Study On The Isolation,identification And Bioactivity Of Endophytic Fungi From Panax Ginseng C.A. Meyer

Plant endophytic fungi is an important component of the plant microbial community,its abundant secondary metabolites can not only increase the adaptability and resistance of host plant to the adversity stress but also exhibit a range of biological activities,including antibacterial,anti-inflammatory,anti-tumor,antioxidant,antivirus,immune suppression and bio-control ability to the plant pathogens and agricultural pest,etc.As a rare Chinese medicinal material,Panax ginseng has an abundant chemical composition and pharmacological activity.However,high requirements for the growth environment caused Panax ginseng easy to be infected by pathogens,which seriously affects its quality and yield.This study selected Panax ginseng C.A.Meyer as the research object,isolated and identified the endophytic fungi of its root tissue.Screened strain with bio-control potential and cultured strain by mass fermentation.Separated and purified secondary metabolites of strain fermentation.Identified the structure of the monomer compounds and evaluated the pathogenic fungi inhibitory activity,the results of the study are as follows:The endophytic fungi from healthy P.ginseng root tissues were isolated by tissues isolation method,and three endophytic fungi strains GS1,GS2,GS3 were obtained,three endophytic fungi strains were identified by molecular biological identification methods,finally,GS1,GS2,GS3 were identified as Fusarium tricinctum,Botryotrichum murorum,Doratomyces columnaris.Four plant pathogens: Rhizoctonia solani,Fusarium solani,Botrytis cinerea,and Sclerotinia sclerotiorum were used as test strains,endophytic fungi with inhibitory effect on four phytopathogens were screened by confrontation culture,and according to the results of the confrontation culture,the GS1 strain(Fusarium tricinctum)was selected as the research object because of its inhibitory effect on all four phytopathogens,and its secondary metabolites were separated and purified.Oat liquid mediums were used to ferment fungi strain in large quantities,and the activity of fermentation liquor and the mycelial extract was tracked,it was found that both parts had significant inhibitory effects on four plant pathogens.Combined of fermentation liquor and the mycelial extract.Extracted fermentation and continued to be tracked for the inhibitory effect of obtained extract on four plant pathogens.The chemical components of the petroleum ether and ethyl acetate phase were separated and purified by various chromatographic techniques,spectroscopic analysis was used to determine the structure of the compounds,and finally,six metabolites were obtained,six metabolites were(1R,2R,4a S,8a S)-Decahydro-1-(6-hydroxy-3-methyl-3-hexen-1-yl)-2,5,5,8a-tetramethyl-1-naphthalenol,enniatin I,2-Hydroxy-6(Z)-hexadecenoic acid,terretonin,lupeol,and 3β-hydroxy-cholesta-5-ene,respectively.Evaluated the pathogenic fungi inhibitory activity of the compounds,in vitro test showed that compound 2 has an excellent anti-pathogenic fungi effect on R.solani,F.solani,B.cinerea,S.sclerotiorum.At the concentration of 25 μg/m L,the inhibition rate of S.sclerotiorum reached 83.41 ± 4.66%,and at the concentration of 50 μg/m L,the inhibition rate of B.cinerea reached 81.83 ± 5.07%,and the inhibition activity of F.solani and R.solani was moderate.Other compounds showed no activity.This study enriched the diversity of endophytic fungi of P.ginseng and provided reference for the application of endophytic fungi and their metabolites in the biological control of the plants.