Optimization Of Fermentation Technology Of Bacillus Subtilis BS501A And Its Control Effect In Greenhouse

Rice blast is a fungal disease caused by Magnaporthe grisea. Because of its serious effect on rice production, and the easy-loss of the resistance of the rice varieties to rice blast, and the environmental pollution caused by chemical compounds, the study of the biological control of M. grisea is important. Bacillus subtills strain BS501a which was screened from the soil of a rice field has strong antagonistic activity to M. grisea. So it has a great biocontiol potential. Optimization of fermentation technology of B. subtills BS501a to improve the antagonistic activity of the fermentation supernatant and reduce the production cost of fermentation could lay a foundation for submerged fermentation.Fermentation medium and conditions were optimized for B.subtills BS501a fermentation technology. Firstly, soybean meal and peanut cake as the optimal carbon and nitrogen source were screened using one-factor-at-a-time. Then a Plackett-Burman (PB) design was used to find the significant factors affecting antagonistic metabolites production. The result of PB showed that soybean meal, peanut cake and KH2PO4were the significant factors on the production of antagonistic metabolites of B.subtills BS501a. A central composition design (CCD) was used to optimize the significant factors screened from PB design. Finally, The optimal fermentation medium formula and fermentation conditions were determined, i.e, soybean meal3.2%, peanut cake1.21%, yeast extract0.5%, KH2PO40.07%, K2HPO40.2%, MgSO4·7H2O0.05%, MnSO4-H2O0.01%, FeSO4-7H2O0.01%, CaCl20.15%, initial medium pH of7.2, inoculums volume5%, cultivation temperature37℃, liquid volume in flask100mL/500mL. Under the optimal fermentation technology, the hypha growth inhibition rate of B. subtilis BS501a filtered fermentation supernatant was91.6%, increased26.7%than that of the pre-optimization and6%than that of initial medium, but the fermentation cost decreased RMB900per ton then the initial medium.The stability of the antagonist of the fermentation supernatant of B. subtilis BS501a was studied. The antagonistic activity was stable in the temperature less than45℃, the hyphal growth inhibition rate of the fermentation supernatant only decreased2.3%after being stored for10d in the temperature of45℃, but the hyphal growth inhibition rate decreased27.3%after stored for10d in60℃. The antagonistic activity was strong in pH7-9, the antagonists in fermentation supernatant were soluble in water in alkaline conditions, but would precipicate in acid conditions. The hyphal growth inhibition rate only decreased0.7%after10d under the light intensity of4500±5001x. The antagonists could not be digested by proteinase K. These results showed that the anagonistic metabolites had physical and chemical stability. The inherited stability was studied by transfering B. subtilis BS501a from one plate to another and the hyphe growth inhibition rate of the fermentation supernatant of different generations of B. subtilis BS501a were studied. The result showed that the hyphe growth inhibition rate of the fermentation supernatant of the10th generation of B. subtilis BS501a only decreased0.3%than that of the1st generation. So, B. subtilisBS501a has a good inherited stability.The control effect of the fermentation supernatant of B. subtilis BS501a to M. grisea in greenhouse was studied. The result showed that the biological control efficiency of B. subtilis BS501a was63.8%. So B. subtilisBS501a is a potential bacterial strain for biological control.