Newly Designed Fluorescence In Situ Hybridization Probes Reveal Previously Unknown Endophytic Abilities of Tuber magnatum in Herbaceous Plants

Tuber magnatum Picco (the Italian white truffle) is the most valuable and widely appreciated truffle. It is an ectomycorrhizal fungus known to associate with many broadleaf tree species. However, its mycorrhizae are rarely observed in the field, suggesting possible alternative symbiotic strategies, such as endophytism with non-ectomycorrhizal plants. In order to test potential endophytic interactions of T. magnatum with wild plants, a combination of polymerase chain reaction (PCR) and Fluorescence In Situ Hybridization (FISH) approaches were used. Specific FISH probes for T. magnatum were designed, tested in vitro on hyphae and/or ectomycorrhizae, and selected for their specificity. These probes were then used on a wide variety root samples of wild plants collected from three T. magnatum production areas in Italy and previously tested for the presence of T. magnatum mycelium using PCR-specific primers. Molecular analyses detected the presence of T. magnatum in 21 of 100 plant samples analyzed. FISH analysis confirmed the extracellular presence of active T. magnatum hyphae inside the root system of Carex pendula Huds plant. This study provides the first evidence of T. magnatum acting as an endophyte in an herbaceous plant. The newly designed, highly specific T. magnatum FISH probes can be used for further investigations to confirm the endophytic tendencies of T. magnatum and to understand their influence on the life cycle and biology of this fungus.