Screening And Identification Of Antifungal Strains Against Carya Cathayensis Canker And Exploration Of Antagonistic Mechanism

The canker disease of Carya cathayensis caused by Botryosphaeria dothidea is one of the diseases that result in serious damage and even death to C.cathayensis in China.So far,the area of C.cathayensis forest received damage has been expanded.This study more than one hundred of strains were isolated and purified previously from soils from Phyllostachys praecox stands in Zhejiang Province,China.Our results demonstrated that:(1)Screening of the inhibition strain.We successfully obtained a Bacillus strain named WK1 that showed a strong inhibition activity in vitro against the B.dothidea.by screening with plate dual-culture method.The strain of WK1 was designated as Bacillus amyloliquefaciens subsp.plantarum via morphological,biochemical and physiological assay as well as phylogenetic analysis based on 16 S rDNA gene sequence.(2)Exploring of the inhibitory mechanism.The inhibitory mechanism of strain WK1 against B.dothidea was obtained by screening with the dual-culture method and cultured in medium mixtures with ratios of sterilized fermentation supernate to PDA being 1:9,2:8,and 5:5(volume ratio).Then,the inhibitory mechanisms of strain CGMCC 11640 against Botryosphaeria dothidea were further explored by scanning electron microscopy(SEM).Results revealed that the Botryosphaeria dothidea strain growth rate for the three volume ratio mediums were restrained by 62.53% for 1:9,77.29% for 2:8,and 90.45% for 5:5.The SEM results showed that both bacterial cells and their sterilized fermentation supernate exhibited strong inhibition activity in vitro against Botryosphaeria dothidea and that the hyphae and spores of Botryosphaeria dothidea exhibited atrophy and introcession.Thus,CGMCC 11640 inhibited Botryosphaeria dothidea by destroying cell membranes(e.g.punching holes in them)and walls of hyphae and spores resulting in leakage of plasmatic material from the inside of the cells.(3)Idetification of the antimicrobial lipopeptides.The lipopeptide antibiotics of WK1 was extracted from the filtrate of culture liquids by acid precipitation and methyl alcohol extract.The characteristics of crude lipopeptides of WK1 was determined via HPLC and LC-MS by comparing the molecular weight of the compoundsin crude lipopeptides with theoretical value of relative molecular mass of the lipopeptide.The results showed that the crude lipopeptides contained three lipopeptideantibiotics,namely fengycins,surfactins and iturins,with fengycins being the dominant components.The fengycins were further identified by LC-MS with single mass spectrum peak identification,which suggested that the fengycins were a mixture of C15,C16,C17 and C18 fengycin A and their isomers(normal-,iso-,and anteiso-).(4)Antimicrobial spectrum of the antimicrobial lipopeptides.By using disc agar diffusion method,the crude lipopeptides were shown antagonistic against Fusarium oxysporum f.sp.cucumerinum,Rhizoctoniasolani,F.oxysporum f.sp.vasinfectum SF2,F.oxysporum f.sp.cucumis melo L.,Botrytis cinerea,Verticillium dahliae,Pythiummyriotylum drechsler and F.oxysporum Schlecht.The antagonistic effect was increased with increasing concentration of lipopeptides.At the concentration of 0.625 mg/ml,the lipopeptides showed strong antagonistic effect on F.oxysporum f.sp.vasinfectum SF2,F.Oxysporum f.sp.Cucumerinum and F.oxysporum Schlecht..At the concentration of 10 mg/ml,the lipopeptides showed strongest effect on F.Oxysporum f.sp.Cucumerinum among the pathogenic bacterium with the diameter of zone of inhibition being 21 mm,while showed weak effects on Rhizoctoniasolani and Pythiummuriotylum rechsler with the diameter of zone of inhibition being 14.16 mm and 13.65 mm,respectively.