Addition of fungal inoculum increases seed germination and protocorm formation in a terrestrial orchid

Because orchid mycorrhizal fungi are important for orchid seed germination, addition of germination-supporting fungal inoculum to the soil can be an efficient way to improve in situ seed germination of terrestrial orchids, but empirical evidence is still largely lacking. In this study, we used seed inoculation experiments to test the hypothesis that addition of fungal inoculum increases seed germination of the terrestrial orchid Gymnadenia conopsea under natural field conditions. In China, this species has suffered dramatic declines in abundance due to habitat loss, grazing and over-collection, while observational studies have shown very low recruitment of seedlings into natural populations. Previous research has shown that the fungal strain GS2 (Ceratobasidiaceae) was able to support germination under in vitro conditions. Seed packages were used to bury orchid seeds in the soil with and without fungal inoculum and to assess seed germination. In total, 11.96 ± 2.65 % of seeds had the testa ruptured when fungal inoculum was added to the seed packages, 10.63 ± 3.32 % of seeds germinated to the protocorm formation stage, 5.24 ± 2.71 % of the seeds grew to the protocorm differentiation stage, and 3.16 ± 1.63 % of seeds finally formed early seedlings. In contrast, no seed germination was observed in the control groups without fungal inoculum. We isolated the fungi associating with the germinating seeds and confirmed that seed germination was supported by fungal strain GS2. In addition, several other fungi were isolated from germinating seeds, including four other Ceratobasidiaceae strains that are known to support seed germination in vitro. Our results highlight the potential of fungal inoculum to induce seed germination of terrestrial orchids under field conditions and may be useful when developing more efficient symbiotic seed germination protocols for the restoration and conservation of terrestrial orchids, especially in situations where natural germination is severely limited.