Objective: In the present study,Gentiana rigescens was screened for fungi communities to clarify their diversity and community assemblage in hosts.Meanwhile,the identification and activity assays of the strains were also conducted.Methods: By culture-dependent (endophytic fungi isolations from plant sections) and culture-independent (metagenomic library and cloning from plant sections) techniques,fungi communities were studied.The metagenomic library was generated using direct DNA isolation of whole plants,plant radixes,plant stems,plant leaves,plant flowers and soils around the plant.Meanwhile,endophytes were isolated from all parts ofG.rigescens plants.After fermentation of the fungi isolations,all the isolates were evaluated for their cytotoxicity against four kinds of human cancer cell lines (HCT116,BEL7404,A549,MDA-MB-231).Results: Eventually,200 strains were isolated and 103 strains were further identified through the internal transcribed spacer (ITS,ITS1 and ITS2 regions) sequence by using the universal primers ITS5 and ITS4.A total of 59,106 fungal sequences corresponding to 374 putative operational taxonomic units (OTU) were identified by 454 pyrosequencing.Through 454 pyrosequencing,the main fungal genera were Sebacina,Botrytis,Mycosphaerella,Boletus and Gibberella,and the major fungal genera which were directly isolated were Aspergillus,Fusarium,Penicillium and Alternaria.Activity assays showed strains 5-26 (Aspergillus sp.)and 6-2 (Fusarium avenaceum) had the outstanding cytotoxicity to all the tested cell lines with IC5o values ＜5 μg/mL.Conclusion: This study revealed the abundance of endogenetic fungal resources and a variety of genetic information in G.rigescens by high-throughput 454 sequencing technology and fungi isolation methods.Activity assays indicated that endophytes were a promising natural source of potential anticancer agents.