title: Development of a bacterial cell enrichment method and its application to the community analysis in soybean stems.
abstract: A method was developed for enriching bacterial cells from soybean stems which was recalcitrant for a culture-independent analysis of bacterial community due to the interference with plant DNA. Stem homogenates were fractionated by a series of differential centrifugations followed by a Nycodenz density gradient centrifugation. The efficiency of bacterial cell enrichment was assessed by ribosomal intergenic spacer analysis (RISA). The intensity and the number of bacterial amplicons of RISA were markedly increased in the DNA extracted from the enriched bacterial cells compared to that in the DNA directly extracted from soybean stems. The phylogenetic diversity of the enriched bacterial cells was evaluated by analyzing a clone library of 16S rRNA gene in comparison with those of the culturable fractions of the enriched and non-enriched stem-associated bacteria, endophytic bacteria, and epiphytic bacteria. The results indicated that the method was able to enrich both endophytic and epiphytic bacteria from soybean stems, and was useful to assess the bacterial diversity based on a 16S rRNA gene clone library. When the sequence data from all clones (1,332 sequences) were combined, 72 operational taxonomic units were affiliated with Proteobacteria (Alpha-, Beta-, and Gammaproteobacteria), Actinobacteria, Firmicutes, and Bacteroidetes, which also provided the most comprehensive set of data on the bacterial diversity in the aerial parts of soybeans.
authors: Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai, Miyagi, 980-8577, Japan. sikeda@ige.tohoku.ac.jp
authors: Seishi
authors: S
authors: Ikeda
authors: Takakazu
authors: T
authors: Kaneko
authors: Takashi
authors: T
authors: Okubo
authors: Lynn E E
authors: LE
authors: Rallos
authors: Shima
authors: S
authors: Eda
authors: Hisayuki
authors: H
authors: Mitsui
authors: Shusei
authors: S
authors: Sato
authors: Yasukazu
authors: Y
authors: Nakamura
authors: Satoshi
authors: S
authors: Tabata
authors: Kiwamu
authors: K
authors: Minamisawa
mesh_terms: D001419:Bacteria / Q000235:genetics / Q000254:growth & development*; D001431:Bacteriological Techniques / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D015723:Gene Library; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D013025:Glycine max / Q000382:microbiology*
chemical_list: D004269:DNA, Bacterial; D021903:DNA, Ribosomal Spacer; D012336:RNA, Ribosomal, 16S