Isolation Of Antagonistic Strain Against Verticillium Dahliae Dm-54 And Purification Of Antifungal Protein

Cotton Verticillium wilt is one of the most dangerous fascicular disease caused by Verticillium dahliae Kleb. V. dahliae caused serious losses to cotton production in China, known as the "cancer of cotton". Biological control which people pay more attention now is a highly effective measure for management of cotton Verticillium wilt.Eight hundred ninety one strains having antagonistic activities against V. dahliae Kleb were isolated and screened from the soil of cotton field from several different regions.89 strains had antagonistic activity.10 strains had antagonistic activity after secondary screening. A strain named DM-54 strain with a rather higher antagonistic activity was obtained. The experiment result showed that the antifungal substance of DM-54 is protein. The antifungal protein showed antibiosis activity to 9 pathogenic fungis, such as V. dahliae, Colletotrichum gloeosporioides Penz, Ceratosphaeria phyllostachydis, Colletotrichum lagenarium, Fusarium moniliforme Sheld and so on.Thus the morphology characteristics, physiological and biochemical properties and 16S rDNA sequence of this strain were further studied. Through 16S rDNA sequence comparison analysis as well as the physiological and biochemical properties analysis referring to Bergey's Manual of Determinative Bacteriology and Manual of systematic and Determinative Bacteriology, the DM-54 strain was finally identified as a kind of Bacillus amyloliquefaciens.The further studies on the antifungal protein-producing conditions of the strain was carried out. Through single factor experiment and orthogonal experiment, the optimal shaking flask fermentation conditions were determined as follow:media composed of 5% dextrin,3% soy peptone,0.02% MgSO4,0.3% CaCl2, initial pH 7.0 and 10% inoculum volume, media volume 30/250(mL/mL), fermentation temperature 32℃, rotating speed 200r/min, fermentation time 48h. A distinguished elevation of the antagonistic activity was observed with about 39.9%. This essay paves a bright way for the large scale production of the antagonistic protein.Spore is the best preparation form of microbial agents. Strain DM-54(Bacillus amyloliquefaciens) which was isolated and screened by our lab had better antagonistic activity against V. dahliae. In order to improve the formation rate and spore quantity of antagonistic bacterium strain DM-54. We investigated the main factors influencing sporulation using shake-flask fermentation method. Through single factor experiment and orthogonal experiment, the optimal shaking flask fermentation conditions were determined as follow: media composed of 0.5% lactose,2% soybean meal,0.3% MnSO₅·H₂O, initial pH 7.5, incubation time 18 h and bottle filling capacity 50 mL/250mL,10% inoculum volume, fermentation time 36h, fermentation temperature 30℃, rotating speed 220r/min. Under this optimum condition, the spore formation rate achieved 98.3%. Biomass achieves 2×109 spore /mL.Colonization of antagonistic bacteria DM-54 in root soil of cotton was studied through pot experiment of sterilized and non-sterilized soil, part and all mixed soil three kind of different methods. The results showed that antagonistic bacterium DM-54 could colonize in the sterilized and non-sterilized under laboratory conditions,the population in the sterilized and non-sterilized soil was 106cfu/g,and still kept the high bacteriostatic activity. The numbers in the non-sterilized soil were less than those in the sterilized soil. The colonization activity of all mixed soil is the highest. The results showed that antagonistic bacterium DM-54 could colonize in root of cotton on 30th day after treatment.An antagonistic protein was purified from the fermented broth of strain DM-54 by ammonium sulphate fractional precipitation, exchange chromatography on DEAE-Sepharose Fast Flow and preparative PAGE. The protein was a single chain polypeptide with a predicted molecular weight of 47 kDa in SDS-PAGE. We have done a preliminary research of the antifungal protein nature. The results showed that the optimum temperature of the antifungal protein was 28℃and the activity of antifungal protein increased with the temperature dropped. The optimum pH was 5.0, and Cu2+ was completely inactivating the antifungal protein, Mn2+ could apparently activate the antifungal protein.