A Study On The Endophytic Fungi From Stephania Kwangsiensis And Their Antimicrobial Ingredients

In this paper,endophytic fungi were isolated and identificated from Stephania kwangsiensis,Among them two strains were choosed for fermentation,and the fermentation extracts were obtained by extracted with organic solvent.Antimicrobial activities of fermentation extracts were preliminary screened against ten kinds of plant pathogenic fungi and fifteen kinds of animal pathogens.mycelium growth rate method and poison plate method were used to determinate the antimicrobial activities of fermentation extracts.By bioassay-guided fractionation,active ingredients were isolated and their structures were identified by organic solvent extraction,silica gel column chronmatography and thin-layer chronmatography combined with msgnetic resonance spectrum,and the antifungal mechanism of antimicrobial ingredients was also preliminarily studied.The main results are as follows:1.Twenty-three strains of endophytic fungi(named DBR-1 to DBR-23)were isolated from the earthnuts of Stephania kwangsiensis by tissue culture.Among them,eight strains were identified by morphological and molecular biology methods.DBR-3(Albonectria rigidiuscula),DBR-4(Cladosporium),DBR-5(Nigrospora sp.),DBR-9(Penicillium citrinum),DBR-11(Myrothecium verrucaria),DBR-13(Phomopsis sp.),DBR-17(Paraphoma radiccina)and DBR-19(Pestalptiopsis sp.).Antimircobial activities of the eight endophytic fungi were preliminary screened.The results show that DBR-3,DBR-5,DBR-9,DBR-11 and DBR-13 has strong antimircobial activity,and DBR-17 had some antimircobial activity.2.Selecting DBR-9 and DBR-11 for further study.Fermentation products of the two endophytes fungi preliminary divided into ethyl acetate extract,butyl alcohol extract and raffinate by liquid-liquid extraction method.The their antifungal activities were respectively tested by mycelium growth rate method against ten types of plant pathogenic fungi.The results indicated that the inhibition rate were exceed 90%with the concentrations at 5 g/L of ethyl acetate extracts from the two endophytes fungi fermentation products.Other extract of the two endophytes fungi fermentation products had almost no antifungal activities.Further test of the ethyl acetate extracts against the 10 plant pathogenic fungi.The results showed that,EC50 value of DBR-9 and DBR-11 at 0.0203 g/L-0.3633 g/L,and 2 g/L-0.4465 g/L,respectively.Poison plate method was used to determinated the antibacterial activity of different extracts from the two endophytes fungi fermentation products against fifteen kinds of animal pathogens.The results showed that,ethyl acetate extract of DBR-9 fermentation products completely inhibited the growth of five kinds of animal pathogens at 72 h,agaisnt Micrococcus lysodeikticus at 48 h,agaisnt Micrococcus luteus,Staphyloccocus aureus and Shigella dysenteriae at 24 h.The MIC of the fethyl acetate extract against eight animal pathogens was further determined at 24 h,with the MIC values of 0.25 g/L-1 g/L.Ethyl acetate extract of DBR-11 fermentation products completely inhibited the growth of seven kinds of animal pathogens at 72 h,agaisnt Proteusbacillus vulgaris at 24 h.The MIC of the fethyl acetate extract was further determined at 24 h against eight animal pathogens,and the MIC values at 0.0625 g/L-0.25 g/L.It suggested that inhibition of the active ingredients mainly remain in the ethyl acetate extracts.3.Active ingredients were isolated and purified from the ethyl acetate extracts of two endophytes fungi fermentation products by silica gel column chromatography and thin-layer chromatography combined with bioassay-guided method,and three active compounds DBR-9a,DBR-9b and DBR-lla were obtain.By nuclear magnetic resonance(NMR)analysis,the active compounds were identified as Janthinoe(Abbreviated 9a),emodin(Abbreviated 9b)and Roridin A(Abbreviated 11a).4.The antifungal activity of the three compounds against ten types of plant pathogenic fungi were tested.The results showed that when the concentration was 0.1 g/L,the antifungal broad-spectrum was 11a>9b>9a.The pathogenic fungi,for which their growths were inhibited more than 60%by the three compounds,were selected to further test the toxicity of the three compounds agaisnt these plant pathogenic fungi.The results showed that:(1)The EC50 values of 9a agaisnt Phytophthora parasitica,Exserohilum turcicum and Alternaria oleracea were 0.266 5 g/L,0.121 4 g/L and 0.156 8 g/L,respectively.(2)The EC50 values of 9b agaisnt six tested pathogenic fungi were 0.0030 g/L-0.1410 g/L.(3)The EC50 values of 11a to seven tested pathogenic fungi were 0.0055 g/L-0.1330 g/L.The antifungal activity of the three compounds against fifteen types of animal pathogens were tested.The results showed that when the concentration was 0.1 g/L,they had no inhibitory effect.5.The mechanism of three compounds against Phytophthora parasitica was preliminary studyed.The results showed that Phytophthora parasitica post-treated by the three compound had thinner,flatter and some distortion hyphae compared with the controls,and its hyphae surface was broke with some inclusions leaking.The permeability of its hyphae membrane was also changed,and leaded to extravasating of inclusions and increasing the conductivity of hyphae broth.The content of soluble protein was declined and eventually the celles of fungi were death.The paper on Stephania kwangsiensis antibacterial activity of endophytic fungi findings provided the basis for the development of related microbial origin antibacterial agents.