Effects On Soybean Rhizobial Diversity And Nitrogen Fixation Under Different Nitrogen Levels

In this study, strains of soybean rhizobia were isolated and verified from7different fertilizertreatments in “Long-term fertility experiments of black soil”, that was found by Heilongjiang Academyof Agricultural Sciences in1979. Genetic diversity and phylogeny of these rhizobia with referencestrains were analyzed and a superior nitrogen-tolerance rhizobia strains was screened. Proteomics wasused to analyze the mechanisms of tolerance to high nitrogen. The results were great meaningful toreveal the effect on genetic diversity and abundance of soybean rhizobia, and the mechanisms ofrhizobia strains growing under high nitrogen levelin black soil. Conclusions were showed as follows.In this paper,254soybean strains were isolated. The genetic diversity and phylogeny wereanalyzed by BOX-PCR, IGS PCR-RFLP and16S rDNA gene sequencing methods. The resultsindicated that, all the tested soybean strains belonged to Bradyrhizobium, and Bradyrhizobiumjaponicum was the dominant species. Under similarity level of59.24%in BOX-PCR and87.42%inIGS PCR-RFLP, rhizobia were divided into5and3groups, respectively. Correlation coefficient ofcorrelation analysis was0.524(p<0.05), indicating the clustering and location fertilization of isolateswere significantly related. Further analysis was carried out baced on the analysis of the similarity andSimpson diversity index. The results showed that, with the raise of nitrogen content, the similarity toCK of treatments was lower, and the single application of nitrogen fertilizer could reduce the diversityof rhizobia. The rhizobia population in CK, N1, N2treatment was tested by MPN methods, the resultsshowed that, application of nitrogen fertilizer could significantly reduce rhizobia population, and themore the notrogen fertilizer is, the smaller the rhizobia population would be. Correspondence analysisbetween soil properties and population structure of rhizobia indicated that, long-term application ofnitrogen fertilizer resulted in an increase of available nitrogen and decrease of pH, which was the maincause for the changes of population structure of rhizobia.The superior nitrogen-tolerance rhizobia strains were screened form254strains mentioned aboveby adopting high-concentration nitrogen plate method and vermiculite pot. The result showed that, theincreasing urea concentration had an increased bacteriostatic effect. Urea concentration which couldseriously inhibit growth of strains was5g·L-1, only11strains could grow in this urea concentration,which were all isolated from continuous application of nitrogen fertilizer. In the subsequenthigh-concentration nitrogen vermiculite pot experiment, the best strain of5841were selected accordingto the dry weights and numbers of nodules, dry weights and total N contents, which had the ability ofnodulation nitrogen fixation in high-concentration nitrogen.Proteomics was followed to analyze the mechanisms of different tolerance to high nitrogenbetween B. japonicum5841and5777. The results showed that, B. japonicum5841had11up-regulatedand5down-regulated proteins; B. japonicum5777had10up-regulated,10down-regulated and9vanished proteinswhen treated by additional nitrogen source.Different expressed protein spots were identified by MOLDI-TOF mass spectrum and the resultsshowed that, the different regulation mechanism of adapting to the high nitrogen environment was different for two strains. B. japonicum5841upregulated important defense protein methionine sulfoxidereductase B to against oxidative stress, and many metabolic-related proteins were up-regulated. B.japonicum5777increased cell viability in stress environment by the up-regulatedglutathione-S-transferase, and the expression of many important proteins associated with metabolicappeared down-regulated or disappeared. The expression of ABC transporter proteins involved in themetabolism of amino acids or monosaccharide transportation,and regulatory protein two-componentregulatory factor were also down-regulated. Therefore, different defense mechanisms and metabolicadaptation may be the main reason resulting in two strains of rhizobia survival and nitrogen fixationability differences in high nitrogen conditions.