| Watermelon fusarium wilt also known as Fusarium wilt, a worldwide distributed of soil-borne disease, is caused by Fusarium oxysporum. The incidence of watermelon fusarium wilt is generally 10%-30%, particularly serious in successive cropping melonfield. The disease leads to 20%-50% yield reduction, and in severe cases even to complete loss in harvest, which causes huge economic losses. At present, the screening of antagonistic microorganisms from soil to prevent and treat Watermelon fusarium wilt caused by Fusarium oxysporum, has gradually become one hot topic of research. Research was done to screen Bacillus bacteria with better biological control potential of Watermelon fusarium wilt caused by Fusarium oxysporum, in order to prevent and treat Watermelon fusarium wilt in watermelon farming.In order to obtain antagonistic bacteria against Fusarium oxysporum, strains were isolated and screened with the improved agar plate diffusion method using the soil from different districts. The antagonistic activity of strain HD-5, strain CZ-10 and strain BY-1 against Fusarium oxysporum was tested via the pot test for preventing and treating of Watermelon fusarium wilt in the laboratory. A strain HD-5, exhibiting the strongest antagonistic activity against Fusarium oxysporum was obtained. The morphological characteristics, physiological and biochemical properties and 16S rDNA sequence of this strain were studied to determine the species. The similarity of the 16S rDNA sequences between strain HD-5 and Bacillus amyloliquefaciens was up to 99.78%. Finally, the strain HD-5 was identified as Bacillus amyloliquefaciens.In order to increase the quantity of antifungal protein from Bacillus amyloliquefacien HD-5, the strain was cultured under different conditions. Through single factor experiments, the suitable carbon source, nitrogen source and inorganic salt were chosen. The optimal composition of media was determined by orthogonal experiments. The optimization of fermentation factors, such as initial pH, fermentation temperature and fermentation time, was made by single factor experiments. By the orthogonal design, the optimal proportions of fermentation medium were glucose 5.0%, soybean peptone 5.0%, MnSO4 0.05% , MgSO4·7H2O 0.05%. By single factor experiments, the optimal fermentation condition was initial pH 7.5 and inoculum age of 14~16 hours, shaking flask fermentation temperature is 30℃for 48 hours, under the listed conditions, the highest quantity of antifungal protein was obtained. After optimization, diameter of inhibition zone was increased by 65.0%In order to improve the production rate and spore quantity of antagonistic strain HD-5 of Bacillus amyloliquefacien against Fusarium oxysporum. The main factors influencing sporulation were investigated using shake-flask fermentation method. Through the single factor experiment and orthogonal experiment, the optimal shaking flask fermentation condition was determined as follow: media composed of 0.5% corn meal, 0.5% soybean peptone, 0.05% MgSO4·7H2O, 0.05% MnSO4, initial pH at 7.5, 5% of inoculum size, fermentation for 48h at 37℃with rotating speed 180r/min. Under this optimum condition, the spore quantity was 1.2×10~9 spore /mL, and spore production rate achieved 89.62%.For field application testing and extension application of B. amyloliquefaciens strain HD-5, fermentation parameters of strain HD-5 in large-scale production were investgated and obtained by trial production in 2 m3 fermenter. After fermentation, the fermentation broth of bacteria concentration was up to 1.0×109 cfu/mL and the sporulation rate up to 85.0%. Finally, 140 kg inocula solids was prepared by drying through adding sawdust. The viable cell number was determined to be 7.0×10~9 cfu/g. |
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