Screening Of Antagonistic Bacteria Against Sporisorium Scitanminea And Determination Of Antimicrobial Compounds

Sugarcane is an important commercial crop worldwide and one of the principal sources of sugar.China is the third largest sugar producer in the world.Sugarcane smut is a fungal disease caused by Sporisorium scitanminea.It seriously affects the yield and quality of sugarcane.It causes huge losses to the entire sugarcane industry.The traditional fungicides cause great pollution to the environment,yet have poor prevention effect because they are difficult to enter sugarcane.The main objective of this study is to develop the biological control technology of sugarcane smut,by inhibiting the growth or sexual mating of sugarcane smut.Antagonistic strains were isolated from rhizosphere soil and endophytic region of sugarcane.Bioactive substances were isolated and identified.The sesquiterpene compound farnesol was selected from 14 small molecular libraries biological sources.The synergism of farnesol with three kinds of absorbant fungicides was tested.The main experimental findings are as follows:Total 150 bacteria were isolated from the sugarcane rhizosphere soil by serial dilution technique.A strain of Pseudomonas.spp was isolated by 4 rounds of screening ang named LC4-1.The ethyl acetate extract from fermentation broth were inhibiting the sexual mating and hyphal extension of the sugarcane rhizome.In addition,the strain also produced volatile bacteriostatic substances.A single active compound was obtained by separating and purifying the crude extract.It was identified as phenazine-1-carboxylic acid(C₁₃H₈N₂O₂)by NMR analysis and mass spectrometry comparison.The yield of the compound was 40 mg/L from the bacteria cultured in LB medium at 28℃for 24 hours.Total 80 endophytic bacteria were isolated from sugarcane stalks by gradient culture.A novel strain displaying a significant inhibitory effect was named A52-1,and idetified as Bacillus.its were selected on the basis of antagonistic effect on Sporisorium scitanminea.It was obtained by three times screening.The ammonium sulfate precipitation of fermentation broth and ethyl acetate crude extract both were inhibited the growth and sexual mating of Sporisorium scitanminea.It was found that extraction component activity was stable.The reference molecular formula of C₁₇H₁₈N₂was identified by mass spectrometry and nuclear magnetic resonance（NMR）analysis.Through the screening of substances from known biological sources of small molecular library,we found farnesol.It was found that farnesol were showing a significant inhibitory effect on different stages of the life cycle of in sugarcane smut.The effect of 0.5μM farnesol on germination rate of teliospores was decreased by50%while survival rate of basidiospores decreased by 20%.The effect of 0.04μM farnesol treatment on sexual mating of S.scitanminea was significantly inhibited.Also,the formation of hyphae was significantly reduced,which has proven that it is a potential fungicidal agent for sugarcane smut fungus.The synergistic effect of farnesol was tested with three commonly used fungicides azoxystrobin,difenoconazole and hexaconazole.The synergistic ratio of 0.15μM farnesol and pyridamole reached 34times while,the synergistic ratio with dimethyclozole and hexazolol reached 74 times and 59 times respectively,which significantly improved the efficacy of fungicides and thus significantly reduced the dosage of fungicides to reach the same antifungal effect.