Isolation And Identification Of Soil Free Living Nitrogen Fixation Bacteria And Their Nitrogen-fixation And Plant Rowth-promotion Effect

Free-living nitrogen fixation bacteria are important plant growth-promoting rhizobacteria (PGPR), and they can supply plants with N by bio-nitrogen fixation. This reseach began with isolating and screening free-living nitrogen fixation bacteria. 26 strains from different colonies were isolated from Ashby medium. After cultivating them on Ashby plate for several generation, we got 14 strains which could grow well on the plate. Then total nitrogen was determined by using of Ashby liquid medium of a 12d culture to evaluate nitrogen fixation activity of each strain. The results showed that strains R6, MR4, MR8, V1 had comparatively high amounts of fixed nitrogen which were 2.7, 4.0, 3.0, 2.7 mg L^-1, respectively. A pot experiment was conducted by inoculating soils with the cultures of R6, MR4, MR8, V1, in which wheat plants were grown. The results showed that inoculation with R6 and MR4 significantly increased the biomass and total nitrogen of wheat plants. By 16S rDNA technique and phenotypic feature analysis, MR4 and R6 were identified and named as Phyllobacterium brasssicacearuvm MR4 and Rhodococcus ruber R6.The potential of the two strains Rhodococcus sp and Phyllobacterium sp as PGPR were further checked by a laboratory mediaum test and a greenhouse pot experiment. In laboratory experiments, inoculation of cucumber seedlings with LB or Ashby liquid medium growing with MR4 and R6 significantly increased total root length and biomass of the plants in comparison with controls. In greenhouse experiments, biomass and nitrogen uptakes of tomato plant increased with the increase of inoculation rates of both strains, indicating that Rhodococcus sp and Phyllobacterium sp are PGPRs. Denaturing gradient gel elecrophoresis (DGGE) results showed that application of organic manure or the two bacterial agents increased the DGGE bands, implying that the soil bacterial diversity was enhanced.Bacterial nitrogenase activities of P. brasssicacearuvm MR4 and R. rubber R6 were measured by acetylene reduction assay (ARA). P. brasssicacearum MR4 had similar nitrogenase activities in comparison with reference Azospirillum brasilense, whereas the nitrogenase activity of the R. rubber R6 was not detected. The mechanism of growth on nitrogen-free medium and plant growth-promoting effect of the Rhodococcus sp needed to be further investigated.We concluded that P. brasssicacearum MR4 and R. ruber R6 strains have significant effect on plant growth-promoting and a great potential to improve crop production.