Isolation and Reproductive Structures Induction of Fungal Pathogens Associated with Xylem and Wood Necrosis in Grapevine

Grapevine (Vitis vinifera L.) trunk diseases (GTDs) are considered a disease complex including five diseases: esca, Petri disease, black-foot disease, Botryosphaeria dieback, and Eutypa dieback. The main symptom is a general decline in affected plants, which show xylem necrosis and discoloration or sectorial necrosis in the wood. Their diagnosis is tedious due to four main reasons: (i) the wide diversity of internal symptoms that we can find; (ii) the great diversity of fungi that are associated with them; (iii) the high frequency of co-infections in the same plant; and (iv) the different behavior that the fungal species associated with GTDs show in vitro. Here, we describe a detailed protocol to isolate the different fungal trunk pathogens associated with GTDs as well as methods to induce sporulation and formation of fruiting bodies (pycnidia) to make easier their morphological characterization.