Endophytic and ectomycorrhizal, an overlooked dual ecological niche? Insights from natural environments and Russula species.

Ectomycorrhizal fungi (EcMF) are key components of temperate ecosystems, and recent studies suggest that they can also inhabit non-EcM plant roots as endophytes. We aimed to (1) provide new evidence of EcMF colonization of non-EcM hosts, (2) offer direct microscopic confirmation of such endophytism and (3) assess factors influencing colonization, including plant identity, site and potential root filtering. Using ITS2 amplicon sequencing, we characterized root fungal communities in 66 non-EcM plant species sampled across nine French sites. At two sites, we compared rhizosphere and root communities to test for a root filtering. We also investigated endophytism in Russula species at two Russula-rich sites via FISH and confocal microscopy. We found substantial but variable proportions of EcMF sequences in non-EcM roots, particularly near EcM trees, indicating that endophytism may serve as a secondary ecological niche. Although EcMF were more abundant in the rhizosphere than in roots, community composition was similar, suggesting limited root filtering. Microscopy revealed metabolically active Russula hyphae within the apoplast of two non-EcM plant species. Our results confirm a dual EcM/endophyte niche for Russula spp., echoing patterns observed in other EcMF. The ecological implications of this duality remain to be explored.