Hygrocybe virginea is a systemic endophyte of Plantago lanceolata

Species of Hygrocybe (waxcaps) are mostly colorful mushrooms, which are characteristic of undisturbed grasslands. These fungi are endangered in many places worldwide, but their biology remains a mystery: while isotopic signatures indicate that waxcaps are neither mycorrhizal nor saprotrophic, they were recently observed in plant roots and molecularly detected in aboveground tissues. We aimed to establish a model system of Plantago lanceolata plants colonized by H. coccinea for future detailed studies of the plant-fungus association, and species-specific primers were designed to control infection success and screen environmental samples for waxcaps. The experimentally treated plants grown from surface-sterilized seeds were indeed colonized by waxcaps after 22 weeks of incubation. However, the fungal infection was independent from the experimental treatment and apparently resulted from infected seeds. Screening of field material confirmed that at least one species, i.e., H. virginea, is a maternally transmitted endophytic fungus associated with P. lanceolata. In the experiments, it obviously expanded to the roots during or after seed germination. The endophytic growth is also consistent with the carbon isotopic signature of Hygrocybe, which deviates less from the host plants' signature than known from ectomycorrhizal associations. However, waxcaps obviously acquire nitrogen (N) from a source outside the plant, like mycorrhizal fungi do. The extensive root system of P. lanceolata is hypothesized to facilitate reaching of nitrogen sources for Hygrocybe which are enriched in the heavier (15) N isotope.