Even though attempts have been made to use endophytic diazotrophic bacteria as biofertilizers for rice cultivation, their community structure and complexity in rice tissues is not well understood. In this study, the diversity and the community structure of endophytic diazotrophs as well as the nifH gene expression within each plant part and growth stage of rice under different soil conditions were investigated. The population of viable endophytic diazotrophic bacteria in cultivated rice (Oryza sativa L. cultivar KDML-105) was investigated under different soil conditions and the N2-fixing ability of selected consortia and single isolates was assessed. Single isolates from each diazotrophic consortium were shown to be capable of both the inhibition and promotion of N2-fixation. Some isolates were closely related to Enterobacter dissolvens, Brevundimonas aurantiaca, Pantoea agglomerans, Pseudomonas spp., and Enterobacteriaceae. Carbon source utilization, and production of IAA, pectinase and cellulase indicated high diversity in the rice tissues. The presence of diazotrophic bacteria was detected in roots, stems and leaves on the basis of the GUS reporter gene. PCR-DGGE analysis conducted directly on rice tissue samples using 16S rDNA primers was used to elucidate the structure of the endophytic bacterial communities. Nested PCR-DGGE analysis with nifH primer demonstrated less diazotrophic bacterial diversity in the roots of rice cultivated in paddy soil amended with nitrogen fertilizer than in unfertilized and previously uncultivated soil. Plant tissue type was found to dictate the endophytic diazotrophic community structure rather than the type of soil or fertilizer amendment. In order to detect the nifH gene expression which implies the nitrogen fixing activities of the diazotrophic bacterial community, the RT-PCR approach was used. The results demonstrated that nifH gene expression could be differently detected in each part and growth stage of rice plants as well as being influenced by soil nitrogen status. The expression level of the nifH gene in all roots from plants grown in N-fertilized soil was the lowest among the treatments studied. The results confirm the complexity of the endophytic diazotrophic bacterial community, and indicate that the type of plant tissue seems to influence the community structure.
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