Polyphasic Taxonomy Of Rhizobia Isolated From Mimosa Spp. In Hainan Province

A total of 104 rhizobial isolates from 54 root nodules gathered from geographically separated populations of leguminous plant Mimosa spp. in Hainan Province were obtained. And they were analyzed by polyphasic taxonomy such as 16S rDNA PCR-RFLP, SDS-PAGE of whole cell protein, BOX-PCR fingerprinting, the sequences analysis of 16S rRNA gene together with 6 reference strains.The results of 16S rDNA PCR-RFLP indicated that all the examined strains could group together at the similarity of 61%, and further three groups were obtained at 71%: the genera Cupriavidus,Burkholderia inβ-Proteobacteria and the genus Rhizobium inα-Proteobacteria, and all the examined strains could group with the corresponding reference strains.The 104 isolates were divided into 35 BOX-PCR genotypes and 61 SDS-PAGE phenotypes, respectively, derived from analysis of BOX-PCR fingerprinting and SDS-PAGE of whole cell protein that were used to identify the strains further. Based on the results above, all the examined strains were classified into 69 different strains, which revealed that there existed abundant biodiversity both on phenotypic and genotypic.The phylogenic relationship constructed by the 16S rRNA gene full sequences suggested that bacteria in the genus Cupriavidus were the predominant root nodule symbionts for Mimosa spp. in Hainan Province; while 5 isolates were placed in the genus Burkholderia, the representative strain HBU08040 had a 16S rDNA sequence 99% similarity to Burkholderia mimosarum PAS44T isolated from Mimosa pigra in Taiwan.Furthermore, 8 strains within isolates belonged to Rhizobium,the representative strains HBU08051 and HBU08115 had a maximum similarity of 96.8% to all the validly published species in the genus Rhizobium, consequently, these strains maybe represent a single, novel species of the genus Rhizobium, and needed further analysis such as G+C mol% and DNA-DNA hybridization to confirm further.In view of the results above, the inoculation test and SDS-PAGE of whole cell protein analysis confirmation were done to test the nodulating ability of 11 selected strains, 9 of 11 selected strains were confirmed that they had the ability of nodulation with Mimosa pudica, and the other two strains (HBU08001 and HBU08149) which gained different SDS-PAGE patterns between inoculated and re-isolated strains should be studied further through other researches to illustrate. Based on these, 5 strains with the nodulation ability were conducted a follow-up experiment to directly compare number of nodules and dry weight for plants interacting with these different rhizibial strains. As a result, the rhizobial strains were all of great advantages to the growth of the plants; meanwhile, the Cupriavidus strains showed more effective.