Evaluation of the Nitrogen-fixing Ability of Endophytic Clostridia based on Acetylene Reduction and Reverse Transcription-PCR Targeting the nifH Transcript and Ribosomal RNA

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To examine whether plant-derived clostridia and their consortium fix nitrogen in plants, an inoculation system was developed using the grass Miscanthus sinensis under aseptic conditions. Among 13 clostridial strains previously isolated from M. sinensis, Clostridium sp. strain Kas107-1 was selected as the best colonizer in the plant with the non-diazotrophic bacterium Entrobacter sp. strain B901-2. Nitrogen-fixing (acetylene-reducing) activity was not observed in the plants inoculated with Kas107-1 without a carbon source. On the other hand, nitrogenfixing activity was detected when carbon sources were supplied to the roots. To confirm the endophytic nitrogenfixing activity, we cloned nifH genes and monitored their expression in strain Kas107-1. Although this bacterium possessed at least two copies of nifH (nifH1 and nifH2), the nifH1 transcript was exclusively detected in free-living cells and endophytic cells in the plants by reverse transcription (RT)-PCR analysis. RT-PCR analysis of ribosomal RNA suggested the endophytic colonization of the plants by Kas107-1. These results indicate that Clostridium sp. strain Kas107-1 can potentially fix nitrogen in plants. A RT-PCR analysis targeting both functional gene transcripts and the ribosomal RNA molecule is useful for researching endophyte ecology, because their function and colonization in plants can be examined simultaneously with a single preparation of RNA.