Analysis Of Microbial Species Diversity In Kumtag Desert And Screening Of Antagonistic Strains Against Colletotrichum Gloeosporioides

In this study, strains were isolated from the soil samples of Kumtag desert with the method of dilution butteron on plate. Purification and determination of 16 S r DNA sequence were conducted according to the difference of colony morphology on the plate. The bacterial species of the strains were identified and their population diversity were analyzed. Then a strain named N147-1-3 with stronger antagonism against the Cymbidium(Colletotrichum gloeosporioides) was obtained with the methods of plate confrontation and diffusion from the strains separated, and sequence of 16 S r DNA was analyzed and physiological and biochemical identification were conducted. Finally, inhibitory substances were preliminarily explored and properties of the strain were analyzed. The results were as follows:Strains were isolated from 9 soil samples of Kumtag desert using two mediums(R2A, 1/4R2A), and 190 strains were obtained. The strains were mainly distributed in the Firmicutes, Proteobacteria and Actinobacteria. A total of 30 genera and 57 species were isolated in this desert area, among them there were 119 bacillus strains which were the predominant strains accounted for 63% of the total strains. Potential new species of 22 strains needed further research, which the similarity was below 97.5% compared with the sequence similarities known on the Ez Bio Cloud wet. The four parameters of population diversity were: R2A: richness(S) was 12.37±56.55, dominance index 0.2913±0.0536, hannon-Wiener index(H) 1.3262 ± 0.3612, evenness(E) 0.6034 ± 0.0312; 1/4R2A: richness(S) was 10.62±41.41, dominance index 0.3566±0.0544, hannon-wiener index(H) 1.3025±0.3576, evenness(E) 0.6066±0.0476.An antagonistic strain with higher activity against Cymbidium anthracnose pathogen named N147-1-3 was obtained from the isolated strains, and was identified as Bacillus methylotrophicus according to the results of 16 S r DNA sequence analysis and physiological and biochemical test. The optimum fermentation conditions of strain were 5.0% sucrose, 2.0% Na NO3, 0.05% Ca Cl2 and 0.3% KNO3, p H 7.0, fermentation time 48 h, inoculation size 5.0%, liquid volume 125 m L broth in 250 m L flask. Results showed that crude protein of the antagonistic bacteria reached the highest antibacterial activity by adding 60% ammonium sulfate to fermented liquid. The supernatant of the strain had no antimicrobial activity after extraction with chloroform. The antagonism effect of the strain was disappeared after treatments with high temperatures or proteinase K. It was finally identified as proteinaceous material. The antibacterial substances was sensitive to the temperature. As the temperature rised, the activity decreased. The activity reached the maximum at p H 6.0. The dependence of antibacterial substances on the metalions was not strong.