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BackgroundMycoheterotrophic plants are one of the most difficult plant groups to conserve because they are entirely dependent on symbiotic fungi. Establishment of viable culture systems would greatly aid their conservation. We describe a simple culture system for the mycoheterotrophic orchid, Gastrodia pubilabiata, that does not require laboratory facilities. The orchid is symbiotic with leaf-litter-decomposing fungi.Results Gastrodia pubilabiata seeds were incubated in plastic boxes or glass bottles filled with leaf litter collected from the natural habitat of the species. Seed germination was observed after 35 days and seedling development followed. Fungal isolates from seedlings were identified as Mycenaceae (Basidiomycota), a leaf-litter-decomposing mycorrhizal fungus of Gastrodia species.ConclusionOur method can be used to conserve endangered mycoheterotrophic plants associated with leaf litter-decomposing fungi efficiently, and can also serve as a model system for physiological and molecular studies of such plants.Electronic supplementary materialThe online version of this article (10.1186/s40529-017-0214-6) contains supplementary material, which is available to authorized users.

Primer bias toward Tulasnellaceae fungi during PCR is a known issue with metabarcoding analyses for the assessment of orchid mycorrhizal communities. However, this bias had not been evaluated for the fungal communities of epiphytic orchids, which account for 69% of all orchid species diversity. We compared the mycorrhizal communities detected using two primer pairs, a fungal universal primer pair (ITS86F/ITS4) and Tulasnella-specific primer pair (5.8STulngs/ITS4-Tul2), using a mock community of fungal isolates from epiphytic orchids and also environmental samples, including orchid roots and a tree bark tip from the host tree of an epiphytic orchid collected. The detected mycorrhizal communities differed widely depending on the primer pairs used. The fungal universal primer pair successfully identified Ceratobasidiaceae and Serendipitaceae fungi but did not reflect Tulasnellaceae diversity. Tulasnellaceae fungi were mainly detected using the Tulasnella-specific primer pair. These tendencies were observed in both the mock community and environmental samples. These results strongly suggest that the use of a Tulasnella-specific primer in combination with a fungal universal primer is essential for assessing the mycorrhizal communities of orchids through metabarcoding analysis, especially in epiphytic orchids. Our study contributes to further understanding of the diversity of mycorrhizal fungi in orchids.

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.

PREMISE Orchids depend primarily on mycorrhizal fungi to obtain nutrients throughout their life cycle. Epiphytic orchids account for 69% of orchid diversity. The unstable availability of water and nutrients in their arboreal habitats often results in severe water and nutrient stresses. Consequently, mycorrhizal associations may be important for the survival of epiphytic orchids, but our understanding thereof remains limited. Here, we investigated the mycorrhizal community in a single epiphytic orchid species, using more samples than in any previous study. METHODS We assessed the mycorrhizal communities of Thrixspermum japonicum, one of the most common epiphytic orchids in the temperate region of Japan. In total, 144 individuals were collected from 28 host tree species at 20 sites across 1300 km. The mycorrhizal fungi were identified based on nuclear ribosomal DNA internal transcribed spacer sequences and assigned operational taxonomic units (OTUs) based on 97% sequence similarity. RESULTS We obtained 24 OTUs; 9 belonged to the Ceratobasidiaceae and 15 to the Tulasnellaceae. These OTUs are widely distributed throughout the phylogenetic trees of the two fungal families. However, a single Ceratobasidiaceae OTU accounted for 49.7% of all fungal sequences and was predominant in samples from 15 host tree species and 12 sites. CONCLUSIONS Our results imply that despite having a broad range of mycorrhizal partners, T. japonicum was predominantly associated with a single fungal taxon at most of the sites among the host‐tree species investigated. These findings contribute to elucidating mycorrhizal symbiosis in epiphytic habitats.

In vitro symbiotic culture of Gastrodia pubilabiata seeds were conducted with the fungal isolates from G. pubilabiata roots. We obtained five fungal isolates, which belonged to Mycena, Marasmiaceae and Omphalotaceae. Firstly, optimal temperature and culture medium for subculture of these fungal isolates were examined. All five isolates grew the fastest on malt extract agar medium. Mycelial growth rate was highest at 25 °C between 10 °C and 40 °C. Secondly, we evaluated suitable culture vessels and organic materials for symbiotic culture. Seeds germinated well in petri dishes with Quercus leaf disc on water agar medium, and the seed germination process was well observed without dense mycelium. The most developed seedlings were found in glass bottles filled with Japanese cedar leaves, but densely grew mycelium prevent accurate seedling counts. Leaves of Quercus, Japanese cedar and bamboo were used as organic materials for symbiotic culture. All three leaves induced seed germination with Mycena and Marasmiaceae fungi, but material types affected subsequent seedling growth. Our method will contribute to understanding the mycorrhizal association of Gastrodia species and also other mycoheterotrophic plants.

Mycorrhizal specificity, i.e., the range of fungi allowing mycorrhizal partnerships, differs among orchid species, but that at early developmental stages is unclear. We investigated whether mycorrhizal specificity during seed germination and seedling development differs among three Dendrobium species, D. officinale, D. okinawense and D. moniliforme, in vitro. Nine mycorrhizal fungal strains were obtained from the roots of these species and cultured with a seed of each Dendrobium species. Five to eight fungal strains stimulated seed germination, whereas one to four fungal isolates significantly promoted protocorm development in the three species. To evaluate effects on leafy seedling growth, seedlings obtained from asymbiotic culture were cultured with nine fungal isolates. D. officinale and D. okinawense showed specificity for a single Serendipitaceae or Tulasnellaceae isolate, whereas D. moniliforme exhibited specificity for three isolates of Serendipitaceae and Tulasnellaceae. Therefore, the three Dendrobium species had a growth bottleneck from seed germination to the protocorm stage, and mycorrhizal specificity of protocorm growth and seedling development in vitro varied among the species. Our findings imply divergent mycorrhizal specificity in Dendrobium species at early developmental stages. This study provides insights into the diversity of orchid mycorrhizal specificity, as well as valuable information for conservation of endangered orchids.

An endangered orchid, Cephalanthera longifolia, recently expands their habitat to developed lands, such as parks and landfills. This shows that developed lands can be available for the habitat of the endangered orchids, that is an interesting model for considering the ecosystem in urban area. However, the factors that allow this orchid to colonize in developed lands still remain unclear. This orchid, which is so-called partially mycoheterotrophic plants , depends on the symbiotic mycorrhizal fungi for their nutrient supply, especially in germination stage. To clarify which fungal groups induce its seed germination and are involved in its colonization in developed lands, we conducted in situ seed germination method in the landfill site and identified the symbiotic fungi of the seedlings. Three Thelephoraceae fungi were mainly involved in the seed germination of C. longifolia. These fungi were found from various growth stages of the seedlings and distributed across the study site regardless the soil depth and the inoculation point, suggesting that they play a key role in the seed germination and development of the seedlings. Thelephoraceae fungi have ectomycorrhizal association with the Pinaceae and Fagaceae trees. Stable symbiotic relationships were developed between these planted trees and Thelephoraceae fungi, and these relationships allowed this orchid to colonize in the developed land.