A Study On The Inhibitory Activity And The Active Ingredients Of Endophytic Fungi From Sarcandra Glabra

Sarcandra glabra（Thunb.）Nakai,as a traditional medicinal plant in my country,has significant biological activities in antiviral,antibacterial,anti-tumor and anti-thrombocytopenia,During long-term co-evolution,endophyte fungi can produce the same or similar active substances as host plants.Based on the recognized antibacterial activity of Sarcandra glabra.,the purpose of this study is to find antimicrobial active substances from its endophytic fungi,which is of great significance for the development of green and efficient plant-derived antimicrobial agents.In this study,healthy Sarcandra glabra.were collected from Fuchuan County,Hezhou City,Guangxi Province,and endophytic fungi were isolated and purified,and the antimicrobial activity of each strain was preliminarily determined.The endophytic fungi with strong antimicrobial activity were selected for fermentation,and the antimicrobial active substances were isolated and purified from the fermentation products.The main findings are as follows:1.Sarcandra glabra.was collected from Fuchuan County,Hezhou City,Guangxi,and 61 endophytic fungi were isolated from the plants by tissue block separation method.Among them,10 endophytic fungi（numbered G-1～G-10）were isolated from roots,36endophytic fungi（numbered J-1～J-36）were isolated from stems,and 15 endophytic fungi（numbered Y-1～Y-15）were isolated from leaves.2.Take the strain with Athelia rolfsii and Fusarium solani,the antifungal activity of61 strain coral endogenous fungi was determined by plate confrontation.It was found that strains J-9,J-10 and G-4 had a strong inhibitory effect on the mixture of Athelia Rolfsii and Fusarium Solani.3.The antifungal activity of fermentation product of J-9,J-10,J-12,J-20,G-4,J-10,J-12,J-20,G-4,G-5 and Y-8 against Athelia rolfsii and Fusarium solani was determined by mycelial growth rate method.It was found that the antifungal activity of the fermentation product of strain J-10 was the best.When the concentration was 2 mg/m L,the antifungal rate of the ethyl acetate extract and the mycelium methanol extract of the fermentation product were both 100%against Athelia rolfsii,the antifungal rates against the Fusarium solani were 88.51%and 85.17%.4.The toxicity of the fermentation product of J-10 strain to Exserohilum turcicum,Pesta-lotiopsis theae,Colletotrichum capsica,Alternaria citri,Alternaria oleracea,Ceratocystis paradoxa,Fusarium solani and Athelia rolfsii was further determined.The results showed that with the EC₅₀value of the ethyl acetate extract of J-10 fermentation products against 8 pathogenic fungi ranged from 0.1995 mg/m L to 1.1722 mg/m L,the extract had the highest toxicity to Exserohilum turcicum;the extract had the least toxicity to Fusarium solani.The EC₅₀values of J-10 mycelium methanol extracts against 8pathogenic fungi ranged from 0.1088 mg/m L to 9.4687 mg/m L,the extract had the highest toxicity to Athelia rolfsii;the extract had the least toxicity to Fusarium solani.5.The MIC of the ethyl acetate extract,n-butanol extract and mycelium methanol extract of J-10 fermentation product to 10 species of animal pathogenic bacteria such as Proteus vulgaris,Escherichia coli,Micrococcus lyssodeikticus,Salmonella typhi,Bacillus subtilis,Bacillus cereus,Bacillus megaterium,Bacillus anthraci,Micrococcus luteus,Staphyloccocus aureus.The results showed that the mycelium methanol extract and ethyl acetate extract had strong antibacterial effects,and the MIC values were 0.125 mg/m L～5mg/m L and 0.125 mg/m L～2.5 mg/m L,;n-butanol extraction the antibacterial effect of the substance is poor.6.Using silica gel column chromatography,thin layer chromatography and other separation techniques,combined with activity tracking,one active compound was isolated from the methanol extract of J-10 mycelium,and identified for lasiodiplodin by NMR and MS.7.The toxicity of lasiodiplodin to 8 species of plant pathogenic fungi and 10 species of animal pathogenic bacteria was determined,and it was found that the EC₅₀values of lasiodiplodin to 8 species of phytopathogenic fungi were 0.0155 mg/m L～0.2491 mg/m L,among them,the toxicity of E.turcicum is the strongest,and the toxicity of F.solani is the weakest.The MICs of lasiodiplodin against Proteus vulgaris,Escherichia coli,Salmonella typhi and Bacillus cereus were all 0.25 mg/m L,and the MIC values for the other six animal pathogenic bacteria were all greater than 4 mg/m L.8.Preliminary research on the antimicrobial mechanism of lasiodiplodin and found that it has an effect on the morphological structure of the mycelium of E.turcicum,destroys the cell structure,and makes the intraspore material of the mycelial cells leak out.9.Through morphological observation and molecular identification,the endophytic fungus J-10 of Sarcandra glabra.was identified as Lasiodiplodia pseudotheobromae.Through the observation of its biological characteristics,it is found that the endophyte is suitable for growth in the environment of light,temperature of 30℃and p H of 5.5.10.Taking the yield of lasiodiplodin as the index,the fermentation conditions of L.pseudotheobromae were preliminarily explored.The results showed that when the fermentation temperature was 28℃,the p H was 7,the fermentation time was 20 d,the liquid volume was 300 m L,and the carbon-nitrogen ratio was（sucrose/yeast extract）1/0,the yield of lasiodiplodin in the fermentation product was the highest.In this artical,strains with antibacterial activity were screened of endophytic fungi from Sarcandra glabra.,and the experimental results of their antibacterial active substances were studied,which provided a basis for the development of relevant microorganism-derived antibacterial agents.