Changes in the Bacterial Community of Soybean Rhizospheres during Growth in the Field

Yazaki K (2014) Changes in the Bacterial Community of Soybean Rhizospheres during Growth in the Field. PLoS ONE 9(6): e100709. doi:10.1371/journal.pone.0100709 Changes in the Bacterial Community of Soybean Rhizospheres during Growth in the Field Akifumi Sugiyama 0 Yoshikatsu Ueda 0 Takahiro Zushi 0 Hisabumi Takase 0 Kazufumi Yazaki 0 Matthew W. Fields, Montana State Univeristy, United States of America 0 1 Research Institute for Sustainable Humanosphere, Kyoto University , Uji, Kyoto , Japan , 2 Faculty of Bioenvironmental Science, Kyoto Gakuen University , Kameoka, Kyoto , Japan Highly diverse communities of bacteria inhabiting soybean rhizospheres play pivotal roles in plant growth and crop production; however, little is known about the changes that occur in these communities during growth. We used both culture-dependent physiological profiling and culture independent DNA-based approaches to characterize the bacterial communities of the soybean rhizosphere during growth in the field. The physiological properties of the bacterial communities were analyzed by a community-level substrate utilization assay with BioLog Eco plates, and the composition of the communities was assessed by gene pyrosequencing. Higher metabolic capabilities were found in rhizosphere soil than in bulk soil during all stages of the BioLog assay. Pyrosequencing analysis revealed that differences between the bacterial communities of rhizosphere and bulk soils at the phylum level; i.e., Proteobacteria were increased, while Acidobacteria and Firmicutes were decreased in rhizosphere soil during growth. Analysis of operational taxonomic units showed that the bacterial communities of the rhizosphere changed significantly during growth, with a higher abundance of potential plant growth promoting rhizobacteria, including Bacillus, Bradyrhizobium, and Rhizobium, in a stage-specific manner. These findings demonstrated that rhizosphere bacterial communities were changed during soybean growth in the field. - Funding: This study was partly supported by a Kyoto University Step-Up Grant for Young Scientists (AS) and a grant from the Ministry of Agriculture, Forestry, and Fisheries of Japan (Genomics-based Technology for Agricultural Improvement, SFC2001) (AS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The rhizosphere is the small region around the roots, defined as the zone of soil surrounding the root which is affected by it [1,2], where plants and millions of microbes interact with each other [3]. Rhizosphere microbes were shown to have intense activity and to be important for plant health and growth [4]. For example, mycorrhiza and rhizobia provide phosphorous and nitrogen, respectively, and microbes called plant-growth-promoting rhizobacteria (PGPR) exert both direct and indirect effects on plant growth, such as the prevention of colonization by pathogens and modulation of plant immunity [57]. These rhizosphere microbes are regarded as prominent components of sustainable agriculture that reduce the use of fertilizers and pesticides [8]. Plants have been shown to accommodate rhizosphere microbes by providing a platform and nutrients mainly at the root exudates, which account for up to 40% of photosynthates [912]. In addition to the climate and chemical properties of soils, resident plants exert influence on rhizosphere microbial communities. Microbial communities have been found to depend on the plant species grown in the same type of soil [1316], demonstrating a tight interaction between plants and rhizosphere microbial communities [17]. Because rhizosphere microbial communities are important in plant growth and performance, these communities have been extensively studied using both culture-dependent and cultureindependent methods [4,17,18]. Recent advances in next generation sequencing methods have enabled in-depth analyses of rhizosphere microbial communities. Arabidopsis root bacterial communities have been investigated comprehensively [1921], and the analysis of soils collected under Arabidopsis plants in their natural habitats indicated possible interactions between Arabidopsis growth and the microbial communities in these soils [22]. However, despite increases in community-based analyses of rhizosphere bacterial communities [4,17], it remains unclear how plant and bacteria communicate to form rhizosphere bacterial communities from reservoir bulk soil. In particular, few studies have comprehensively analyzed rhizosphere microbial communities during growth using next generation sequencing [16,23,24] although growth-dependent analyses of rhizosphere microbial communities have been performed using methods such as automated ribosomal interspacer analysis (ARISA) and denaturing gradient gel electrophoresis (DGGE) [2528]. Legume plants include important crop species such as soybeans (Glycine max), which supply nutrients rich in protein and oil for human consumption. Legume plants have been used to investigate plant-microbe interactions in the rhizosphere, due to their agricultural importance and ability to form symbiotic relationships with rhizobia and arbuscular mychorrhizal fungi (AMF) [2931]. Genetic analysis using model legume plants such as Lotus japonicus and Medicago truncatula revealed pathways leading to symbiosis. Despite the large number of reports analyzing the components of legume-rhizobia and legume-AMF symbiosis, the broad range of rhizosphere microbial species in soil, which could affect legume interactions with rhizobia and AMF, have not been characterized in depth. The mechanisms underlying legume plant interactions with various soil microbes during growth in the field remain especially elusive. Understanding the composition of rhizosphere microbial communities during growth in the field could provide a basis for optimizing agricultural utilization of rhizosphere microbes. For example, DGGE showed that the composition of the soybean rhizosphere changed during growth with alterations in the relative contributions of various phyla, including Proteobacteria, Acidobacteria, Bacteroidetes, Nitrospirae, Firmicutes, Verrucomicrobia and Acidobacteria [27]. To enhance understanding of legume-microbe interactions in the field, and to obtain basic information on soybean rhizosphere bacterial communities for further research, bacterial communities of rhizospheres were analyzed together with bulk soil during soybean growth in a field in Kyoto Prefecture, Japan. The physiological properties of the bacterial communities were analyzed by community-level BioLog substrate utilization assays. In addition, the 16S ribosomal RNA (rRNA) gene, which is the most important target in the study of bacteria, was analyzed by PCR amplicon pyrosequencing. The results of this study suggest that plant growth could affect the composition of rhizos (...truncated)