Polyphasic Taxonomy And Phylogeny Of Rhizobia Isolated From Tree Legumes Robinia Sp., Dalbergia Spp. And Albizia Spp.

This thesis reviewed the historical development, the current taxonomic status and the phylogeny of rhizobia, and compared the main methods applied in rhizobial polyphasic taxonomy. Rhizobia isolated from tree leguminous Dalbergia spp., Robinia pseudoacacia, Albizia spp. in various geographical regions of China were studied together with the reference strains of some recognized rhizobial species by performing polyphasic taxonomy, including numerical taxonomy, 16S rDNA PCR-RFLP, BOX-PCR fingerprinting, AFLP fingerprinting, DNA base composition and DNA-DNA hybridization. The results of 129 phenotypic characteristics showed that most strains could grow on YMA with 5μg/ml, even 300μg/ml antibiotics. Nineteen strains can grow on YMA with pH12.0. Eight strains grow well on YMA at the temperature of 40℃. Five groups could be clustered at the similarity level of 85% on the basis of numerical taxonomy. Group 1, 2, 3 and 5 each consisted of one or two known reference strains; however, group 4, which maybe a new taxon, didn't contain any known reference strains. The analysis of 16S rDNA PCR-RFLP described 30 different genotypic characteristics and 7 phylogenetic branches. The dendrogram derived from PCR-RFLP of 16S rDNA showed that the clusters of above 86% of strains agreed with that of numerical analysis of phenotypic characteristics. BOX-PCR fingerprinting is more appropriate using as a method revealing genotypic diversity within a rhizobial species. The fingerprintings of some highly related strains were identical and grouped within one group. Most of reference strains including one recognized rhizobial species were not grouped together. Six groups obtained from AFLP fingerprinting at the similarity level of 78%. Each group correlated well with the results of numerical taxonomy and 16S rDNA PCR-RFLP with exception of some little differences. The congruence of the results between genetics and phenotypes means that AFLP fingerprinting is a better method for identification and grouping of rhizobial species. Sequences of 16S rDNA of four strains were analysized for the study of phylogenetics. CCBAU41044 and CCBAU25087 were clustered within the phylogenetic branch of Rhizobium, the other strains, CCBAU25126 and CCBAU23046, were in the Mesorhizobium and Bradyrhizobium, respectively. The DNA G+Cmol% contents of the four strains CCBAU 41044, CCBAU25087, CCBAU25126, CCBAU23046 were 56.6, 56.8, 60.8, 62.3 (Tm), respectively. The DNA homology between CCBAU25087 and the other strains in the same group 4 of numerical taxonomy was above 70%. The homology between CCBAU25087 and the known reference strains of Rhizobium was lower than 70%, this indicated that group 4 was a new DNA relatedness group different from known rhizobia species.