Screening And Identification Of Antagonistic Bacteria For Alternaria Alternate And Preliminary Study On Its Biocontrol Mechanism

Tobacco brown spot is a one of the most important leaf diseases on tobacco,which occurs in many tobacco plant areas around the world and seriously affects the yield of flue-cured tobacco.At present,the chemical control is the preferred method to prevent and restrict tobacco red star disease in most plant areas.Compared with other metheds,pharmaceutical control is a fast and highly effective method.With the great attention to environmental pollution and pesticide residues,it is urgent to find suitable biocontrol agents to replace chemical pesticides.In this study,the screening and identification of antagonistic bacteria and the biocontrol effect of Alternaria alternate were preliminarily studied.The main results are as follows:Two antagonistic bacteria strains YC30 and YK2,which were isolated from the rhizosphere soil,were screened and obtained.The inhibitory rates of fermentation of YC30and YK2 on A.Alternate reached 96.21%and 96.00%and made the mycelia of A.alternate enlarged,deformed and cutting-edge abnormal.Moreover,the results showed that the EC₅₀ of Dimethachlon and Fluazinam were 2.48 mg/L and 0.65 mg/L,while the EC₅₀ of YC30 and YK2 were 1.6 m L/L and 12.3 m L/L.The results indicated that the inhibition effect of the two strains were good.Based on the evidence of morphological and phylogenetic,the srain YC30 was indentified as Bacillus velezensis and strain YK2 was identified as Bacillus amyloliquefaciens.The optimal growth conditions of the two strains were determined as follows.The most optimum culture condition was 28℃;the most optimum culture time was 48 h;the most optimum culture condition pH was 6 for YC30 and 8 for YK2.The 10% fermentation of two antagonistic bacteria strains showed effective inhibitory activity on 12 pathogens in this study,and the control effect on Sclerotinia sclerotiorum was the best,reaching 100%.By testing the stability,volatile substances,protein extracts and lipopeptide extracts of the two strains,it was found that the stability of YC30 strain was better than that of YK2 strain.Under the same treatment,the activity of YC30 strain fermentation was better than that of YK2 strain.The inhibitory rate of volatile substances from the two biocontrol strains on A.alternate respectively was 58.77%and 66.72%.Both crude protein extracts and lipopeptide extracts extracted from the two biocontrol strains could inhibit the mycelia of A.alternate,and the lipopeptide extract had better antifungal activity than the protein extract.When adding the amount of lipopeptide extract of YC30 and YK2 strains were5%,the antifungal rates were reached 90.56%and 90.45%,which had an effective inhibitory activity on A.alternate.The compounds with different molecular weights in the metableolites of the two strains were analyzed by GC-MS.According to the matching degree comparison with the database,the results showed that:the main metableolites,including acetic acid,cesium acetate and other compounds.Acetic acid and cesium acetate had good effective in inhibiting A.alternate.The EC₅₀ of exogenous cesium acetate was 23.19 mmol/L.When the concentration was 100 mmol/L,the inhibition rate of A.alternate reached 82.26%.The EC₅₀of exogenous acetic acid was 14.63 mmol/L.When the concentration was 40 mmol/L,the inhibition rate of A.alternate reached 83.67%.Both compounds tested had effective inhibitory activity on A.alternate.