Narrow mycorrhizal specialization and its effect on early development in the Mediterranean orchid Orchis italica

The contribution of individual orchid mycorrhizal (OrM) fungi to orchid germination is poorly understood. We isolated Tulasnella OrM fungi from 11 populations representing nine Italian regions associated with the orchid Orchis italica. We performed in vitro germination experiments using both single and two OrM isolate combinations of Tulasnella and Ceratobasidium isolates found in O. italica and co-occurring orchids. We performed an in situ reciprocal translocation experiment of O. italica seeds in two populations and we experimentally planted seeds into two un- occupied sites to test whether the presence of suitable OrM may limit its distribution. In vitro, Tulasnella isolates initiated germination, but only two isolates resulted in seedling development, while seeds did not germinate in the presence of Ceratobasidium isolates. Combinations of two Tulasnella isolates had lower germination rates compared to single isolates. Reciprocal translocations revealed that seeds from the same population had higher germination rates. Protocorms developed in one unoccupied site, with the associated fungus identified as Tulasnella spp. These results suggest that Tulasnella vary in their efficacy in seed germination and O. italica has a narrow specialization on Tulasnella. Such information is important for understanding the range limits of orchids and their ecological dependency on OrM fungi.