| This study collected three species root segments of Cypripedium tibeticum,Cypripedium flavum and Cypripedium bardolphianum in Sichuan Huanglong Valley Nature Reserve.Free-hand section-cutting method was used to observe the structure of mycorrhiza.We combined small piece culture medium of peloton suspension,root segment of mycorrhizal section and coating plate of of mycorrhizal section approaches to isolate endophytic fungi and identifed the isolated strains by observation of cultured morphology of endophytic fungi and ITS information of isolated strains.Species diversity,Simpson diversity index and Pielou evenness index were used to analyze the diversity of endophytic fungi in three species.The main results were as follows:1.Endophytic fungi enter the root of Cypripedium tibeticum,Cypripedium flavum and Cypripedium bardolphianum through the outer layer of the epidermis,but the intruding mycelium was not observed in vascular area.2.The method of small pieces of culture medium of peloton suspension was better than the others for obataining the endophytic fungi in this study.3.Molecular identification shows that 5 isolated strains were Tulasnella sp.,1 strain was Epulorhiza sp.,2 strains was Irpex sp,m 1 strain was Hypoxylon sp.,1 strain was Gibberella sp.,1 strain was Hypocrea sp.,1 strain was Periconia sp.and 1 strain was Nectria sp.;There were 10 genera,15 strains of endophytic fungi in total,which Tulasnella sp.accounted for 33.3%of the total isolates.Root endophytic fungi isolated from three species of Cypripedium were different,which may be caused by different habitat and growth cycles of Cypripedium.4.Among the three species,the diversity and evenness of root endophytic fungi of Cypripedium flavum were the highest,followed by Cypripedium tibeticum,then the Cypripedium bardolphianum.It indicates that the ability of Cypripedium flavum in adapting to the external environment is better than Cypripedium tibeticum and Cypripedium bardolphianum. |
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