Screening, And Isolation Of Antifungal Protein Against Fusarium Oxysporum

Watermelon fusarium wilt is a fungal vascular system disease which is infested byFusarium oxysporum f.sp.niveum.It's a devastating disease. Watermelon fusarium wilt causedserious losses to waermelon production in China.And it severely restrict the development ofthe watermelon production.In order to identify a strain which is effective in controlling the Fusarium oxysporum,346 strains were isolated and screened from the soil of the watermelon root. A strain named5-20 strain with a rather higher activity was obtained. Though the morphology characteristics,physiological and biochemical properties and 16S rDNA sequence of this strain, the 5-20strain was finally identified as a kind of Bacillus velezensis.The further studies on the antifungal protein-Producing conditions of the strain wascarried out. Through single factor experiment and orthogonal experiment, the optimal shakingflask fermentation condition was determined as follow: media composed of 3% cornmeal, 3%peptone, 0.05% CuSO₄·7H₂O, 0.05% ZnCl2, initial pH 7.0 and 10% inoculum volume, mediavolume 50/250(mL/mL), fermentation time 36h. A distinguished elevation of the antagonisticactivity was observed with about 39.9%.The difference in activity is obvious: the area ofinhibition zone was 509.25mm2 for those optimized and 379.94mm2 for those common.Spore is the best preparation form of microbial agents. Strain B.velezensis5-20 whichwas isolated and screened by our lab has better antagonistic activity against Watermelonfusarium. In order to improve the formation rate and spore quantity of antagonistic bacteriumstrain 5-20,We investigated the main factors influencing sporulation using shake-flaskfermentation method.The optimal shaking flask fermentation condition was determined asfollow: media composed of 0.5% cornmeal, 2% soybean meal, 0.3% MnSO4,0.3%FeSO4·5H2O. Initial pH 7.0, incubation time 18 h and bottle filling capacity 75 mL/250mL,10% inoculum volume, fermentation time 48h, fermentation temperature 30℃, rotating speed220r/min.Under this optimum condition, the spore formation rate achieves 96.75%. Biomassachieves 2×10⁹ spore /mL. The proteins were extracted by ammonium sulphate precipitation from the fermentedbroth of B.velezensis5-20. Assays for proteins activity were done on plate. The antifungalprotein was stable at pH6.0¹0.0, the optimal reaction temperature and pH value were 25℃and pH 7.0 respectively. The activity was enhanced by the addition of Mn²⁺and Mg²⁺.