Polyphasic Taxonomy And Phylogeny Of Rhizobia Isolated From Desmodium Spp. And Glycyrrhiza Spp. In Chinese Different Regions

The rhizobial taxonomy and methods used for polyphasic taxonomy were reviewed. Seventy isolates from nodules of some species in Desmodium and Glycyrrhiza grown in different geographic regions of China, which are important pioneer plants used as Chinese traditional medicines, were analyzed by polyphasic taxonomy (phenotypic and genetic analysis such as numerical taxonomy, the SDS-PAGE of whole protein, 16S rDNA PCR-RFLP, BOX-PCR fingerprinting, IGS PCR-RFLP, and analysis of DNA homology, etc.) and phylogenic study (the sequences analysis of 16S rDNA gene and symbiotic gene).The physiological and chemical characteristics of all isolates were tested and the results indicated that thirteen strains could grow in culture medium of pH4.0 and four strains isolated from Glycyrrhiza spp. resisted 5% NaCl. Twenty five bradyrhizobia from different desmodia species could grow in the YMA with 3% NaCl. The bradyrhizobia associate with Desmodium spp. had more resistance for tetracycline than fast-growing rhizobia that eight slow-growing strains could grow in the concentration of 100μg/ml ,while the fast-growing rhizobia could not grow in the 5μg/ml.All the test isolates were divided into 8 phena at the level of 82%~83% by numerical taxonomy. The phenon 1-4 were bradyrhizobia clusters and phenon 5-8 were fast-growing clusters. The phenon 5 may be a new group because in this cluster there were no reference strains included. The phenon 6 clustered with type strains of Rhizobium and the phenon 7 and 8 were clustered with reference strains of Sinorhizobium and Agrobacterium, respectively.Thirty-two genotypes of seventy isolates were obtained in analysis of 16S rDNA PCR-RFLP. The fifty isolates associate with desmodia had 20 genotypes and twenty isolates from Glycyrrhiza had fourteen genotypes. There were 7 genetic groups at 96% similarity level. Most isolates were clustered according to different host genus. Eight strains of Glycyrrhiza spp. were separated with reference strains and twelve strains belonged to Rhizobium, Sinorhizobium, Agrobacterium and Mesorhizobium.Twenty nine strains of Desmodium spp. located in the Bradyrhizobium and eighteen were fast-growing rhizobia that located in the Rhizobium, Sinorhizobium, Agrobacterium and Mesorhizobium.The genetic diversity of all test isolates from desmodia and glycyrrhiza were characterized at theintraspecies level by BOX-PCR fingerprinting and 18 genetic groups were obtained. Forty-four rhizobia from Desmodium were divided into 8 clusters by SDS-PAGE analysis of the whole cell protein. The analysis of IGS PCR-RFLP described the genetic diversity of desmodia rhizobia and forty test isolates were clustered into 9 different genetic groups.The phylogenetic positions of representative strains symbiotic with desmodia were revealed by 16S rRNA gene sequences. It indicated that all strains separated into Bradyrhizobium, Sinorhizobium, Rhizobium-Agrobacterium and Mesorhizobium..The fast-growing representative isolates CCBAU65255, CCBAU33220a, CCBAU23084 and CCBAU53010 from Yunnan, Jiangxi, Anhui, and Guangxi provinces, respectively, were located in the subbranch of Rhizobium-Agrobacterium.The slow-growing strains CCBAU23174, CCBAU23230 and CCBAU65199 from Anhui and Yunnan provinces distributed in the Bradyrhizobium branch, and CCBAU13090 isolated from Jilin province in the Mesorhizobium genus, CCBAU33036a of Jiangxi province in the Sinorhizobium branch. The results of phylogeny study indicated that the rhizobia symbiotic with Desmodium spp. had great diversity at generic level.The phylogenetic positions constructed by the sequences of nitrogen genes nifH had a consistency with 16S rRNA gene sequences, while the nodulation genes nodC existed discrepancy with 16S rRNA gene sequences, which revealed a relativity with the nodulation range of rhizobia.The DNA relatedness between Bradyrhizobium sp. CCBAU23174 and B.elkanii USDA76~T was 100%, which demonstrated that this group reprensentive by CCBAU23174 belonged to B.elkanii. And the DNA homology of representative strains CCBAU23230 and CCBAU65199 with...