The Study On Biodiversity And Phylogenesis Of Rhizobia Isolated From Trifolium And Medicago

Fifty-five isolates were obtained from root nodules of clover and alfalfa. The assay techniques of numerical taxonomy,BOXAIR-PCR, ERIC-PCR, 16S rDNA-RFLP, 16S rDNA sequence was used to study their phenotypic and genotypic diversity, and to determine some unknown strains' phylogenesis and taxonomic position of the isolates.The phenotypic study showed that strains isolated in Sichuan province and Chongqing region possessed prominent traits tolerating high concentration antibiotics and salinity (5% NaCl), high temprature(60 C), low temprature(4C), acid condition(PH4.0) and alkaline condition (pH11.0). According to numerical taxonomy, all strains tested were divided into 13 phenotypic groups at 81% similarity level. All isolates showed great phenotypic diversity. As a whole, all rhizobial strains are accordant in the cluster of the genus, but they didn't manifest the difference based on the geography. The strains from clover revealed the geographical difference in the specific level.rep-PCR fingerprinting included BOX-PCR and ERIC-PCR. At 87% and 89% similarity, BOX-PCR, ERIC -PCR and the multiple dendrogram were composed of 27, 33, and 37 groups respectively. The relationship between genus and species in the three trees was confused, and the clusters did not correlated well with the geographical and cultivar origins. The strains belonged to one phenon in the numerical taxonomy distributed into several rep-PCR groups. These results showed that rep PCR fingerprinting is an effective method to study the diversity in the interior of one species, and rep-PCR fingerprinting also had higher resolution, and at the same time, the multiple tree can indicates more difference between specieses and strain's idiosyncrasy.All strains had 27 genetic types in 16S rDNA PCR-RPLP. The same result as the numerical taxonomy was that all isolates had not the same genotype and phenotype as Agrobacterium. Except for IAM13129, B33 and 711, at the similarity level of 75% dendrogram derived from 16S rDNA RFLP-PCR showed that all isolates were devided into two phylogenetic branches: Rhizobium and Sinorhizobum and unknown one. At the similarity level of 91%, most strains from alfalfa clustered into 17 groups belonged to Sinorhizobum phylogenetic branch, while some strains from clover from Yaan and Xich-ang belonged to Rhizobium phylogenetic branch. It was similar to the results of numericaltaxonomy. The phylogenesis and taxonomic relationships of the other 17 strains were not confermed. Synthetically, results implied that they had great genetic diversity.That some strains were not clustered with known rhizobial strains indicated new taxonomy units may be found.According as the results above, 16 rRNA genes of TH62 and WZ421 were sequenced completely. The phylogenetic tree of 16S rRNA genes showed that TH62 representing group 7 was related to species of Rhizobium at 99.6% identity, and WZ421 representing group 8 was similiar to species of Sinorhizobium at 99.2% identity.