Screening Of A Biocontrol Bacteria LYM3Against Banana Anthracnose, Optimization Of Fermentation Conditions And Preliminary Study On Its Antifungal Substances

Banana anthracnose is a common disease of banana fruit in plantation. Colletotrichum musae is the pathogen of Banana anthracnose. Chemical control and biological control are the common methods to defeat the disease. The long-term use of chemical pesticides has already led to the resistance of C. musae that forced farmers to use more chemical pesticides and scientists to find new alternatives. The vicious circle accelerated the resistance of C.musae. Biological control is more friendly environmental and sustainable. Searching the antifungal substance form endophyte in Southern Chinese Herbs is one of the research focuses. An endophytic bacteria LYM3isolated form Rauwolfia was screened by growth confrontation method which had a strong antagonistic activity to C. musae, and its antifungal substance was also analyzed preliminarily. The concrete restults is as follows.(1) The strain LYM3has broad spectrum anti-fungal activities to10pathogen fungus. It was selected by growth confrontation method in plate. Inhibition rate of LYM3for these pathogen fungus were more than60%. According to the results of morphology physiology biochemistry experiments and16S rDNA sequence analysis characteristics of colonies and cells, strain LYM3was identified as Bacillus subtilis.(2) The fermentation conditions of LYM3producing antifungal substance was optimized base on LB medium. The Restults showed that the optimal culture medium was2%soluble starch,1%tryptone,0.8%yeast extract,1%NaCl, and the optimal initial pH was6. After culturing for60h, the inhibition zone diameter was up to34.2mm.(3)Physiological and biochemical tests about the supernatant fermentation showed that Proteinase K, UV, high temperature, organic solvent and metal ions had little effect on the antifungal substance of LYM3. Flocculent precipitate will appear when the pH of LYM3the supernatant fermentation below4. Leaching precipitation by methanol, the lost of antifungal substance was the least.(4)The antifungal substance of LYM3was identified as lipopeptide. The existence of biosurfactant in the supernatant fermentation of LYM3was identified by drain circle method. Color reaction and TLC of crude extract proved it had peptide bond and closed circular structure. The detection conditions of HPLC HPLC was UV205? m,80%acetonitrile,30?. The restult of LCMS showed that A might be C15IturinB, B might be C14SurfactinB and C15BacillomycinD, E might be C14SurfactinA or C15SurfactinB or C14SurfactinC.