| Plant diseases are among the main constraints affecting the production and productivity of crops both in terms of quality and quantity.Biological control of plant disease on the crop has a good control effect,people have paid more attention to it.However,the commercial use and application of plant disease biological control agents have been very slow,and it is still a challenge to obtain broad-spectrum and efficient biocontrol agents that inhibit plant diseases.we screened an antagonistic bacterium AnH,which had broad spectrum antifungal properties against 12 plant pathogens.It was identified as Bacillus amyloliquefaciens based on the results of 16S rRNA gene sequence analysis,physiological and biochemical characteristics.Some basic properties of antimicrobial substances in fermentation were studied.The results showed that they had a high stability in circumstance,such as high temperature,strong acid and alkali,strong light and ultraviolet radiation.They were insensitive to protease K,and three months preservation had little effect on the activity of antimicrobial substances.Which laid the foundation for future field experiments.Studies on the ability of AnH to secrete fungal cell wall degrading enzymes had shown that they secreted proteases and cellulases.The medium compositions for AnH strain to produce the bioactive substances were optimized and the results were as follows:15g/L corn flour,15g/L yeast extract,2g/L magnesium sulfate(Diameter of inhibition zone was 18.1 mm).Organic solvent extraction method,ammonium sulfate precipitation method and hydrochloric acid precipitation method were used to extract antagonistic substance.The results showed that the antimicrobial substance was water-soluble.Further studies showed that the crude extracts of lipopeptides had broad-spectrum antifungal properties.The bioactive substances produced by AnH strain was purified by gel filtration.The LC-MS analysis results showed that the molecular masses werel042.5,1056.5 and 1070.5 and were 14 Da different,which was the size of a-CH2 group.These results suggested that the antimicrobial substance isolated from the strain AnH were the Iturin homologues.Cherry trees in Bai Lu Yuan in Shaanxi Province,China,are susceptible to a serious disease.The pathogens causing the "black pimple" disease,are currently unknown.This disease spreads quickly,and the chances of infection are high.Local farmers use a variety of chemical treatments for "black pimple" disease are often ineffective.In this study,10 strains of cherry tree disease were isolated and cultured by microbiology separation technique.A total of one hundred and thirty-five strains of bacteria were isolated and then combined into different 27 strains by morphological and physiological and biochemical reactions.16S rRNA sequencing results showed that twenty three strains belonged to Enterobacteriaceae,including eleven strains of Pantoea sp.,six of Enterobacter sp.,three of Salmonella sp.and three of Escherichia sp..The dominant bacteria in the diseased tissue are Enterobacteriaceae.8 fungi strains were isolated and the ITS sequencing results showed that two strains of Wickerhamomyces,two strains of Meyerozyma,one strain of Botryosphaeria dothidea,two strains of Fungal sp.,one strain of Gibberella.Botryosphaeria dothidea can cause a variety of fruit trees disease.More attention should be paid to Botryosphaeria dothidea in future studies of the causes of cherry tree "black pimple" disease.Antimicrobial experiments were carried out on the bacteria and fungi isolated from the diseased tissue by the lipopeptides and protein crude extracts of the antagonist AnH.The results showed that the liposomes of the antagonistic bacteria AnH had good bacteriostatic effect.The above studies had shown that antagonist AnH had a good potential for application,which provides a scientific basis and theoretical basis for controlling the occurrence of cherry tree "black pimple disease" and plant disease. |
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