Abstract

Putative endophytic nitrogen-fixing bacteria that actively express nitrogenase gene in rice were determined using reverse transcription-PCR (RT-PCR) technique. The amplified portion of dinitrogenase reductase genes (nifH) from mRNA of rice roots and stems were cloned and sequenced. Phylogenetic analysis of deduced amino acid sequences of isolated nifH clones (117 sequences) revealed that majority of these sequences, around 71.8% of all examined clones, formed a distinct and deeply branching assemblage. None of these sequences were closely related to any sequences from known diazotrophes extracted from GenBank/DDBJ databases (DNA data bank of Japan), although it gathered with Geobacter sulfurreducens. This novel set of strains was frequently recovered from stems of used cultivars more than the roots. The other expressed nifH sequences (around 28.2%) were either clustered with known members of division Proteobacteria or with different lineage of the known and unknown anaerobes and sulfur reducing bacteria. These results indicate that the active nitrogen-fixing community associated with rice was consisting mainly of uncultured organisms that are at least specific to rice ecosystem and yet uncharacterized.

Key words: nifH sequence, nitrogenase expression, phylogenetic tree, rice.