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Neuwiedia singapureana is a rare and endangered plant of the Apostasioideae subfamily. The Apostasioideae subfamily has a unique evolutionary status, as it is considered to be the most primitive group forming the base of the Orchidaceae evolutionary tree. Therefore, N. singapureana has high scientific research and conservation value. The endophytic fungal communities associated with orchids are rich and diverse, but few studies have investigated the endophytic fungi of Neuwiedia orchid plants. In the present study, the aim was to examine the endophytic fungal community structures associated with wild N. singapureana rhizomes and normal roots in the ground and with bare prop roots in the air at two sampling sites in China. High-throughput sequencing of nuclear ribosomal DNA fragments of the internal transcribed spacer regions was conducted, and cultivable methods were adopted. A total of 2161 endophytic fungal operational taxonomic units (OTUs) were obtained at a 97% sequence similarity threshold. The endophytic fungal diversity differed among the samples but not significantly. There were many more non-mycorrhizal endophytic fungal than orchid mycorrhizal (OM) fungal species detected in the N. singapureana orchid, about 98.33% OTUs of non-mycorrhizal fungi contrasting with 1.67% OTUs of potential orchid mycorrhizal fungi, among which Ceratobasidiaceae, Russulaceae, and Thelephoraceae were the dominant orchid mycorrhizal fungi. One culturable OM fungal Epulorhiza sp. isolated from the rhizome was capable of significantly promoting the seed germination and seedling growth of Dendrobium officinale and Epidendrum secundum orchids, respectively, with different efficiencies. These endophytic fungal strains with growth-promoting functions will provide materials for orchid conservation and for the study of the mechanisms underlying orchid symbiotic associations. Full article