Isolation Of Antagonistic Bacteria Against Fusarium Gramineae And Study On Its Antagonistic Mechanism

Fusarium graminearum is the main pathogen of Fusarium Head Blight,which not only causes wheat yield reduction,but also produces deoxynivalenol(DON),nivalenol(NIV)and zearalenone(ZEN)and other mycotoxins.Wheat scab can affect the quality and safety of wheat,cause economic losses and food safety problems.In recent years,the use of microorganisms that are not toxic to animals and plants to inhibit Fusarium graminearum has received increasing attention.In this study,the antagonistic bacteria of Fusarium graminearum were screened,and the antagonistic related compounds were isolated.In addition,we also tried to locate antagonistic genes of antagonistic bacteria.The main results are as follows:(1)In this study,105 antagonistic bacteria that inhibit Fusarium graminearum GZ3639 were screened from wheat grain and wheat field by plate sputum test,and an antagonistic library was constructed.The 16 S rDNA identification of these 105 antagonistic bacterium showed that there were 102 strains of Pantoea,2 strains of Bacillus amyloliquefaciens and 1strain of Bacillus polymyxa.(2)Six strains of antagonistic bacterium were selected Pantoea agglomerans HP3,Bacillus amyloliquefaciens BP17,Bacillus amyloliquefaciens BL13,Paenibacillus polymyxa FA1,Pantoea agglomerans HL2 and Pantoea agglomerans BL1.The in-situ biocontrol experiment of wheat was carried out,and the control effect of the fermentation broth and crude extract of fermentation broth was preliminarily investigated.The incidence of wheat scab.The crude extract of antagonistic bacteria was used to treat the spores of Fusarium graminearum GZ3639.The results showed that the crude extract of antagonistic HP3 and BP17 fermentation broth could effectively inhibit the germination and hyphal growth of Fusarium graminearum GZ3639.(3)The active substance in the antagonistic BP17 fermentation broth was purified,and the antagonistic crude extract was obtained by extracting the antagonistic BP17 fermentation broth with n-butanol.The silica gel column was used to separate the molecular polarity based on the molecular polarity of the substance,and the dextran gel was used.The column methodis based on the molecular size of the material,and the substance is purified by high performance liquid chromatography.Finally,a pure active substance is separated and purified from the antagonistic BP17 fermentation broth,and identified by nuclear magnetic resonance and flight time pristine detection Cyclo-(L-phenylalanine-L-valine).(4)Using the EZ-Tn5<R6K?ori/KAN-2>transposon mutation,102 resistant inactivating mutants and 113 resistant mutants were obtained.The transposon insertion site was flanked using plasmid rescue technology,and transposon insertion sites of 8 mutants were obtained.These insertion sites are the coding regions of the Pantoea agglomerans orf04350(52),orf03816,orf03689(369),orf03584(935),orf02567(682),orf00203,orf00165 and orf00164 genes,respectively.These genes encode proteins containing amino acid adenylation domain,transcriptional regulators,bifunctional aspartate kinase/homoserine dehydrogenase I and other functional proteins,which are related to substance expression and synthesis.