| Gray mold caused by Botrytis cinerea infection can harm a variety of fruits andvegetables, causing serious economic losses. Long-term use of chemical pesticides tocontrol gray mold would bring great harm to the ecological environment and humanhealth. Biocontrol is an environment-friendly,safe and effective control technology.In order to obtain satisfactory biocontrol bacteria for service in production directly,the paper studied the biocontrol gray mold of efficient broad-spectrum antagonisticbacteria systemly in antagonistic bacteria isolation, screening, identification,fermentation condition, the extracion of antibacterial substance, prevent disease andpromote growth and so on.264Bacillus strains were isolated from the soil samples in Hebei, Shandong,Changchun, Nanchang, Lanzhou and other places. With Botrytis cinerea as the testedpathogen,4efficient antagonistic bacteria which can metabolism to produceantibacterial substances was screened out by the antagonistic assay in plates. Afterdetermining passage stability and antibacterial spectrum, the strain ch2-22with higherantagonistic activity, genetic stability and broad-spectrum were selected foridentification. It was identified as Brevibacillus brevis based on the results ofmorphologic characteristics, physiological and biochemical properties andphylogenetic analysis of16S rDNA sequence and were named for ch2-22.The optimal liquid fermentation medium of Brevibacillus brevis ch2-22wascomposed of beef extract0.5%, peptone1%, NaCl0.5%, glucose2%. The optimalculture time of seed solution and producing antifungal substance respectively were14hours and60hours. Other optimal fermentation conditions were initial pH7.0-7.5,inoculation quantity4%with50mL medium in250mL flask,30℃,200r/min. Theproperties of the antibacterial substance of strain ch2-22were studied. The resultssuggested that it had high stability against heat, acid, alkali and irradiation of glareand UV, and it was unsensible to Trypsin, Proteinase K, Pepsin and chloroform, andit’s antibacterial activity would not change by freezing and thawing repeatedly and saving long-term, and it had the ultraviolet absorption peak in196nm.The organic phase from the fermentation filtrate of strain ch2-22extracted withorganic solvents had no antagonistic activity, the antibacterial substance waspreliminarily determined as water-soluble substance. The supernatant withantagonistic activity could be extracted with concentrated hydrochloric acid and coldethanol. Both retentate and filtrate which could be obtained by ultrafiltration with a3kD hollow cellulose membrane had antibacterial activity. This shows that there are avariety of antibacterial substance. The molecular weight of the antibacterialsubstances form retentate and filtrate were determinated by Tricine-SDS-PAGE. Theresults show that there were four kinds of antibacterial substances in retentate andtheir molecular weight respectively are about15kD,11kD,7.5kD and3kD, therewas only one kind of antibacterial substances in filtrate and it’s molecular weight isabout3kD.The study of antimicrobial mechanism demonstrated that the strain ch2-22couldsecrete proteinase which could digest the protein in the fungal cell wall so that thefungal hyphal were destroyed and its growth was inhibited. The fermentation filtrateof the strain ch2-22could strongly inhibit mycelia growth and spore germination ofBotrytis cinerea. The defence enzymes such as POD、PPO、PAL、CAT and SODandthe pathogenesis-related protein such as β-1,3glucanase and chitinase in cucumberwere induced by the fermentation filtrate of the strain ch2-22, and their enzymeactivity increase could improve disease resistance.The cucumber leaves in vitro was treated by different dilution times offermentation filtrate of the strain ch2-22and tested control rate against Botrytiscinerea. Control efficacy of the filtrate diluted100times was100%. In greenhouseexperiment, the filtrate diluted100times showed stronger antagonism against Botrytiscinerea, with control efficacy of87%and therapy efficacy of70%, and diseaseprevention was better than treatment. Different dilution times of fermentation filtrateof ch2-22had a certain growth-promoting effect on cucumber by soaking cucumberseeds. The filtrate diluted50times and100times could improve seed germination,root and shoots growing, and increase plant height, stem diameter, the number of expland leaves, leaf area, fresh and dry weight, and the content of chlorophyll, solubleprotein and soluble sugar. It provide favorable physiological functions,increase theyield and quality. |
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