Mycorrhizae are an important energy source for orchids that may replace or supplement photosynthesis. Most mature orchids rely on mycorrhizae throughout their life cycles. However, little is known about temporal variation in root endophytic fungal diversity and their trophic functions throughout whole growth periods of the orchids. In this study, the community composition of root endophytic fungi and trophic relationships between root endophytic fungi and orchids were investigated in Bletilla striata and B. ochracea at different phenological stages using stable isotope natural abundance analysis combined with molecular identification analysis. We identified 467 OTUs assigned to root-associated fungal endophytes, which belonged to 25 orders in 10 phyla. Most of these OTUs were assigned to saprotroph (143 OTUs), pathotroph-saprotroph (63 OTUs) and pathotroph-saprotroph-symbiotroph (18 OTUs) using FunGuild database. Among these OTUs, about 54 OTUs could be considered as putative species of orchid mycorrhizal fungi (OMF). For both Bletilla species, significant temporal variation was observed in the diversity of root endophytic fungi. The florescence and emergence periods had higher fungal community richness of total species and endemic species than did other periods. Both Bletilla species were dominated by Agaricomycetes and Basidiomycota fungi throughout the whole year; however, their abundances varied between two Bletilla species and among phenological stages. Meanwhile, the ranges of 13C and 15N natural abundance were also highly dynamic across all growth stages of Bletilla species. Compared with the surrounding autotrophic plants, significant 13C enrichments (ε13C) were found across all phenological stages, while significant 15N enrichment in the florescence period and strong 15N depletion during the fruiting period were found for both Bletilla species. We can deduce that both Bletilla species obtained carbon from root endophytic fungi during the whole year. Additionally, the temporal varying tendency of root endophytic fungal diversity was consistent with 13C enrichments, which was also accord with the nutritional requirement of plant.

Orchids of the genus Bletilla are well-known ornamental plants and sources of traditional medicine in Asia that rely on the symbiotic relationship with root endophytic fungi throughout their whole life cycle. However, little is known about their fungal partners, infection pattern, and pathways of carbon gain. We investigated carbon and nitrogen stable isotope patterns in different organs of three Bletilla species, identified the root endophytic fungal community composition, and determined mycorrhizal colonization rates. The three Bletilla species were comprised by a polyphyletic group which belongs to different trophic modes, such as saprotroph, pathotroph, and symbiotroph; however, the dominant species and their abundances varied among Bletilla spp. Mycorrhizal infection rates also varied among Bletilla species, with B. striata (65% ± 25%) being significantly higher than those of B. formosana (35% ± 16%) and B. ochracea (22% ± 13%). Compared with surrounding autotrophic plants, all Bletilla spp. were significantly enriched in 13C with B. striata to a significantly higher level than other two Bletilla species. Among different organs, stems had higher δ13C values, while leaves and flowers had higher δ15N and total N content values across all three species. Our results indicate that the symbiotic relationship of Bletilla and its root endophytic fungi is not strictly specific. Although mycorrhizal infection rates were highly variable, the three Bletilla species had the same infection pattern with hyphae penetrating the cortex cell by the pathway cell. Different Bletilla species have different strategies for C allocation among plant organs. These findings provide new insights into the ecological adaptation of orchids and will contribute to Bletilla germplasm conservation and sustainable utilization.