The endangered epiphytic orchid Dendrobium okinawense has a highly specific mycorrhizal association with a single Tulasnellaceae fungus

Abstract

Epiphytic orchids, which account for 68% of vascular epiphytes, contribute substantially to plant species diversity in forest canopies. Many orchid species are naturally rare and have limited geographical distributions. All orchids rely on mycorrhizal fungi for nutrient acquisition during seed germination and seedling establishment, and many species retain these associations throughout their lifecycle. Due to their reliance on mycorrhizal fungi, epiphytic orchid species with highly specific mycorrhizal associations may be geographically restricted and are often rare. Dendrobium okinawense is a rare epiphytic orchid endemic to the Yambaru Forest in Japan and to southern Taiwan. We hypothesized that this species exhibits high mycorrhizal specificity and tested this hypothesis via identification of the species' mycorrhizal associates. We collected root samples of this orchid from four tree species and assessed mycorrhizal fungi in 25 root fragments harvested from 10 plants. Fungi were identified using molecular methods based on fungal isolates and DNA extracted from mycorrhizal roots. Detected fungi were assigned to 21 operational taxonomic units (OTUs) based on 97% sequence similarity of nuclear ribosomal DNA internal transcribed spacer region sequences. TU11, an OTU belonging to Tulasnellaceae (Basidiomycota), was the most frequently detected OTU, occurring in 20 root fragments from all 10 sampled individuals. Our results indicate that Dendrobium okinawense has a highly specific association with TU11. Further, this specificity may be high relative to the Dendrobium species assessed in other studies. These findings support the hypothesis that Dendrobium okinawense exhibits high mycorrhizal specificity.

Dominant Dendrobium officinale mycorrhizal partners vary among habitats and strongly induce seed germination in vitro

“…Although D. officinale is associated with a wide range of basidiomycetous mycorrhizal partners, its main fungal partners may be widely shared among D. officinale populations. In Dendrobium okinawense, 11 mature plants from four sites were predominantly associated with a single Tulasnellaceae OTU (Rammitsu et al, 2021). Such high specificity is also found in Dendrobium fimbriatum, which was associated with only two OTUs in 15 root samples from two sites (Xing et al, 2013).…”

“…The fungal isolates obtained in this study were deposited in the Biological Resource Center of the National Institute of Technology and Evaluation (NBRC) (Table 2). 10.3389/fevo.2022.994641 Molecular identification of mycorrhizal fungi DNA was extracted from root samples as described previously (Rammitsu et al, 2021). Samples were crushed with forceps to disperse hyphal coils into TE buffer.…”

“…These primer pairs failed to amplify sequences of Tulasnellaceae, which is a dominant mycorrhizal fungal family associated with orchids. Therefore, we also used the Tulasnellaceae-specific primer pairs ITS5/ITS4-Tul2 (White et al, 1990;Oja et al, 2015) and 5.8S-Tulngs/ITS4-Tul2 (Oja et al, 2015;Rammitsu et al, 2021). PCR amplification was PCR amplification was performed with the following cycling parameters: initial denaturation at 98 • C for 2 min, followed by denaturation at 98 • C for 10 s, annealing at 58 • C for 15 s, extension at 68 • C for 40 s, for a total of 35 cycles.…”

“…Mycorrhizal associations with Dendrobium species have also been investigated for the propagation of medicinal species and conservation of endangered species (Chen et al, 2021). However, most such studies have focused on symbiotic culture with fungal isolates from roots, seeds, or seedlings (Nontachaiyapoom et al, 2011;Mala et al, 2017;Maharjan et al, 2020), whereas mycorrhizal associations among wild orchid populations remain poorly understood, although a few studies have revealed in situ associations with several wild populations (Xing et al, 2013;Rammitsu et al, 2021).…”

Ecological management of insular forests: conservation of endangered species and native ecosystems in Ryukyu Archipelago

“…The next study deals with an endangered epiphytic orchid, Dendrobium okinawense. Rammitsu et al (2021) investigated the symbiotic fungi of D. okinawense and found that this orchid has highly specific symbiotic fungi. The degree of symbiotic specialists is likely to relate closely to the forest health, suggesting that it is important to protect oldgrowth forests in Yambaru from anthropogenic disturbances for the conservation of this species.…”