Two ectomycorrhizal truffles, <i>Tuber melanosporum</i> and <i>T. aestivum</i>, endophytically colonise roots of non‐ectomycorrhizal plants in natural environments

<jats:title>Summary</jats:title><jats:p> <jats:list list-type="bullet"> <jats:list-item><jats:p>Serendipitous findings and studies on <jats:italic>Tuber</jats:italic> species suggest that some ectomycorrhizal fungi, beyond their complex interaction with ectomycorrhizal hosts, also colonise roots of nonectomycorrhizal plants in a loose way called endophytism. Here, we investigate endophytism of <jats:italic>T. melanosporum</jats:italic> and <jats:italic>T. aestivum</jats:italic>.</jats:p></jats:list-item> <jats:list-item><jats:p>We visualised endophytic <jats:italic>T. melanosporum</jats:italic> hyphae by fluorescent <jats:italic>in situ</jats:italic> hybridisation on nonectomycorrhizal plants. For the two <jats:italic>Tuber</jats:italic> species, microsatellite genotyping investigated the endophytic presence of the individuals whose mating produced nearby ascocarps. We quantified the expression of four <jats:italic>T. aestivum</jats:italic> genes in roots of endophyted, non‐ectomycorrhizal plants.</jats:p></jats:list-item> <jats:list-item><jats:p><jats:italic>Tuber melanosporum</jats:italic> hyphae colonised the apoplast of healthy roots, confirming endophytism. Endophytic <jats:italic>Tuber melanosporum</jats:italic> and <jats:italic>T. aestivum</jats:italic> contributed to nearby ascocarps, but only as maternal parents (forming the flesh). Paternal individuals (giving only genes found in meiotic spores of ascocarps) were not detected. Gene expression of <jats:italic>T. aestivum</jats:italic> in non‐ectomycorrhizal plants confirmed a living status.</jats:p></jats:list-item> <jats:list-item><jats:p><jats:italic>Tuber</jats:italic> species, and likely other ectomycorrhizal fungi found in nonectomycorrhizal plant roots in this study, can be root endophytes. This is relevant for the ecology (brûlé formation) and commercial production of truffles. Evolutionarily speaking, endophytism may be an ancestral trait in some ectomycorrhizal fungi that evolved from root endophytes.</jats:p></jats:list-item> </jats:list> </jats:p>