Diversity And Phylogeny Analysis Of Rhizobia Isolated From Lespedeza Floribunda In Shannxi Shangluo Area Of China

Fifty-nine bacterial isolates obtained from root nodules of the Lespedeza floribunda which grew in Shaanxi Shangluo of China, were studed by 16S rDNA PCR-RFLP, the sequences analysis of 16S rDNA and numerical taxonomy.Phylogenetic position of rhizobial from Lespedeza floribunda had been Initially identified.Based on the 16S rDNA PCR-RFLP and sequences analysis, the tested strains were divided into nine different genotypes, in which six genotype falling into Rhizobium, one genotype belong to Sinorhizobium,The strains CCNWSX0906 and CCNWSX0884, each represented 9 Rhizobium stains according to genotypes of 16S rDNA PCR-RFLP , may be putative novel species becaues they could not fully come together with other strains in Rhizobium brouches of the 16S rDNA sequence phylogenetic tree. R. leguminosarum was first isolated from root nodules of the Lespedeza floribunda in this study. In addition, the test first isolated two types of non-rhizobia from the root nodules of the Lespedeza floribunda.They were respectively the Paenibacillus amylolyticus and the Stenotrophomonas maltophilia,each of which included three strains.Through further analysis of the geographical origin of isolated strains, it can be found that although there are significant geographical differences at each collection points, the unknown bacterium was not distinguished in accordance with the geographical origin.Strains collected from different sites have the same genetic types, and strains collected from the same sites have differences in the genetic types.Especially different strains even had been isolated from the same root nodule.59 unknown strains were analyzed with 125 phenotypic features and numerically classified. The result showed that the unknown strains had more diverse phenotypic characteristics.They could use the majority of all the carbn and nitrogen sources and adapt to a wide range of temperature and PH, which are conducive to their wide application in different geographical environments. The proportion of unknown strains which could be resistant to antibiotic, salt, dye, heavy metals and could produce oxidase was relatively high. Some strains had the properties of endurance to high concentrations of salt and alkali,in which about 67% strains had properties resistant to 3.0% NaCl, 25% of strains could grow in YMA medium with pH12.According to dendrogram of numerical taxonomy, in addition to strain CCNWSX0922,other test strains were clustered into eight clusters at the similarity of 80.5%, clusterâ… was Mesorhizobium including 6 unknown strains, clusterâ…¡was Sinorhizobium including 19 unknown strains and it could be further divided into 4 subgroups, clusterâ…¢consisted of 1 unknown strain, 2 Rhizobia and 1 Agrobacteria,clusterâ…£and clusterâ…¤were Rhizobium. Clusterâ…¥, clusterâ…¦and clusterâ…§may be a group of novel species or non-rhizobia because in this cluster there were no reference strains included. Their taxonomic status need to be further confirmed.By comparing it can be found that the clustering results of numerical taxonomy largely consistented with that of 16S rDNA PCR-RFLP. Numerical taxonomy also indicated that the phenotypic characteristics of unknown bacterium was not distinguished in accordance with the geographical origin. The strains in same group could collected from different collection points, and the same collection point could isolate the strains which were classfied into different groups.Conclusions can be reached from experimental results as follows: Shangluo Lespedeza floribunda has more rich diversity of rhizobia; which R. leguminosarum from Lespedeza floribunda was proved to be a new biovar of R. leguminosarum,the potential new species of rhizobia may nodulate with Lespedeza floribunda,endogenous non-rhizobia as well as exists in the nodules.In addition, the results further show there is no obvious correlation between the grouping of Lespedeza rhizobia and the geographical environment, and the geographical environment is not the main factor determining the diversity formation of rhizobia. We should do a deep study in symbiotic choice evolution between the rhizobia and its host plant, especially from the horizontal transfer of genes in symbiotic system to probe the causes leading to diversity of rhizobia.