Endophytic Bacteria And Root-Associated Bacteria With Rice By Culture-independent And Culture-dependent Approaches

The rice endophytic bacteria and root-associated bacteria were explored with culture-independent approaches. The diversity and population dynamics of endophytic bacteria and root-associated bacteria of two rice cultivars (Oryza saliva L. japonica 90-3 and Oryza sativa L. japonica 90-12) grown in the same paddy field were studied by using both 16S rDNA cloning, sequencing and denaturing gradient gel electrophoresis (DGGE) as well as traditional cultivation technique.The method of amplifying endophytic bacterial 16S rDNA from rice roots directly was established. A pair of bacterial 16S rDNA primers (799f-1492r) was selected to amplify bacterial sequences directly from rice root tissues by PCR for exclusion of chloroplast DNA. The mitochondrial sequence from rice was separated from the PCR-amplified bacterial 16S rDNA sequence by the different size of fractionations. Sequence analysis of clones in the 16S rDNA library of rice endophytic bacteria indicated that this pair of primers was available for the study of endophytic bacteria communities associated with rice plant. To our knowledge, this is the only one report of the primers used for culture-independent approaches on the analysis of endophytic bacterial communities associated with rice plants.Among the 196 positive clones in the 16S rDNA library of endophytic bacteria, 52 OTUs were formed according to the similarity of the banding patterns obtained by amplified DNA restriction analysis (ARDRA). The sequence analysis revealed a broad phylogenetic spectrum of bacteria, including α , β, γ, δ and ε subgroup of the Proteobacteria, low G+C gram-positive bacteria, and some microbes belonging to the Cytophaga/Flexibacter/Bacteroides (CFB) phylum in the library. The most dominant group was y - Proteobacteria (25% of the total). 28.64% of the total clones showed the highest similarity to uncultured bacteria. To our knowledge, it is the first report that archaea was identified as endophytic bacteria associated with rice by the culture-independent approach. It is suggested that a abundant diversity of endophytic bacterial community occurred within rice roots.The structure of bacterial community associated with rice roots was studied with both construction of 16S rDNA clone library and culture-dependent approache. ARDRA patterns showed 43 OTUs in the 16S rDNA clone library as well as 17 OTUs in the bacterial isolates. Y...