Purification Of Two Antimicrobial Substances Produced By Bacillus Subtilis Strain B11 And Consruction DNA Library Of The Strain

Bacillus subtilis strain B11, isolated from soil in Guangxi, was strongly inhibitory to the pathogen of watermelon wilt. Produced antimicrobial substances by this strain was one of the antagonistic mechanisms against the pathogens. In this paper, two antimicrobial substances produced by strain B11 were purified by successive steps of chromatography, then their characters and antagonistic mechanism were tested in the experiments. Meanwhile, a DNA library of this strain was constructed. The results of this research are as follows:Two antimicrobial substances produced by Bacillus subtilis strain B11, were purified through successive steps of boiling, DEAE 52 anion exchange chromatography and aluminum oxide adsorption chromatography. These purified substances showed only one spot on silicon thin layer chromatography (TLC). These antimicrobial substances were named as antimicrobial substance A and antimicrobial substance B, respectively.These antimicrobial substances were resistant to proteinase K and pepsin; antimicrobial substance A was also resistant to trypsin; but antimicrobialsubstance B was sensitive to trypsin. Both of these two antimicrobial substances were thermostable (121℃, 20 min).These antimicrobial substances could inhibit the growth of Fusarium oxysporum f. sp. niveum, Xanthomonas oryzae pv. oryzae strain 13751, Ralstonia solanacearum strain P13 and Rhizoctonia solani; but antimicrobial substance A could not inhibit the growth of Pyricularia grisea. However, antimicrobial substance B could inhibit the growth of Pyricularia grisea. Treated with 4.92 μg/mL of antimicrobial substance A 120 h, the inhibitory rate of the hyphal growth and conidiation were 52.5% and 98.61%, respectively. Meanwhile treated with 5.08 (μg/mL of antimicrobial substance B 120 h, the inhibitory rat of the hyphal growth and conidiation were 51.22% and 94.93%, respectively.The inhibitory mechanism of antimicrobial substances produced by strain B11 to the conidia of F. oxysporum f. sp. niveum was detected under microscope. Treated with 30% (V/V) of antimicrobial substance A, 40% of the conidia were abnormal in 12 h, all the conidia were abnormal in 24 h; the morphology of the conidia was bulgy, and its bioplasm was condensed in 48 h. Meanwhile treated with antimicrobial substance B the conidia's bioplasm of the pathogen was condensed in 36 h. Electronic microscopic scanning the spores treated with these two antimicrobial substances revealed that these two antimicrobial substances deformed conidia of F. oxysporum f. sp. niveum and produced holes on the conidia, the results of the experiment indicated thatthese two antimicrobial substances acted on the thalli membrane of F. oxysporum f. sp. niveum.A DNA library contained about 6,700 clones of strain B11 was constructed by meaning of successive steps of digesting the total DNA of the strain B11 by EcoRV enzyme, recovering 48Kb DNA fragment followed end-repair DNA, ligating insert DNA with cosmid vector, packaging the ligated insert DNA, and adding the packaged cosmids to Escherichia coli EPI100 host cells. The result of detecting the quality of the library by BamHI digesting showed that the insert DNA fragments of the library were random, and average length was 37Kb.