Evaluating diazotrophy, diversity, and endophytic colonization ability of bacteria isolated from surface-sterilized rice

Information about the nitrogen fixing potential, diversity and sites of colonization of endophytic bacteria from rice are needed to expand our understanding of a new area of microbial ecology and plant microbe interactions and lay the foundations for future studies aimed at using biologically fixed nitrogen to replace nitrogen fertilizers. A collection of 142 bacteria isolated from mechanically-abraded, surface-sterilized rice roots was studied. Polymerase Chain Reaction (PCR) mediated gene amplification using degenerate primers derived from highly conserved regions of the nitrogenase nifD gene revealed 20 isolates harboring nifD gene sequences. Southern hybridization analysis confirmed the presence of nif genes in 19 of these isolates. The diazotrophic nature of these 19 isolates was confirmed using Acetylene Reduction Assays (ARA). Examination of genetic diversity using amplified ribosomal DNA restriction analysis (ARDRA) fingerprints and rep-PCR genomic fingerprints with Gelcompar software revealed 56 unique ARDRA fingerprints and 71 unique rep-PCR genomic fingerprints. Clusters of similar combined fingerprints, consisting of 37, 15, 12, and 9 non-diazotrophic bacteria, as well as two clusters each containing 4 diazotrophic bacteria, were found. Analysis of partial Small Subunit (SSU) ribosomal RNA (rRNA) gene sequences revealed the presence of isolates with similarity to strains from the alpha-, beta-, and gamma subdivisions of the Proteobacteria, and to members of the Bacillaceae and Microbacteriaceae. Many of the ARDRA fingerprints and/or SSU rRNA gene sequences of these bacteria were highly similar to those of other bacteria previously isolated from the rhizosphere of rice. Two isolates from the collection and Sinorhizobium meliloti , a control, were tagged with the biomarker gus or gfp, and the colonization of rice tissue was examined. In situ visualization of colonization of three week old inoculated rice seedlings revealed no endophytic colonization of rice tissue by these bacteria. However, clumps of bacteria, as well as individual cells, could be visualized on the surface of the roots. On very rare occasions an isolated epidermal cell filled with bacteria was observed.