Study Of Biocontrol Bacterium And Isolation And Purification Of Antifungal Protein Of Antagonistic Strain Bacillus Subtilis 7-19 To Tomato Early Blight

Early blight of tomato is one of the most constant disease caused by A.solani(Ell&Mart).sorauer. It is also a worldwide disease.Which caused serious losses to tomato production in China as meanwhile in the world. At present, biological control method which has most development potentiality is regarded as a highly effective measure for the preventionand cue of early blight of tomato.Three hundreds and sixty-two strains having antagonistic activities against early blight of tomato were isolated and screened from the soil of tomoto field and disease tissues of tomoto plants from several different regions. One hundred and twenty strains have antagonistic activity. Five strains have antagonistic activityfter secondary screening. A strain named 7-19 strain with a rather higher antagonistic activity was obtained. The experiment result shows that the antifungal substance of 7-19 is protein.Thus the morphology characteristics, physiological and biochemical properties and 16S rRNA sequence of this strain were further studied. Through 16S rRNA 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 7-19 strain was finally identified as a kind of Bacillus subtilis.The further studies on the antifungal protein-producing conditions of the strain B. subtilis 7-19 was carried out. Through single factor experiment and orthogonal experiment, the optimal shaking flask fermentation condition was determined as follow: media composed of 3% maize meal, 3% soybean cake powder, 0.1% MgSO₄, initial pH 8.0 and 6% inoculum volume, media volume 50/250(mL/mL), Seed age 16 h,fermentation temperature 30℃, rotating speed 180 r/min, fermentation time 48 h. A distinguished elevation of the antagonistic activity was observed. This essay paves a bright way for the large scale production of the antagonistic protein.An antagonistic protein was purified from the fermented broth of strain B. subtilis 7-19 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 23 kDa in SDS-PAGE. We have done a preliminary research of the antifungal protein nature. The results shows that the optimum temperature of the antifungal protein is 42℃and the activity of antifungal protein increased with the temperature dropped. The optimum pH was 5.0, Cu²⁺ is completely inactivating the antifungal protein, Mn2+ can apparently activating the antifungal protein. Spore is the best preparation form of microbial agents. Strain B. subtilis 7-19 which was isolated and screened by our lab has better antagonistic activity against Verticillium dahliae. In order to improve the formation rate and spore quantity of antagonistic bacterium strain B. subtilis 7-19. We investigated the main factors influencing sporulation using shake-flask fermentation method. Through single factor experiment and orthogonal experiment, the optimal shaking flask fermentation condition was determined as follow: media composed of 1% Corn flour, 1% Soybean peptone, 0.1% MnSO4,0.051% CaCl₂. Initial pH 6.0, incubation time 20 h and bottle filling capacity 30 mL/250 mL, 8% inoculum volume, fermentation time 60h, fermentation temperature 30℃, rotating speed 220r/min. Under this optimum condition, the spore formation rate achieves 98.56 percentages. Biomass achieves 3.5×10⁹ spore /mL.Colonization of antagonistic bacteria 7-19 in root soil of cotton was studied through pot experiment of sterilized and non-sterilized soil,and Soaking of Seed,part and all mixed soil three kind of different methods,The results showed that antagonistic Bacterium 7-19 can colonize in the sterilized and non-sterilized under laboratory conditions,the population in the sterilized and non-sterilized soil was 106 cfu/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 highest was proved.The results showed that antagonistic Bacterium 7-19 can colonize in root of cotton on 30th day after treatment.