Addition of fungal inoculum increases germination of orchid seeds in restored grasslands

Grasslands restored on arable land often retain high residual nutrients, modified soil biota, and lower plant species diversity. Establishment of rare plant species with complex multitrophic interactions, typical of undisturbed nutrient-poor environments, may be hindered by the absence of interacting organisms. We hypothesised that the addition of a mycorrhizal symbiont improves the seed germination of orchids that crucially depend on fungi. We focused on grasslands restored on arable land 1–15 years ago featuring residual mineral nutrients and low organic matter contents compared to semi-natural grasslands and on four orchid species differing in the level of mycorrhizal specificity: high – Anacamptis pyramidalis and Orchis mascula – and low – Platanthera bifolia and Gymnadenia conopsea. Five fungal isolates obtained from non-green underground mycorrhizal orchid seedlings (protocorms) or adults' roots were tested for orchid-fungus compatibility under conditions in vitro. Orchid seeds inserted in retrievable seed packets were subsequently co-introduced with selected fungal isolates grown either on agar or sterilized hay into the soil of nine restored grasslands and incubated for twelve months. The identity of mycorrhizal fungi in retrieved protocorms was verified by molecular methods. The isolates that supported protocorm establishment in vitro enabled also protocorm formation in situ, but success rates differed among orchid species. While mycorrhizal specialists produced most protocorms after inoculation, the mycorrhizal generalists took advantage of naturally occurring fungi and produced some protocorms both in inoculated and uninoculated treatments. We showed that the addition of mycorrhizal fungi enhanced protocorm formation regardless of the modified soil environment, especially in mycorrhizal specialist orchids. This method may help to restore populations of native orchid species in their former distribution ranges, including farming-altered habitats.