title: Isolation and genetic characterization of Aurantimonas and Methylobacterium strains from stems of hypernodulated soybeans.
abstract: The aims of this study were to isolate Aurantimonas and Methylobacterium strains that responded to soybean nodulation phenotypes and nitrogen fertilization rates in a previous culture-independent analysis (Ikeda et al. ISME J. 4:315-326, 2010). Two strategies were adopted for isolation from enriched bacterial cells prepared from stems of field-grown, hypernodulated soybeans: PCR-assisted isolation for Aurantimonas and selective cultivation for Methylobacterium. Thirteen of 768 isolates cultivated on Nutrient Agar medium were identified as Aurantimonas by colony PCR specific for Aurantimonas and 16S rRNA gene sequencing. Meanwhile, among 187 isolates on methanol-containing agar media, 126 were identified by 16S rRNA gene sequences as Methylobacterium. A clustering analysis (>99% identity) of the 16S rRNA gene sequences for the combined datasets of the present and previous studies revealed 4 and 8 operational taxonomic units (OTUs) for Aurantimonas and Methylobacterium, respectively, and showed the successful isolation of target bacteria for these two groups. ERIC- and BOX-PCR showed the genomic uniformity of the target isolates. In addition, phylogenetic analyses of Aurantimonas revealed a phyllosphere-specific cluster in the genus. The isolates obtained in the present study will be useful for revealing unknown legume-microbe interactions in relation to the autoregulation of nodulation.
authors.affiliation: Graduate School of Life Sciences, Tohoku University, Sendai 980–8577, Japan.
authors.forename: Mizue
authors.initials: M
authors.lastname: Anda
authors.forename: Seishi
authors.initials: S
authors.lastname: Ikeda
authors.forename: Shima
authors.initials: S
authors.lastname: Eda
authors.forename: Takashi
authors.initials: T
authors.lastname: Okubo
authors.forename: Shusei
authors.initials: S
authors.lastname: Sato
authors.forename: Satoshi
authors.initials: S
authors.lastname: Tabata
authors.forename: Hisayuki
authors.initials: H
authors.lastname: Mitsui
authors.forename: Kiwamu
authors.initials: K
authors.lastname: Minamisawa
mesh_terms: D020561:Alphaproteobacteria / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003470:Culture Media; D014644:Genetic Variation; D020580:Methylobacterium / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D009586:Nitrogen Fixation / Q000235:genetics; D010802:Phylogeny; D055170:Plant Root Nodulation; D018547:Plant Stems / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D013025:Glycine max / Q000382:microbiology*; D013559:Symbiosis / Q000235:genetics / Q000502:physiology
chemical_list: D003470:Culture Media; D012336:RNA, Ribosomal, 16S