Genetic Diversity And Phylogeny Of Rhizobia Isolated From Six Genera Of Legume, And Determination Of The Taxonomy Of Rhizobia From Pueraria Spp. In Sichuan Province, P.R. China

The genetic diversity of 103 rhizobial strains from six genera of legume plants of Kummerowia spp., Vxgna spp., Pueraria spp., Albizia spp., Campylotropis spp., Acacia spp., in different regions of Sichuan Province, together with 17 reference strains, were analyzed by AFLP, BOX-PCR, 16S and 23S ARDRA, and the phytogeny of the representative strains were performed by 16S rRNA sequences. Furthermore, the taxonomic position of rhizobial strains from Pueraria spp. was determined.The results showed that, there was high genetic diversity among these strains. Analysis of AFLP, BOX-PCR divided the 103 strains into 26 AFLP genotypes and 18 BOX genotypes, respectively, and most of the strains grouped with the host plants. 16S and 23S ARDRA analysis revealed that the 56 representative strains formed 13 genotypes, and most of them belonged to the genus of Bradyrhizobium, but distinguished from the type strains of B. japonicvm USDA 6T and B. elkanii USDA 76T.The phylogenic relationship constructed by the 16S rRNA gene full sequences suggested that these rhizobial strains distributed into three branches of Rhizobium-Agrobacterium-Allorhizobium Mesorhizobiwn and Bradyrhizobium. Of them, Rhizobium sp. {Kummerowia) CCBAU 61054 located in the phylogenic sub-branch of Rhizobium-Agrobacterium; Bradyrhizobium sp. {Kummerowia) CCBAU 61030 Bradyrhizobium sp. {Vigena) CCBAU 61073, Bradyrhizobium sp. {Pureraria) CCBAU 61106 and CCBAU 61116 were in the branch of Bradyrhizobium. While, Mesorhizobium sp. {Pueraria) CCBAU 61095 was within the range of Mesorhizobium phylogenic branch.Furthermore, the numerical taxonomy, AFLP, BOX-PCR, 16S and 23S ARDRA, IGS PCR-RFLP and DNA-DNA hybridization were used to determine the taxonomic position of 23 rhizobial strains from Pueraria spp., and the results of numerical taxonomy with 131 physiological and biochemical characters divided the 22 strains (CCBAU 61099 was not available because of contamination), at the similarity of 81%, into three distinctive clusters. 16S, 23S ARDRA showed that these strains were in Mesorhizobium and Bradyrhizbium branches, respectively; whilst they were different from the type strains of the recognized species in the related genera, which was good agreed with those of numericalanalysis. The results of AFLP, BOX-PCR, and 1GS ARDRA analysis of 23 rhizobia were more diverse than those of numerical taxonomy and 16S, 23S ARDRA analysis, and divided these strains into 7, 5 and 8 genotypes, respectively.The 16S rRNA full sequence analysis of the center strains CCBAU 61106, CCBAU 61116 and CCBAU 61095 were performed. The similarities of 16S rRNA sequences between CCBAU 61106 and B. japonicum USDA 6T, B. elkanii USDA 76T, B. liaoningense USDA 3622T, B. yuanmingense CCBAU 10071T were 99.8%, 99.7% 99.5% and 97.1%; and of those between CCBAU 61116 and the bradyrhizobial type strains were 97.5% 97.4% 97.0% 99.1%, respectively. The similarities of the 16S rRNA full sequences between Mesorhizobium sp. {Pueraria) CCBAU 61095 and M. huakuii CCBAU 2609T, M. amorphae ACCC 19665T were 99.8% and 99.4%.The DNA relatedness between Mesorhizobium sp. {Pueraria) CCBAU 61095 and Mesorhizobium amorphae ACCC 19665T was 80.4%, and the G+C mol% of CCBAU 61095 was 60.9% (within the range of 59%~64%), which demonstrated that CCBAU 61095 belonged to Mesorhizobium amorphae. The G+C mol% contents of CCBAU 61106 and CCBAU 61116 were 62.8% and 61.5%, and the DNA homology between CCBAU 61106, CCBAU 61116 and type strains of B. japonicum USDA 6T, B. elkanii USDA 76T, B. liaoningense USDA 3622T, B. yuanmingense CCBAU 10071T ranged from 0 to 29.4%, which suggested that CCBAU 61116 and CCBAU 61106 should be two new species in the genus of Bradyrhizobium.