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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 previ- ously 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, nitrogen- fixing activity was detected when carbon sources were supplied to the roots. To confirm the endophytic nitrogen- fixing 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-liv- ing cells and endophytic cells in the plants by reverse transcription (RT)-PCR analysis. RT-PCR analysis of ribo- somal 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 func- tional 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.

microbes and environments

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