Samples of T. melanosporum FBs were collected at different developmental stages in the field near the city of L’Aquila (Italy), using the methods described by Pacioni et al.28 in addition to the traditional use of trained dogs. According to the season climatic conditions, in the years of this research, specimen of stage 3 were collected between July and August, of stage 4 in August–September, of stage 5 in September–early November, and of stage 6 in late November to March (four biological replicates for each stage). All of the ascocarps were analyzed morphologically and microscopically and classified by maturation stage according to the criteria defined by Zarivi et al.29. For structural analysis and maturation stage assessment, 8–10 µm cryostat sections of each FB were observed after Giemsa staining (1 min).