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Orchids produce small seeds with no energy reserves, relying entirely on orchid mycorrhizal fungi (OMF) for germination. This process, known as symbiotic germination, can lead to different outcomes depending on abiotic factors, such as nutrient availability. Previous studies have shown that nutrient addition has a negative effect on the outcome of symbiotic germination. However, if this effect varies across OMF species, and if it is related to a fungal response to nutrients, remains unknown. This paper examines the effect of fungal identity and nutrient addition (nitrogen and phosphorus) on the germination of the orchid Bipinnula fimbriata using seven OTUs of mycorrhizal fungi from the families Tulasnellaceae and Ceratobasidiaceae. We also evaluated the effect of nutrient addition on mycorrhizal fungi growth rates. Results showed that the nutrient effect on symbiotic germination varied depending on fungal identity. While there was a strong negative effect on symbiotic germination with all Tulasnella OTUs and two Ceratobasidium OTUs, less or no effect was observed on the other two Ceratobasidium OTUs. Further studies are needed to understand the mechanism underlying this variation and how variable is the effect of nutrient addition on symbiotic germination in Orchidaceae and OMF species.

Several species of the family Orchidaceae have limited distribution and small populations, in which the probability of extinction is much higher than in broadly distributed species. As orchid seeds are very small and lack energy reserves, they require association with compatible mycorrhizal fungi that provide nutrients and carbon needed for germination and early development. Therefore, identification of putative orchid mycorrhizal partners is essential for the design of conservation strategies for these species. We investigated the mycorrhizal associations of two endangered rare species of orchids, Bipinnula apinnula and B. volckmannii, and compared our results with the mycorrhizal associations of two broadly distributed species of Bipinnula. We isolated mycorrhizal fungi and directly amplified DNA from colonized root segments and found an extremely low diversity of mycorrhizal fungi. The rarefaction curves of rare species populations saturated at a single OTU, showing a highly specialized association and considerably lower diversity than the other two species of Bipinnula. Nevertheless, symbiotic germination experiments with fungal strains of this specific fungal OTU only reached the stage of seed coat rupture in both orchid species, without formation of protocorms. Our results suggest that seeds may require additional treatment for successful germination and that fungal partners isolated from adult plants may not be the same that support the germination of seeds. Further studies are needed to improve germination of thiese orchid species and to contribute to their conservation.