Study On The Antifungal Activity Of Endophytic Fungi From Yulania Denudata ’Hailuo’ Against Colletotrichum Orbiculare

Endophytic fungi of medicinal plants have various antifungal activities due to their secondary metabolites,and have great biocontrol potential.In order to find biocontrol fungal strains to control melon anthracnose,the endophytic fungi isolated from Yulania denudata’Hailuo’were used as research materials,and Colletotrichum orbiculare,a melon anthrax pathogen,was used as indicator pathogen to investigate the antifungal activity of the endophytic fungi.The fermentation conditions of the target endophytic fungal strain was optimized to maximize the antifungal activity.The antifungal mechanism of crude extract from fermentation broth against C.orbiculare was studied,and the main active compounds were separated and identified.The strain EF-WCH-51 was identified by morphological characteristics and molecular identification.The following results were obtained:1.An endophytic fungus,EF-WCH-51,with high antifungal activity against C.orbiculare was screened from 7 endophytic fungi strains from Yulania denudata’Hailuo’by using the confrontation method and growth rate method.Based on morphological characteristics and molecular biological identification,the strain EF-WCH-51 was identified as Lecanicillium aphanocladii.2.The fermentation conditions of strain EF-WCH-51 were optimized by single factor experiment combined with response surface optimization design.The optimal culture was PDB medium as the base medium,white granulated sugar as the carbon source and no additional nitrogen source was added.The optimal culture conditions were shaking speed200 r/min,culture temperature 24℃and culture time 108 h.After optimization he inhibition rate of the crude extract against melon anthrax pathogen reached 94.47%.The crude extract from the fermentation broth of strain EF-WCH-51 cultured under the optimal fermentation conditions showed good antifungal activity not only against C.orbiculare,the EC₅₀values were 0.0862 mg/m L,but also against Botrytis cinerea,Fusarium oxysporum f.sp.vasinfectum,C.fructicola,F.oxysporum f.sp.niveum.The EC₅₀values were 0.1140、0.5399、0.5214、1.1908 mg/m L respectively.3.The study on the antifungal mechanism of the crude extracts on C.orbiculare showed that the crude extract increased the permeability of the cell membrane mainly by destroying the cell membrane,and the functional components such as protein and soluble polysaccharides released from cells,thus producing the antifungal effect.The study on the isolation and identification of the active components of the crude extract from the fermentation broth of strain EF-WCH-51 showed that the main active component with great inhibitory effect on C.orbiculare was oosporein4.The results of the bioassay showed that the crude extract from the fermentation broth of strain EF-WCH-51 had 57.54%preventive effect and 63.50%therapeutic effect on C.orbiculare on detached leaves of Cucumis sativus.The crude extract had 50.88%preventive effect and 51.71%therapeutic effect on C.orbiculare on living leaves of C.sativus.The endophytic fungus EF-WCH-51 isolated from Yulania denudata’Hailuo’was identified as Lecanicillium aphanocladii,which showed good antifungal activity against plant pathogens such as Colletotrichum orbiculare.The main antifungal active component in the metabolites of its fermentation broth was oosporein,which showed certain preventive and therapeutic effects in both in vitro and in vivo bioassays of cucumber leaves.This study provided a theoretical reference for L.aphanocladii as a biological control fungus for C.orbiculare.