Isolation,Identification And Antimicrobial Activity Of Endophytic Fungi From Leaves Of Hopea Chinesis Hand.-Mazz.

Hopea chinesis Hand.-Mazz.is a national endangered plant and a natural anticorrosive tree because of its resistance to pests and diseases.The previous study of our experiment team found that different extract layer of H.chinesis Hand.-Mazz.showed antibacterial activity against 21 kinds of plant pathogenic fungi,15 compounds were isolated and identified.At present,there is no report on endophytic fungi of H.chinesis.In this study,endophytic fungi were isolated from leaves of H.chinesis by sterile technique,were screened and evaluated by using opposite culture,growth rates,inhibition zone assay and colony diameter assay methods,and identified based on the morphological characters and analysing ITS sequence.The antifungal activities of different treatments on endophytic fungi fermentation liquid was tested.Fermentation conditions of highly active strain,isolation and identification of secondary metabolites were studied.The main results are as follows:Eight endophytic fungi were isolated,and identified by morphological and molecular biological methods,which belonged to 2 subdivisions,2 clas-ses,5 orders,6 families and 6 genus.The characteristic of morphology and ITS sequences of endophytic fungi suggested that strain H.1,H.2,H.3,H.4,H.5,H.6,H.7,H.8 were identified as:Phomopsis sp.(Accession:KF159989.1);Phomopsis sp.(Accession:KU512713.1);Valsaceae sp.(Accession:A B334109.1);Lasiodiplodia pseudotheobromae(Accession:KY052959.1);Fusa-rium incarnatum(Accession:KF255423.1);Chaetomium sp.C36(Accession:J Q936193.1);Physalospora sp.strain F1080(Accession:KU747723.1);Xylaria sp.F14(Accession:KF675758.1),respectively.The antifungal activities of 8 strains of endophytic fungi against 7 kinds of plant pathogenic fungi(F.oxysporum Race 4、C.musae、C.lunata、A.solani、B.cinerea、A.alternata、P.citricarpa)was determined by the method of opposite culture.The results showed that strains H.2,H.3,H.4 and H.8 had different inhibition to 7 kinds of plant pathogenic fungi,especially the inhibitory effect of strain H.4 against B.cinerea was the highest among all strains with inhibition rates of 73.78%.H.2 and H.3 also had a better inhibitory effect on B.cinerea.H.3 had different inhibition to 7 kinds of plant pathogenic fungi,especially to B.cinerea with inhibition rates of 54.74%.H.8 had good inhibitory effect on B.cinerea,A.solani and P.citricarpa,especially to P.citricarpa.The antifungal activities of fermentation of H.2、.H.3、H.4、H.8 against 7 pathogenic fungi showed that inhibition rate of H.4 fermentation against B.cinerea was only 13.10%.The inhibitory effect of H.2 and H.8 fermentation against pathogenic fungi was not significant.The inhibitory effect of H.3 fermentation against F.oxysporum Race 4,B.cinerea and P.citiricarpa was significant,especially to B.cinerea with inhibition rates of 82.34%.The mycelial growth rate method was used to explore the inhibitory effect of fermentation liquor of different fermentation period of H.3 on B.cinerea,the results showed that the inhibition was increased with the increase of fermentation period of 5-20 days,especially had a strong inhibitory effect to B.cinerea with the inhibition rates of 14、17 and 20 days were 80.87%、83.11%、84.46%respectively and no significant difference between them,indicating active metabolites were stable after 14 days.The antifungal activities of H.3 fermentation liquid extracts against B.cinerea results showed that chloroform and ethyl acetate extract had antifungal activities,especially ethyl acetate extract was best with inhibition rate of 61.40%,indicating bioactive secondary metabolites mainly existed in ethyl acetate layer.Virulence curve of ethyl acetate extract results showed that significant antifungal activity against B.cinerea with EC50 value of 440.64 mg/L.H.3 was cultured in PYG for 16 days within the rotary table at 28℃ and 160rpm,and extracted by using ethyl acetate to accumulate more bioactive secondary metabolites.Two compounds were isolated from ethyl acetate extracts by repeated column chromatography on silica gel,thin layer chromatography(TLC),Sephadex LH-20 and recrystallization.The structures of compounds were identified by means of spectroscopic methods including 1D-NMR(1H-NMR,13C-NMR,DEPT),2D-NMR(1H-1H COSY,HSQC,HMBC)as well as chemical method and comparison with published data.Two compounds were identified as β-sitosterol.This is the first study to describe the isolation and antifungal activities of endophytic fungi from leaves of H.chinesis,the results showed that isolated endophytic fungi and their fermentation liquid had good antifungal activities,was potential active secondary metabolites for pesticide.Metabolites of ethyl acetate extract of H.3 had great potential development in anti-B.cinerea.