Novel endophytic nitrogen-fixing clostridia from the grass Miscanthus sinensis as revealed by terminal restriction fragment length polymorphism analysis
Novel endophytic nitrogen-fixing clostridia from the grass Miscanthus sinensis as revealed by terminal restriction fragment length polymorphism analysis
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DOI：
10.1128/aem.70.11.6580-6586.2004
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发表时间：
2004-11-01
影响因子：
4.4
通讯作者：
Minamisawa, K
中科院分区：
文献类型：
--
作者：
Miyamoto, T;Kawahara, M;Minamisawa, K
由 N-2 固氮梭菌和多种非固氮细菌组成的厌氧固氮菌群先前已从各种禾本科植物中分离出来（K. Minamisawa、K. Nishioka、T. Miyaki、B. Ye、T. Miyamoto、M. You、A. Saito、M. Saito、W. Barraquio、N. Teaumroong、T. Sein 和 T. Tadashi，Appl.微生物学。70：3096-3102，2004）。在这项工作中，通过 16S rRNA 基因靶向末端限制性片段长度多态性 (TRFLP) 分析结合最可能数 (MPN) 计数，评估了日本芒草丛中的梭菌种群及其系统发育结构。 PCR 引物和限制性内切酶针对植物梭状芽胞杆菌的分析进行了优化。在经过严格表面消毒的植物叶、茎和根中检测到梭状芽胞杆菌，数量约为 10(4) 至 10(5) 个细胞/克鲜重；根据与乙炔还原测定相关的 MPN 计数确定，它们在 N-2 固定细菌群体中占很大比例。 MPN-TRFLP分析的系统发育分组表明，梭菌种群属于簇XIVa的II组和簇I的IV和V组；这一结果得到了使用直接从植物中提取 DNA 进行的不依赖于培养物的 TRFLP 分析的支持。当对中华海棠和植物周围土壤的系统发育种群进行比较时，发现第二类梭菌仅存在于中华海棠中。
Anaerobic nitrogen-fixing consortia consisting of N-2-fixing clostridia and diverse nondiazotrophic bacteria were previously isolated from various gramineous plants (K. Minamisawa, K. Nishioka, T. Miyaki, B. Ye, T. Miyamoto, M. You, A. Saito, M. Saito, W. Barraquio, N. Teaumroong, T. Sein, and T. Tadashi, Appl. Environ. Microbiol. 70:3096-3102, 2004). For this work, clostridial populations and their phylogenetic structures in a stand of the grass Miscanthus sinensis in Japan were assessed by a 16S rRNA gene-targeted terminal restriction fragment length polymorphism (TRFLP) analysis combined with most-probable-number (MPN) counts. PCR primers and restriction enzymes were optimized for analyses of the plant clostridia. Clostridia were detected in strongly surface-sterilized leaves, stems, and roots of the plants at approximately 10(4) to 10(5) cells/g of fresh weight; they made up a large proportion of N-2-fixing bacterial populations, as determined by MPN counts associated with an acetylene reduction assay. Phylogenetic grouping by MPN-TRFLP analysis revealed that the clostridial populations belonged to group II of cluster XIVa and groups IV and V of cluster I; this result was supported by a culture-independent TRFLP analysis using direct DNA extraction from plants. When phylogenetic populations from M. sinensis and the soil around the plants were compared, group II clostridia were found to exist exclusively in M. sinensis.