Screening Of High Efficiency Of Biocontrol Strain Of Bacillus Subtilis And Its Optimization Of Fermentation Technology

In production agriculture,Fungal and bacterial plant pathogens infect plants and crops easily and often seriously,causing great economic damage Application of chemical pesticides is the effective measure to control the fungal and bacterial infection and plays an important role in preventive defence.With the further knowledge to chemical pesticides,however,they bring about pollution of soil,water and atmosphere,and imperils human health and life directly,ecological environment. So,new and safe alternative control measures are much sought after.Biological control by natural antagonistic organisms is a potential nonchemical tool for crop protection against phytopathogenic fungi and bacteria and offers a promising alternative to synthetic pesticides.In our study,with Fusarium oxysporum f sp.melonis as the tested strains,17 antagonistic strains were screened out from 482 reserved strains by hyphal extension inhibition assay in the first step.Their inhibition zone width ranged from 8.5 to 26mm. After several reproduction,the antagonistic activity of 10 strains kept stable and their inhibition zone width exceeded 10 mm.Of the 10 strains,BS-3,BS-5 and BS-17 could restrain the growth of many fungal plant pathogens.In greenhouse test,the disease control efficacies of BS-3,BS-5 and BS-17 were 25.70%,81.77%and 41.27%respectively.In outside field test,the disease control effect of BS-5 was examined repeatedly and its efficacy reached 84.58%.In conclusion,BS-5 controled fusarium wilt disease of watermelon effectively,so it was a promising biocontrol stuff. It was identified as Bacillus subtilis by morphological,biochemical and physiological characteristics,as well as the 16 S rDNA sequence analysis.Ammonium sulphate precipitation was employed to separate antagonistic substances and the result showed that antagonistic substances were entirely deposited when the saturation of ammonium sulphate was at the range of 40%～80%.Then the properties of antagonistic substances produced by BS-5 were primarily studied.The results were as follows:1.Its antifungal activity maitained more than 70%after being heated at 100℃for 20 min while decreased to 35.92%after being sterilized 121℃for 20 min;2.A majority of activity was kept while melt in methyl alcohol,absolute ethyl alcohol,isopropyl alcohol,econdgrade nitrile,ormaldehyde,acetone;3.It was not sensitive to UV,proteinase and too many ions;4.It keeps stable at pH 5～8;5. Its ninhydrin reaction and biuret reaction were positive after acid hydrolysis.Results mentioned above indicated that the main activity in the filtrate of Bacillus subtilis strain BS-5 was peptide with the cycle.The medium components and fermentation conditions of the antagonistic bacterium BS-5 were optimized through orthogonal experiment and single factor experiments in the flask.The components of the optimized medium suitable for the growth of BS-5 consisted of 60 g/L glucose,20 g/L soybean,3 g/L yeast extract,1g/L K₂HPO₄·3H₂O and 0.2 g/L M_gSO₄·7H₂O.The optimum culture condition was at temperature 30℃with starting pH 7.5,inoculum volume 2%of seeds at the age of 8 h,medium volume 20mL in 500 mL flask and rotation speed 200r/min.The biomass was up to 6.2×10⁹cfu/mL in the optimum condition.batch fermentation and fed-batch fermentatin experiments were performed in 3 liter fermentor jar.The results showed that the biomass was increased from 4.8×10⁹ cfu/mL to 8.2×10⁹ cfu/mL while the soybean concentration was increased from 20 g/L to 40 g/L at batch fermentation.In the course of fed-batch culture,three modes of glucose feeding strategy were employed,of which the best mode was that the suitable initial concentration of glucose was 20 g/L,when the glucose concentration in broth was below 10 g/L, condensed glucose was fed into the broth twice,the supplying quantity was 20 g/L every time,the biomass was up to 2.8×10¹⁰ cfu/mL.