Polyphasic Taxonomy Of Rhizobia Isolated From Phaseolus Vulgaris, Campylotropis Spp. And Cassia Spp.

Eighty-six isolates were obtained from root nodules of Phaesolus vulgaris, Campylotropis spp., Cassia spp.. A polyphasic approach including both phenotypic and genotypic techniques was used to study their diversity and determine their taxonomic relationships with the known rhizobial strains.The phenotypic study showed that these strains of the three hosts included both fast- and slow-growing kinds. They could grow in yeast mannitol agar (YMA) in the range of pH5.0-11.0, utilize 44 and 13 kinds of carbon and nitrogen source test for growth respectively. 37 strains could tolerate 7 kinds antibiotics tested at 300μg/ml. There were 2 strains which could grow on YMA with 5% NaCl.By numerical taxonomy, all strains were divided into fast- and middle-slow-grower, slow-grower at 67% similarity. 3 groups clustered in the fast- and middle-slow grower at 85% similarity and 3 groups in the slow-grower at 76% similarity. Group 2, 3, 4, 6 might be new taxa because of no clustering with reference strains, but group 1,5 got together with the established species of Agrobacterium and Bradyrhizobium respectively.SDS-PAGE of whole cell proteins' dendrogram showed that fast- and middle-slow-grower could be divided into 5 groups at 68% similarity and slow-grower 3 groups at 59% similarity. Groupl, 2, 3 were analogous to group 1 in numerical taxonomy, group 4, 5 to group 3 and group 6, 7, 8 to group 6, 4, 5 respectively. Group 2 in numerical taxonomy did not cluster together here. It was obvious that the results of both SDS-PAGE of the whole cell proteins and numerical taxonomy were similar, but the former had higher resolution.rep-PCR fingerprinting included BOX-PCR and ERIC-PCR. At 73% and 70% similarity, BOX-PCR and ERIC-PCR dendrogram were composed of 18 groups respectively. The relationship between genus and species in the two trees was confused, but results from the multiple tree set up by combining the data of BOX-PCR and ERIC-PCR showed reasonable. The multiple tree formed 17 groups at 68% similarity. Groupl,3,4,6 in numerical taxonomy distributed in group 1,3, 4, 5, 6,12, 13,14,16 of the tree. These results told that rep-PCR fingerprinting also had higher resolution.All strains had 62 genetic types in 16S rDNA PCR-RFLP. No unknown isolate owned the same type as the known except that 11 strains had genetic types identical to Agrobacterium. It indicated that they had great diversity. Dendrogram derived from 16S rDNA RFLP-PCR showed that all isolates devided into three phylogeneu'c branches at the similarity level of 73%.They areAgrobacterium-Rhizobium-Sinorhizobum-Allorhizobium, Mesorhizobium and Bradyrhizobium branches. Groupl in numerical taxonomy was more related to Agrobacterium than Rhizobium ,Sinorhiz,obium, and Allorhizobium. Group 3 was related closely to Rhizobiwn. As for isolates of group 4 and 6, they were all related to Bradyrhizobium.The 16SrRNA genes of 4 central strains CCBAU 65237, CCBAU 01012 , CCBAU 33061 and CCBAU 33215 of group 1, 3, 4, 6 in numerical taxonomy were sequenced completely. The phylogenetic tree of 16S rRNA genes showed that CCBAU 65237 of group 1 was related to species of Agrobacterium above 99% identity, CCBAU 01012 of subgroup 3 to species of Rhizobium etli above 98% identity, and CCBAU 33061,CCBAU 33215 of group 4, 6 to species of Bradyrhizobium above 96% identity, but formed a separate branch in this genus. These results corresponded to 16S rDNA PCR-RFLP.DNA-DNA hybridization between central strains and other strains in each group of numerical taxonomy showed that group 1,3,4,6 were high DNA homology groups, and the DNA-DNA hybridization among central strains of groupl,3,4,6 and known type strains of known spp. in each phylogenetic branch showed that CCBAU 65237 was identical to Agrobacterium rubi. As to CCBAU 01012, CCBAU 33061 and CCBAU 33215, they were far related to type strains of known spp. in related phylogenetic branches with low identity of 0-36.2%, 0-42.2%, 0-34.8% respectively. It means that three groups represented by these three central strains were new species in Rhizobium and Bradyrhizobium. Genomic DNA G+Cmol% of the four central strains were 61.0,62.7,65.8,62.4, and fell into the same category of Agrobacterium (57-63%), Rhizobium (59-64%) and Bradyrhizobium (62-66%).Results above showed that isolates from Phaesolus vulgaris belonged to 4 genus and 5 species (included 1 new species), those from Campylotropis spp.3 genus and 3 species, those from Cassia spp.2 genus and 3 species(included 2 new species).In conclusion, the polyphasic approach indicated great diversity among isolates from Phaesolus vulgaris, Campylotropis spp., Cassia spp.. Strains in symbiosis with Phaesolus vulgaris, came from our Chinese territory not only distributed in Rhizobium but also in Agrobacterium, Mesorhizobium and Bradyrhizobium. Some isolates even did not clustered with any unexamed or known strains. There was little systematic research of taxonomy on rhizobia from Campylotropis spp. and Cassia spp.. Our study determined that isolates from Campylotropis spp.and Cassia spp. belonged to Agrobacterium, Mesorhizobium, Sinorhizobiwn and Bradyrhizobium. There were 2 new species of Cassia spp. in Bradyrhizobium.