Isolation and Genetic Characterization of Aurantimonas and Methylobacterium Strains from Stems of Hypernodulated Soybeans
Isolation and Genetic Characterization of Aurantimonas and Methylobacterium Strains from Stems of Hypernodulated Soybeans
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DOI：
10.1264/jsme2.me10203
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发表时间：
2011-06-01
影响因子：
2.2
通讯作者：
Minamisawa, Kiwamu
中科院分区：
文献类型：
--
作者：
Anda, Mizue;Ikeda, Seishi;Minamisawa, Kiwamu
本研究的目的是分离在先前的非培养物依赖性分析中对大豆发酵表型和氮肥施用率有反应的橙色单胞菌和甲基杆菌菌株（Ikeda等人，ISME J.4：315-326，2010）。采用两种策略从从田间生长的、高密度的大豆茎制备的富集的细菌细胞中分离：PCR辅助分离橙色单胞菌和选择性培养甲基杆菌。经菌落PCR和16 SrRNA基因测序鉴定，在营养琼脂培养基上培养的768株菌中，有13株为橙色单胞菌。同时，在含甲醇琼脂培养基上分离的187株菌中，126株经16 S rRNA基因序列鉴定为甲基杆菌。对本研究和先前研究的组合数据集的16 S rRNA基因序列的聚类分析（>99%同一性）分别揭示了橙色单胞菌和甲基杆菌的4个和8个操作分类单位（OTU），并且显示了这两个组的目标细菌的成功分离。ERIC-和BOX-PCR结果显示，目的菌株的基因组具有均一性。此外，系统发育分析的橙单胞菌揭示了一个叶圈特定的集群中的属。在本研究中获得的菌株将是有用的，揭示未知的豆类微生物相互作用的自动调节。
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.