under some circumstances.
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              Both leaf properties and microbe-microbe interactions influence within-species variation in bacterial population diversity and structure in the lettuce (Lactuca Species) phyllosphere.

              Morphological and chemical differences between plant genera influence phyllosphere microbial populations, but the factors driving within-species variation in phyllosphere populations are poorly understood. Twenty-six lettuce accessions were used to investigate factors controlling within-species variation in phyllosphere bacterial populations. Morphological and physiochemical characteristics of the plants were compared, and bacterial community structure and diversity were investigated using terminal restriction fragment length polymorphism (T-RFLP) profiling and 16S rRNA gene clone libraries. Plant morphology and levels of soluble carbohydrates, calcium, and phenolic compounds (which have long been associated with plant responses to biotic stress) were found to significantly influence bacterial community structure. Clone libraries from three representative accessions were found to be significantly different in terms of both sequence differences and the bacterial genera represented. All three libraries were dominated by Pseudomonas species and the Enterobacteriaceae family. Significant differences in the relative proportions of genera in the Enterobacteriaceae were detected between lettuce accessions. Two such genera (Erwinia and Enterobacter) showed significant variation between the accessions and revealed microbe-microbe interactions. We conclude that both leaf surface properties and microbial interactions are important in determining the structure and diversity of the phyllosphere bacterial community.
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                Author and article information

                Journal
                BMC Microbiol
                BMC Microbiol
                BMC Microbiology
                BioMed Central
                1471-2180
                2013
                3 January 2013
                : 13
                : 1
                Affiliations
                [1 ]Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK, 74078, USA
                [2 ]Department of Botany, Oklahoma State University, Stillwater, OK, 74078, USA
                Article
                1471-2180-13-1
                10.1186/1471-2180-13-1
                3546043
                23286760
                f9043e47-9fc3-4149-8d79-bd872ef006d1
                Copyright ©2013 Ding et al.; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 18 August 2012
                : 20 December 2012
                Categories
                Research Article

                Microbiology & Virology
                leaf bacterial endophytes,ecology,t-rflp,biodiversity
                Microbiology & Virology
                leaf bacterial endophytes, ecology, t-rflp, biodiversity

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