Novel endophytic nitrogen-fixing clostridia from the grass Miscanthus sinensis as revealed by terminal restriction fragment length polymorphism analysis

Takuya Miyamoto, Makoto Kawahara, Kiwamu Minamisawa

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68 Citations (Scopus)

Abstract

Anaerobic nitrogen-fixing consortia consisting of N2-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 104 to 105 cells/g of fresh weight; they made up a large proportion of N2-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.

Original languageEnglish
Pages (from-to)6580-6586
Number of pages7
JournalApplied and environmental microbiology
Volume70
Issue number11
DOIs
Publication statusPublished - 2004 Nov

ASJC Scopus subject areas

  • Biotechnology
  • Food Science
  • Applied Microbiology and Biotechnology
  • Ecology

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