4), and were further subdivided into five groups. In particular, N 2 fixation by group II clostridia was induced by metabolites of nondiazotrophs in culture, although N 2 fixation in anaerobic nitrogen-fixing consortia is always supported by the elimination of oxygen by the nondiazotrophs (16).The 16S rRNA gene has been used as a molecular marker that allows the phylogenetic assignment of target organisms in natural environments. Popular methods that rely on 16S rRNA gene analysis include terminal restriction fragment length polymorphism (TRFLP) analysis (14) and denaturing gradient gel electrophoresis (17). Recently, Sessitsch et al. (19) monitored endophytic populations in potato plants by TRFLP and found a wide range of organisms that fell into many distinct phylogenetic groups. Molecular population analyses targeting the 16S rRNA genes of clostridia in the environment have been reported for municipal landfill sites (22), human feces (6), and paddy fields (3,23).For this work, we investigated the population levels and phylogenetic structures of clostridia in the grass Miscanthus sinensis by TRFLP analysis targeting the 16S rRNA gene. M. sinensis is a rhizomatous, perennial grass that naturally dominates almost all of the tallgrass-type meadows and wastelands in Japan (13). The plant often grows as a pioneer plant in areas devastated by lahar from volcanic eruptions; in these areas the nitrogen content of the soil is very low. The objective of this work was to determine (i) how the population levels of endophytic clostridia compare with those of culturable diazotrophs and (ii) which are the dominant phylogenetic groups of plant clostridia.
MATERIALS AND METHODSBacterial strains, media, and cultivation. Sixteen strains of a Clostridium sp. that were previously isolated mainly from M. sinensis were used (16). The strain names (and accession numbers of the sequences) were Sukash-1 (AB114226), Kas203-2 (AB114230), Kas401-4 (AB114231), Kas107-2 (AB114232), Kas203-1 (AB114238), Kas104-4 (AB114239), Kas401-3 (AB114240), Kas107-1 (AB114241), Kas301-1 (AB114242), Kas404-1 (AB114243), Kas303 (AB114244), Kas402-3 (AB114249), Kas202-1 (AB114252), Kas201-1 (AB114258), Kas106-4 (AB114263), and B901-1b (AB114264). The type strains of Clostridium aminovalericum, Clostridium intestinali, Clostridium acetobutylicum, and Clostridium beijerinckii were purchased from culture collections of the Riken Institute (Wako, Japan). Rice extract modified Rennie (RMR) semisolid medium (5) and nutrient agar (NA) (Difco, Detroit, Mich.) were used for the enumeration of bacteria and diazotrophs from plants. Viande-Levure (VL) agar and RMR agar plates were used for the cultivation of clostridia. VL medium contained the following components dissolved in 1 liter of water (pH 7.0): nutrient broth (Difco), 8 g; yeast extract (Difco), 5 g; NaCl, 5 g; glucose, 2 g; cysteine-HCl, 0.3 g. Anaerobic cultivation was carried out with the AnaeroPack system (Mitsubishi Gas Chemical, Tokyo, Japan).