Primary Study On Taxonomy And Diversity Of Rhizobia Isolated From Pueraria Spp.

Thirty-two strains isolated from the root nodules of Pueraria spp. in sichuan province,henan province,hunan province et al. were studied together with the reference strains of recognized Rhizobium and Bradyrhizobiwn species by performing polyphasic taxonomy,including numerical taxonomy,REP-PCR fingerprinting,16S rDNA PCR-RFLP.The result show that:The growth rate of rhizobia isolated from the root nodules of Pueraria spp.showed great diversity. CCBAU41147 CCBAU6110 K CCBAU61096 and CCBAU61095 were fast-growing strains,the single colony size was bigger than 1mm after 2 days incubated OQ YMA medium at 28 They can produce acid.The other strains were slow-growing strains,their single colony size was less than 1 mm after 7 days incubated on YMA medium at 28 .They can produce alkali. The result of numerical taxonomy indicated that there were high phenotypic diversity among the strains isolated from the root nodules of Pueraria spp. It can been seen in utilization of carbon and nitrogen sources,antibiotic resistance,resistance to dyes and chemicals,tolerance to acid,acid production,alkali production,nitrate reduction et al. The dendrogram divided all the strains into. 10 phenons at the similarity level of 80%. The fingerprints of REP-PCR which conducted by boxair primer showed that the isolates from the root nodules of Pueraria spp.had great genetic diversity. They were divided into 11 genetic clusters at the similarity level of 81%. Based on the result of numerical taxonomy,16S rDNA PCR-RFLP were applied to 12 isolates and 9 reference strains. The 16S rDNA PCR-RFLP revealed 9 different genotypies among the 12 isolates. The patterns of slow-growers were markedly different to that of fast-growers. CCBAU61116x CCBAU41069 CCBAU23168 had identical restriction patterns.In numerical taxonomy,phenon 1 included 9 new isolated strains and 1 representative strain (USDA761) of B.elkanii,the new isolated strains in this phenon probably are B.elkanii. Phenon 5 included 4 new isolated strains and 3 representative strains (USDA6T,B15,USDA110) of B.japonicum.,The new isolated strains in this phenon probably are B.japonicum. Other phenons did not grouped together with the representative strains . Their exact taxonomic position required further study. In REP-PCR analysis,genetic group 1 included 9 new isolated strains and 1 representative strain (USDA761) of B.elkanii. Genetic group 3 included 2 new isolated strains and 3 representative strains (USDA6T,B15,USDA110) of B.japonicum. Genetic group 7included 4 new isolated strains and 3 representative strains (CCBAU100711,CCBAU10073,CCBAU10040) of B.yuanmingense. Other genetic groups did not included representative strains. Their genetic relatedness needs further study. The result of RFLP analysis of PCR-amplified 16S rDNA revealed that the rhizobia isolated from the root nodules of Pueraria spp.had diversity at the level of species.The slow-growing rhizobia were divided into 7 groups on the similarity level of 90%.Group 1 included 4 new isolated strains and 2 representative strains of B.japonicum .Group 2 included 1 type strain of B.elkanii and 3 new isolated strains. Other groups were identical phylogenetic groups.To determine the exact taxonomic position and phylogenetic position of the rhizobia isolated from the root nodules of Pueraria spp,the DNA G+Cmol% test,DNA-DNA hybridization and 16S rDNA full sequence must be studied.