Research On The Screening And Fermentation Optimization,active Component Separation Of Biocontrol Bacteria For Plant Fungal Diseases

Taking the soil-derived Bacillus and plant-derived pathogenic fungi as the research objects,the bacteriostatic cross-screening was carried out based on the Oxford Cup method.Optimized fermentation conditions for the design test of excellent strains,and separated and purified the extracts according to the activity tracking method.The main work is summarized as follows:1.Based on the Oxford Cup method,bacteriostatic cross-screening of Bacillus and plant pathogenic fungi was conducted.A total of 30 kinds of bacillus extracts were obtained from 10 kinds of bacillus fermentation broth after extraction with petroleum ether,n-butanol and ethyl acetate,and the antibacterial activity against 10 plant pathogenic fungi was investigated using the diameter of the zone of inhibition as evaluation index.Bacillus with antibacterial effects are:The petroleum ether,n-butanol and ethyl acetate extracts of Bacillus drentensis 15600 have inhibitory effects on Fusarium graminearum、Magnaporthe oryzae and Botrytis cinerea respectively.The n-butanol extract of Bacillus bataviensis 15601 has the effects on Magnaporthe oryzae and Gliocladium oryzae.The n-butanol extract of Bacillus cucumis 101566 has an inhibitory effect on Magnaporthe oryzae.The petroleum ether extract of Bacillus tequilensis WRN032 has an inhibitory effect on Magnaporthe oryzae,and its ethyl acetate extract has an antibacterial effect on Fusarium graminearum and Magnaporthe oryzae and Gliocladium oryzae.Among them,the ethyl acetate extract of Bacillus tequilensis WRN032 had the strongest inhibitory effect on Magnaporthe oryzae,and the inhibition zone diameter was 8.78 mm at a concentration of 1.0 mg/m L.2.The fermentation conditions of Bacillus tequilensis WRN032 were optimized Based on Response Surface Methodology.Plot the growth curve of Bacillus tequilensis WRN032,using the absorbance of fermentation broth and the diameter of the inhibition zone of Magnaporthe oryzae as the indicator to design a single factor test,Plackeet-Burmann Design test and Central Composite Design test to optimize the fermentation conditions.The optimized fermentation conditions determined by Response Surface Methodology are:inoculation volume 1%,flask filling volume 40%,glucose concentration 5%,shaker speed 220 rpm,initial p H 6.5,incubation time 48 h,incubation temperature 30°C.Under optimized conditions,the cell biomass was increased by 186.63%,and the diameter of the inhibition zone was increased by154.10%.3.Based on activity tracking method,the ethyl acetate extract of Bacillus tequilensis WRN032 was separated and purified by thin layer chromatography,silica gel column chromatography and semi-preparative chromatography.The eluent system obtained by TLC was dichloromethane:methanol=99:1,99:5,99:10,99:99 and 0:99,and the extract was eluted by silica gel column chromatography gradient to obtain dichloromethane:methanol=99:99（Component A₄）and 0:99（Component A₅）two components have antibacterial effect on Magnaporthe oryzae.Separation of the two components using semi-preparative chromatography to obtain two active fractions R₂and S₂,and S₂ had the best effect on Magnaporthe oryzae.After separation and purification of the extract of Bacillus tequilensis WRN032,the diameter of the inhibition zone of S2 component against Magnaporthe oryzae increased by 37.70%compared with the before.