Genetic Diversity And Characteristics Of Rhizobia With Heavy Metals Resistance In Taibai Mining Tailing Of Shaanxi Province In China

To screen heavy metals resistant rhizobial strains and study their genetic diversity, phylogeny of rhizobia, 108 rhizobial strains were isolated from 8 species of legume plants nodules: Medicago lupulina, Vicia unijuga, Lespedeza cuneata, Indigofera pseudotinctoria, Vicia sepium, Astragalus chrysopterus, Pueraria lobata, Campylotropis macrocarpa, which generated from gold mining tailing of Taibai region in Shaanxi province of China.One heavy metals resistant excellent rhizobium RS33-2 belonged to the genus of Agrobacteria obtained from heavy metals resistant medium screening, which were characterized with Physiological and biochemical characteristics, growth curve by different pH culturing conditions, growth on the presence of different kinds and concentrations with heavy metal elements, heavy metals accumulation, heavy metals resistant genes amplification, 16S rDNA phylogenetic analysis and so on.In test of growth curve in different pH initial culturing conditions, cell biomass was reduced with pH decreasing and not obviously changed in pH of culture medium from 6.5 to 7.0. In growth inhibitation test on the presence of heavy metals, cell biomass was reduced with the concentrations of heavy metal ions increasing, and the order of toxic effect of four kinds heavy metals element was as follow: Cd²⁺>Cu²⁺>Pb²⁺>Zn²⁺. In high concentration of heavy metal ions, cell biomass of optical biosorption was negative at first growth period. In heavy metal ions accumulation test, the accumulation mass order of three heavy metal ions was: Zn²⁺>Cd²⁺> Pb²⁺>Cu²⁺, the accumulation of heavy metal ions were different with the kind of heavy metal elements and treatment time, and the heavy metal ions accumulation of cell-body inner was more than its in cell wall.To deeper study mechanism of heavy metal resistance, heavy metal resistant genes were amplified by PCR, and other heavy metal genes were not obtained except copper resistant gene cop A. Homologous analysis showed copA was 69.6% similarity level with its Caulobacter crescentus CB15 (NP419780).To study genetic diversity and phylogeny of rhizobia in heavy metals polluted region, 55 rhizobia strains which host plants were M. lupulina, L. cuneata, P. lobata, C. macrocarpa, V. sepium were selected to teste using 16S rDNA PCR-RFLP, 23S rDNA PCR-RFLP, IGS PCR-RFLP and 16S rDNA sequence determinarion.16S rDNA PCR-RFLP results that 55 tested rhizobia strains were grouped at 70% similarity level, and classified 3 geneic groups according host plants range. 23S rDNA PCR-RFLP analysis results showed all strains were good grouped at 74% similarity level, and could be formed 6 genetic groups. The results of IGS PCR-RFLP denoted at 66% similarity level, 55 strians were good grouped. Comparing with 16S rDNA PCR-RFLP, 23S rDNA PCR-RFLP, IGS PCR-RFLP, the result of 16S rDNA PCR-RFLP was good accordance with 23S rDNA PCR-RFLP. From the results of PCR-RFLP,the strains isolated from M. lupulina were less genetic diversity and the strains from L cuneata were large genetic diversity.From 16S rDNA PCR-RFLP results, The phylogenetic analysis of 16S rRNA gene sequences of representative strains was revealed. Type strains from Medicago lupulina plant TB99-2 and TB50-1 were closed to Sinorhizobium genus branch, and 99.8% similarity of S. melilotiLMG6133. L. cuneata rhizobial strains had two phylogenetic branches: Agrobacterium and Rhizobium, TB17-1 was located in Rhizobium genus, 98.8% similarity level with R. leguminosarumUSDA2370, RS33-2 was closed to Agrobacterium phylogeny branch, 98.8% similarity level with Ag. tumefaciens LMG196. TB94-1 isolated from Campylotropis macrocarpa was the highest similarity with R. gallicumR602, the value of similarity level was 99.4%.