Biological Control And The Optimization Of Fementation Of Bacillus Subtilis Bs-412

The biological control of plant disease is more safety compare with chemical pesticide to environments human and animal. Bacillus subtilis has a low requirement of nutrition and it can produce spore which can survival from high temperature、 drought and ultraviolet radiation. Some kinds of Bacillus subtilis have broad-spectrum resistance and strong antagonism to pathogen fungi. They have already been produces who achieved good results. So, Bacillus subtilis is an kind of bacterium resources which is worth to study.A broad-spectrum resistance strain was isolated from rhizosphere soil by our laboratory who was identified as Bacillus subtilis. And named it Bs-412. Bs-412has a broad-spectrum resistance on pathogen fungi such as Valsa ambiens, Dothiorella gregaria, Fusarim solani and Botryosphaeria dothidea etc by the experiment of colony resistance method and cylinder-plate method. It also has a broad-spectrum resistance on pathogen bacteria like Escherichia Coli and Staphylococcus aureus. The activity of antagonistic substance in fermentation supernatant is not resistant to storage,hot temperature and acid or alkali, but the light has no influence to its activity. Bs-412showed that it has good genetic stability after inoculation for ten generations.Corn flour and soybean meal were certainly as the most suitable carbon sources and nitrogen sources by single-factor experiments. And we also found CaO3and MnS04were effective for the viable count and the rate of sporulation of strain Bs-412. The finally culture medium of Bs-412was optimized by Plackett-Burman design experiment,steepest descents experiment and Box-Benhnken Design experiment. The composition of culture medium were:corn flour22.80g, soybean meal24.78g, KNO35g, CaCO33.58g, MgSO43g, MnSO40.3g, KH2PO40.3g, water1000mL, pH7.0. The viable count was4.20×10l0cfu/mL after optimization which is increased by52.73%than the first2.75×1010cfu/mL. Though the optimization of shake flask culture conditions, the best shake flask culture conditions as follow:temperature is32℃, liquid volume in flask is75mL/250mL, inoculation amount is 5%, rotation speed is200r/min. In the end,the viable count of fermentation reached4.72×1010cfu/mL.It found that the effect of number3and4of the emulsion preparation is best by different formula of emulsion test. All of the emulsion didn’t meet the test requirrments, but the composition of emulsion had effect to the survival rate of spore. So, it is necessary to choose more emulsion composition to improving the method of experiment.