Polyphasic Taxonomy And Phylogeny Of Rhizobia Isolated From Albizia Spp., Acacia Spp. And Leucaena Leucocephala

Seventy-four bacterial isolates obtained from root nodules of the woody legumes of Albizia spp., Acacia spp. and Leucaena leucocephala mainly grown in the subtropical region of China were characterized by polyphasic taxonomy including numerical taxonomy, 16S rDNA PCR RFLP, SDS-PAGE of whole cell protein analysis, rep-PCR fingerprinting, phylogenic studies (the sequences analysis of 16S rRNA, atpD and gln II genes), determination of DNA base composition and DNA-DNA hybridization. The aims of this research were to check the diversity and to classify the taxonomic position of the isolates by both phenotypic and genotypic analysis.Phenotypic studies showed that several isolates were resistant to some antibiotics of 300μg/ml and 2 strains could grow on YMA plates supplied with 5% NaCl. Two and twenty-two stains could grow on YMA plates at pH 4 and pH 12 respectively. Most strains grew well on YMA at 37 ℃, among which 23 strains could sustain the heat shock of 10 minutes at 60℃.Methods of numerical taxonomy, 16S rDNA PCR RFLP and SDS-PAGE of whole cell protein analysis were used to group the isolates firstly, and the results were quite agreed with each other. All of the isolates and reference strains studied were divided into 8 phena at the similarity of 84% for the fast or moderately growing bacteria and 87.5% for the slow growing ones. Phenon 1, 4, 6 and 7 were clustered together with the reference strains of A. tumefaciens, M. plurifarium, B. japonicum and B. elkanii, respectively. No reference strains were found in Phenon 2, 3, 5 and 8. Sixteen rDNA types were obtained among 74 isolates. All of them were assigned to Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium and Agrobacterium except for 3 strains. All of the bacteria tested were clustered into 11 groups by SDS-PAGE of whole cell protein analysis. In the results of the latter two methods M. plurifarium and B. yuanmingense were clustered together with the strains of Phenon 5 and 8 respectivly and no reference were recognized among the strains of Phenon 2 and 3.Great genetic diversity was found for the isolates at intraspecies level by rep-PCR fingerprinting including BOX-PCR and ERIC-PCR and 28 genetic groups were recovered.Sequences of 16S rRNA gene of some representative strains were analyzed for the study of phylogenetics. CCBAU61139, CCBAU25213, CCBAU25010, CCBAU43060 and CCBAU45226 were located in the branch of Rhizobium-Agrobacterium. CCBAU35204 was assigned to Sinorhizobium. CCBAU51276, CCBAU51471 and CCBAU61158 belonged to the genera of Mesorhizobium. CCBAU35234, CCBAU61178 and CCBAU35085 were in the branch of Bradyrhizobium. These results were consistent with those of numerical analysis, 16S rDNA PCR RFLP and SDS-PAGE of whole cell proteins and were further confirmed by the analysis of partial sequences of atpD and gln II genes.DNA homology analysis showed that strains in Phenon 1 and 2 of numerical taxonomy were A. tumefaciens and A. vitis, respectively. DNA homology between the central strain of Phenon 3 CCBAU61158 and the type strains of the recognized species in Mesorhizobium was lower than 34.1%,which indicated that Phenon 3 represented a new species in Mesorhizobium. Polyphasic analysis demonstrated that strains in Phenon 4, 5 and CCBAU35094 and CCBAU35103 should be classified as M. plurifarium, while the DNA homologies between these strains were 21.4%~85.2%. These results confirmed the reports that strains in M. plurifarium are of great genomic heterogeneity (de Lajudie et al. 1994, 1998; Wang et al. 2003 ) .From the results mentioned above, we concluded that the microsymbionts of Albizia spp. were of great diversity and 31 isolates were distributed into 8 species in Rhizobium, Mesorhizobium and Bradyrizhibium. One novel species was discovered by polyphasic analysis of Albizia kalkora. Acacia and Leucaena leucocephala may harbour some common rhizobial species, but they also have different preferences of the microsymbionts. Thirteen isolates from Leucaena leucocephala were mainly M. plurifarium and Sinorhizobium and only one strain was Bradyrhizobium which was assigned to B. elkanii. While among 16 isolates from Acacia spp., 12 strains belonged to B. elkanii and B. yuanmingense and others were M. plurifarium.