Study On Active Components Of Endophyte Ld421 Against Gentiana Leaf Blight

The endophytic fungus LD421 was isolated from the root of Getiana manshurica Kitag.that was the resistant cultivar of Getiana.The fermentation of LD421 has inhibition growth effect on Alternaria alternate(Fr.:Fr)Keissler that was the pathogen of Gentian leaf blight,and the inhibition rate reached 81.59%.The main content of this paper were to analyze the chemical components of the fermentation,and to determine the active substances against the pathogen.The analytical results of chemical composition of LD421 fermentation systematically indicated that LD421 fermentation contained carbohydrates,proteins(excluding amino acids),phenols or tannins and alkaloids,PH of the fermentation was between 5.5 and 6.5,and the fermentation produced fluorescence under the irradiation of ultraviolet lamp.Organic solvent extraction,alcohol precipitation and sevage method were selected as preliminary separation methods of active components.The antimicrobial experiment results of organic solvent extraction showed that the antimicrobial components mainly existed in ethyl acetate,n-butanol and water extract,and best antimicrobial effect of ethyl acetate was up to 80.75%,while chloroform and petroleum showed weak antimicrobial activity.The antimicrobial experiment results of alcohol precipitation test showed that the inhibition rate of 80%ethanol precipitate was 83.82%,which was significantly higher than that in alcohol soluble part.The result of antimicrobial experiment results of sevage method showed that the active components mainly existed in aqueous phase and organic phase,and best antimicrobial effect of ethyl acetate was up to 92.12%,which superior to the antimicrobial effect of fermentation.The result of antimicrobial experiment results of sevage method combined with grade ethanol precipitation method showed that chloroform-n-butanol,50%ethanol precipitation,65%ethanol precipitation and 80%ethanol precipitation exhibited good antimicrobial activity.The best inhibition rate of chloroform-n-butanol was up to 91.62%.It explained that there were more than one kind of substance with antimicrobial effect in LD421 fermentation.The chloroform-n-butanol extracts with the best antimicrobial activity was further analyzed.The purposes were to determine the chemical composition,the molecular weight and structure of the chemical components were determined by UPLC/Q-TOFMS.There were 13 kinds of compounds received through analyzing on molecular weight and structure of chemical composition,among them,5 kinds of compounds were known.Compound 1 was 5-Hydroxymethyl-2-furaldehyde,compound 3 was 7-Hydroxy-6-Méthoxy-2H-chromen-2-one,compound 8 was Norselic acid B,compound 12 was Daturametelin C and compound 13 was sclareol.The antimicrobial experiment results of 3 kinds of compounds that standards can be obtained showed that the inhibition rate reached 100%when the concentration of 5-Hydroxymethyl-2-furaldehydel was 12.50 mg/ml,the inhibition rate reached 63.72%when the concentration of sclareol was 4.17 mg/ml,the inhibition rate reached 23.10%when the concentration of 6-hydroxy-7methoxy coumarin was 2.83 mg/ml.