Background: Grapevines host diverse microbial communities, including fungal pathogens associated with grapevine trunk diseases (GTDs), which pose a major challenge in viticulture. Despite extensive research, the ecological drivers shaping fungal community composition across different grapevine microhabitats remain insufficiently understood. Using ITS2 metabarcoding, we characterized fungal communities in bark, wood, and soil from Esca symptomatic and asymptomatic vines, and evaluated the effects of season, year, cultivar, microhabitat, and plant health on key functional groups. Results: Seasonal variation had limited effects on fungal richness, whereas interannual differences had a strong impact on functional group diversity. Microhabitat played a key role: plant pathogens, particularly GTD-associated taxa, were most prominent in bark and wood; mycoparasites were dominant in bark; generalist saprotrophs prevailed in soil and bark; and GTD-related wood saprotrophs were most abundant in bark. Soil harbored the highest overall fungal diversity. Plant health status significantly affected only GTD pathogens, which showed higher abundance and diversity in symptomatic vines. Cultivar influenced only the richness of GTD-related wood saprotrophs. Conclusions: Year (vintage) emerged as a major driver of fungal community composition across plant tissues and soil, driving the most pronounced shifts in the mycobiome. Abiotic factors (year and season) were most strongly associated with soil fungal communities, whereas biotic factors (cultivar and plant health) exerted a greater influence on fungi associated with grapevine tissues. These findings underscore the central role of microhabitat and temporal variation in structuring the grapevine mycobiome.

The grapevine microbiome is shaped by a complex interplay of biotic and abiotic factors, affecting microbial community structure and plant health. This study investigates the diversity, composition, and dynamics of fungal communities associated with grapevine (Vitis vinifera) and neighboring cultivated plants, as well as plants from semi-natural vegetation, including pear (Pyrus communis), apricot (Prunus armeniaca), dogrose (Rosa canina), and blackthorn (Prunus spinosa), in a landscape-level habitat matrix. Using metabarcoding techniques, fungal communities from leaves and woody tissues of grapevine and neighboring plants were analyzed over a growing season. Fungal richness and abundance differed significantly among host plants, with woody tissues exhibiting higher diversity. Host plant identity was the primary factor shaping wood-associated fungal communities (15.7% of explained variance), whereas sampling time dominated in leaves (16.3%), with sampling site having a weaker effect in both cases. Pathogenic fungi associated with grapevine trunk diseases, such as Diaporthe, Eutypa, and Phaeomoniella, were identified across grapevine and neighboring plants, suggesting that multiple hosts may act as reservoirs for fungal inoculum. These findings highlight the complex interactions between fungal communities, host plants, and environmental factors, underscoring the need for landscape-level approaches to plant protection that account for both cultivated and surrounding ecosystems.