Optimization Of Submerged Culture Conditions Of Bacillus Subtilis R31 Against Fusarium Oxysporum F. Sp. Cubense Race 4

Banana Fusarium wilt is caused by the fungus, Fusarium oxysporum f.sp. cubense, which is a major limiting factor in commercial banana production. Bacillus subtilis R31 is one kind of the entophyte bacteria found with anti-banana wilt disease.In our research, we studied the correlation between spore number and control efficacy in pot experiments, and then submerged culture medium and conditions for spores production by Bacillus subtilis R31 were optimized by Taguchi orthogonal array (OA) experimental design (DOE) methodology. The scale up test was carried out in 50 L fermentor. The results were as follows:In pot experiment, we studied the correlation between spore number and control efficacy. The results indicated that the number of spore is positively related to control efficiency (R2=0.9428), so spore number was selected as an optimal parameter in the next study.An OA layout of L18 (21×35)was constructed with six most influensive factors on spore formation, such as sucrose, soybean meal, NaCl, MgSO4, CaCO3 and initial medium pH at three levels for the proposed experimental design with Minitab15.0 software. Optimal initial medium pH was 7.0. Optimum cultivation parameters were (g/L): 15 of sucrose,15 of soybean meal,1 of NaCl,3 of CaCO3,2 of MgSO4. The results revealed an enhanced spore number of 2.5 folds more than that of unoptimized medium (from 7.0×108 cfu/mL to 17.5×108 cfu/mL). The scale up test was carried out in 50 L fermentor. According to the control of the critical dissolved oxygen (higher than 30%), we successfully scaled up the fermentation from 250 mL shake flask to the 50 L fermentor. The fermentation period was 30 hours and spore number was 23.5×108 cfu/mL. The powder was acquired by spray-drying process and the obtaining rate was 61.68%.We tried to plant banana seedlings in greenhouse in Wuhan and succeeded by soil culture. The soil contained two components:loess and peat soil. The ratio of them was 6:4. Establishment of the system laid the foundation for future mechanism research.