The Diversity,Antimicrobial Activity And Secondary Metabolites Of Endophytic Fungi In Sophora Tonkinensis Gagnep

The objective of this paper was the diversity,antimicrobial activity and secondary metabolites of endophytic fungi harbored in the Sophora tonkinensis Gagnep.,a well-known medicinal plant of Guangxi province.Endophytic fungi were isolated from different organs and tissues of S.tonkinensis collected from three different localities in Guangxi province:Chengliang village in Jingxi city(J),Longsu village in Tiandeng county(T)and Shanghu village in Daxin county(D).The main results were showed as follows.The root,stem and seed of S.tonkinensis were immensely rich in endophytic fungi.The organs samples including root segments,stem segments and seeds were collected from three different localities in Guangxi province.A total of 1 504 fungal strains were isolated from six tissues including the phloem of the root,the xylem of the root,the bark of the stem,the xylem of the stem,testa and embryo,separated from three organs such as root,stem and seed.The strains were classified as 95 taxa containing 73 identifiable taxa and 22 unidentifiable taxa.The identifiable taxa belonged to 3 phylums with 7 classes,represented by 22 orders with 44 genera.Out of 73 identifiable taxa,19 taxa were accepted at genus level with 54 taxa at species level.The most abundant phylum was Ascomycota with 68 taxa,followed by Basidiomycota with 3 taxa and Zygomycota with 2 taxa.The dominant classes were Sordariomycetes,Eurotiomycetes and Dothideomycetes with the dominant orders Hypocreales,Eurotiales,Capnodiales and Pleosporales,represented by the dominant genera Fusarium,Penicillium,Cladosporium and Colletotrichum with the dominant species Fusarium solani,Fusarium oxysporum,Cladosporium perangustum,Colletotrichum gloeosporioides and Cercospora beticola.The colonization of the endophytic fungal communities harbored in the S.tonkinensis was significantly influenced by geographic localities,organs and some tissues.The colonization frequency(CF)of the endophytic fungal communities harbored in the S.tonkinensis from three localities was significantly different-i.e.,The CF from the locality T(30.57%)was the highest,followed by the locality J(22.07%)and locality D(14.03%).The CF of the endophytic fungal communities was significantly different in different organs of the S.tonkinensis-i.e.,the CF was the highest in the seed(46.67%),followed by the stem(22.40%),and lowest in the root(14.03%).The tissues including the xylem of the root,the phloem of the root,the xylem of the stem and the bark of the stem had no significant influence on the colonization frequency except for testa and embryo-i.e.,the CF was the lowest in the embryo with 0.67%.The composition and distribution of the endophytic fungal communities harbored in the S.tonkinensis were greatly influenced by geographic localities,organs and tissues.The dominant fungi of the endophytic fungal communities harbored in the S.tonkinensis at different taxonomic levels including orders,genera and species were strongly influenced by geographic locality,organs and tissues.The similarity indices of the endophytic fungal communities between two localities were rather low at the range of 0.1067 to 0.2200.The similarity indices of the endophytic fungal communities between two organs or tissues were rather low at the range of 0 to 0.3579.Among 73 identifiable taxa,8 taxa shared among three localities,12 taxa occurred at two localities,4 taxa shared among three organs,13 taxa occurred at two organs,and the others were special to one locality or organ.The fungi had the highest relative frequency in different localities or tissues-i.e.,Phoma sp.,Phialophora mustea and Metarhizium oanisopliae in the locality T;Neopestalotiopsis sp.and Phaeosphaeria sp.in the locality J;Myrothecium verrucaria in the locality G;Cladosporium perangustum in the xylem of the root;Myrothecium verrucaria and Purpureocillium lilacinum in the xylem of the root;Cercospora beticola in the xylem of the stem;Neopestalotiopsis sp.and Phaeosphaeria sp.in the bark of the stem;Colletotrichum gloeosporioides and Alternaria alternata in the testa;Pseudocercospora elaeocarpi in the embryo?The a-diversity of the endophytic fungal community harbored in the S.tonkinensis was very high(S,95;H',3.993 8;1-D,0.974 0).The a-diversity of the endophytic fungal communities harbored in the S.tonkinensis from the locality D was significantly lower than that from the locality T and the locality J.However,the difference was not significant between the locality T and the locality J.Except for the embryo with rare fungi,other organs and tissues with similarly rich fungi,did not affect the ?-diversity of the endophytic fungal communities.The endophytic fungi with abundant antimicrobial strains harbored in the S.tonkinensis.Many endophytic fungi exhibited strongly antimicrobial activity with similar to their host against three human pathogens including Candida albicans,Escherichia coli and Staphylococcus aureus,and five phytopathogens including Xanthomonas oryzae pv.oryzicola,Xanthomonas campestris pv.citri,Alternaria panax,Colletotriehum gloeosporioides and Fusarium solani.The results were as follows.Ninety-five respective fungi were challenged by these pathogens.Twenty-one strains exhibited strongly antimicrobial activity against pathogenic bacteria and C.albicans.Ten strains displayed significant antifungal activity against three fungal phytopathogens of Panax notoginseng.Particularly,the minimal inhibitory concentration values of metabolites from strains GRPH-0 against C.albicans,TRPH-21 against E.coli,TRPH-35 against S.aureus,JXRPH-24 against S.aureus,JSMXY-17 against X.oryzae,TRXY-5 against X.oryzae and JSMXY-17 against X.campestris,were sixteen times,sixteen times,thirty-two times,thirty-two times,sixteen times,sixteen times,sixty-four times higher than that of positive control,and much smaller than that of metabolites from S.tonkinensis,respectively.The antifungal activities of the metabolites from stains TRPH-73,TRPH-87,TRXY-46,TRXY-18-1,TRPH-89,TRPH-13 and TRXY-34-1,against three pathogenic fungi of Panax notoginseng,were almost equal to or more than that of positive control,and were much stronger than that of metabolites from S.tonkinensis,respectively.The minimal inhibitory concentration values of the metabolites from strains TRPH-73,TRPH-87,TRXY-46,TRPH-89,TRPH-13 and TRXY-34-1 against conidia from two pathogenic fungi of Panax notoginseng in the range 1 to 2 mg.mL-1,were only four to eight times higher than that of positive control,and were less than that of metabolites from S.tonkinensis.The strain TRXY-46 contained abundant sterides.This strain was cultured with 150 L to afford crude extracts of 220 g by soak extraction method.The extracts were fractionated with 6 fractions by gradient elute,namely F1,F2,F3,F4,F5 and F6.The chromatographic methods were used to isolate 5 compounds from the fraction F1.Their structures were elucidated as sterides by means of spectroscopic methods,namely 6?-hydroxystigmast-4-en-3-one(compound 1),(22E,24R)-5?,8?-epidioxyergosta-6,9(11),22-trien-3?-ol(compound 2),5,8-epidioxyergosta-6,22-dien-3?-ol(compound 3),(22E,24R)-5a,8a-epidioxy-23-methylergosta-6,22-dien-3?-ol(compound 4),(22E,24R)-3?,5 ?-dihydroxyergosta-7,22-dien-6-one(compound 5).