Study On Optimization Of Bacillus Surfactin High-efficiency Fermentation Medium And Its Control Effect On Rice Diseases

Surfactin is a natural synthetic lipopeptide antibiotic with excellent surface activity,non-specific and broad-spectrum antibacterial effect,and the ability to induce plant disease resistance.It has broad application prospects in industrial and agricultural production.The yield of strains in nature is low,in order to increase the output of Surfactin and reduce the production cost,this study through metabolic engineering and fermentation engineering technology,the construction of high-yield Surfactin engineering strains and optimization of fermentation medium to achieve high production of Surfactin.Rice sheath blight and rice bacterial leaf blight are both important diseases in rice production and cause serious economic losses every year.In order to explore the mechanism and effect of Surfactin in controlling rice sheath blight and rice bacterial leaf blight,this study through the detection of the expression levels of SA and JA pathway marker genes in rice leaves and the determination of indoor virulence,the mechanism of Surfactin’s prevention and control of rice diseases was determined;finally,through potted plant efficacy and field efficacy tests,the efficacy of Surfactin in preventing and controlling rice diseases was clarified.Results were as follows:1.On the basis of Bacillus subtilis B9D,we successfully constructed the double mutant strain B9BD of degU and bmyA genes.Compared with the wild-type strain Bs916,the double mutant strain can synthesize more Surfactin,and the two lipopeptide antibiotics Bacilimycin L and Fengycin were no longer produced,successfully improving the yield and purity of Surfactin.We also found that the biological phenotype of the double mutant strain has undergone tremendous changes:the growth rate has decreased,the ability to form biofilms and swarming motilities were almost lost,and the expression level of the srfAA gene has increased.2.Through single factor screening test,PB test and CCD test results,we simulated the optimized medium:Opt(Glucose 24.27 g/L,Soybean meal 29.05 g/L,Beef extract 3 g/L,NH4NO3 4 g/L,L-leucine 2.36 g/L,KH2PO4 3 g/L,NaCl 3 g/L,FeSO4 0.02 g/L).Strain B9BD produced 934.25 mg/L of Surfactin in Opt medium,which was 2.3 times the production of Surfactin in Bas medium and 5.6 times that in LB medium and Landy medium.Strain Bs916 produced 334.37 mg/L of Surfactin in Opt medium,which was 2.0 times,4.7 times and 4.8 times of Surfactin in Bas medium,LB medium and Landy medium,respectively.3.The Quantitative Real-time PCR results showed that the expression of the SA pathway marker genes OsPALI,OsEDS1 and OsNPR1 increased significantly when the rice was treated with Surfactin for 8 h and 12 h,compared with the water and ethanol controls;after 24 hours of treatment,the expression levels of five genes in the treatment group were significantly higher than those in the control group,and the expression of OsLOX2 and OsAOS2,the marker genes of the JA pathway,was more intense;at 48 h of treatment,the OsEDSl and OsNPR1 genes in the treatment group could maintain a higher expression level.The laboratory virulence test results showed that Surfactin has certain virulence to rice sheath blight fungus and Xanthomonas oryzae pv.oryzae,the EC50 values are 19.76 μg/mL and 16.49 μg/mL,respectively.In the pot plant efficacy test,the Surfactin solution with an effective concentration of 100 μg/mL has 58.29%control effect on rice sheath blight disease,the average control effect on rice sheath blight disease spots was 55.61%,and the inhibitory effect on rice bacterial blight disease spot was 64.21%.In the field efficacy test,Surfactin solution with an effective concentration of 100 μg/mL has an overall control effect of 44.64%against rice sheath blight,and an overall control effect of 49.71%against rice bacterial blight.The above results show that:the engineered strain B9BD was fermented and cultured in the optimized medium Opt,and the output of Surfactin was greatly increased,reaching 934.25 mg/L;Surfactin can induce rice to acquire disease resistance through both SA and JA at the same time,the SA pathway has a longer lasting;Surfactin’s control of rice diseases is achieved by a combination of induction of disease resistance and bacteriostasis;the 100 μg/mL Surfactin solution has a certain control effect on rice sheath blight and rice bacterial leaf blight.