Summary: Grapevine ( Vitis vinifera ) is a reservoir of fungal endophytes that may affect its growth, health status and grape production. Although there is growing interest in comparing fungal communities of mainly red grape varieties across various factors using only high‐throughput sequencing, the small‐scale mycobiome variations in geographically close vineyards need further examination. We aimed to characterize the fungal microbiome of the above‐ground tissues of V . vinifera cv. Furmint in different plant parts, seasons and sites using culture‐dependent and culture‐independent methods, and in planta fluorescent microscopic visualization techniques. Samples were collected from four sites of the Tokaj wine region in Mád and two reference sites in Eger, Hungary, across different seasons for 2 years. Fungal endophytes of young and mature leaves, flowers and grape bunches were collected at different phenological stages. Based on each technique, Aureobasidium pullulans , Cladosporium spp. and the complex species Alternaria alternata dominated the community at every site, season and plant organ. We found no significant difference among communities in distinct neighbouring vineyards, nor when compared with the distant reference sites. We can conclude that the different shoot parts of the Furmint grapevines harbour a common core group of fungal community in these regions.

Improving our knowledge on biotic and abiotic factors that influence the composition of the grapevine mycobiome is of great agricultural significance, due to potential effects on plant health, productivity, and wine characteristics. Here, we assessed the influence of scion cultivar on the diversity and composition of fungal communities in the berries and leaves of three different cultivars. We generated DNA metabarcoding data, and statistically compared the richness, relative abundance, and composition of several functional groups of fungi among cultivars, which are partly explained by measured differences in chemical composition of leaves and berries and physiological traits of leaves. Fungal communities in leaves and berries show contrasting patterns among cultivars. The richness and relative abundance of fungal functional groups statistically differ among berry and leaf samples, but less so among cultivars. Community composition of the dominant functional groups of fungi, i.e., plant pathogens in leaves and saprotrophs in berries, differs significantly among cultivars. We also detect cultivar-level differences in the macro- and microelement content of the leaves, and in acidity and sugar concentration of berries. Our findings suggest that there appears to be a relatively diverse set of fungi that make up the grapevine mycobiome at the sampled terroir that spans several cultivars, and that both berry and leaf mycobiomes are likely influenced by the chemical characteristics of berries and leaves, e.g., pH and the availability of nutrients and simple carbohydrates. Finally, the correlation between fungal community composition and physiological variables in leaves is noteworthy, and merits further research to explore causality. Our findings offer novel insights into the microbial dynamics of grapevine considering plant chemistry and physiology, with implications for viticulture.

Alternaria, a cosmopolitan fungal genus is a dominant member of the grapevine (Vitis vinifera) microbiome. Several Alternaria species are known to produce a variety of secondary metabolites, which are particularly relevant to plant protection and food safety in field crops. According to previous findings, the majority of Alternaria species inhabiting grapevine belong to Alternaria sect. Alternaria. However, the phylogenetic diversity and secondary metabolite production of the distinct Alternaria species has remained unclear. In this study, our aim was to examine the genetic and metabolic diversity of endophytic Alternaria isolates associated with the above-ground tissues of the grapevine. Altogether, 270 Alternaria isolates were collected from asymptomatic leaves and grape clusters of different grapevine varieties in the Eger wine region of Hungary. After analyses of the nuclear ribosomal DNA internal transcribed spacer (ITS) and RNA polymerase second largest subunit (rpb2) sequences, 170 isolates were chosen for further analyses. Sequences of the Alternaria major allergen gene (Alt a 1), endopolygalacturonase (endoPG), OPA10-2, and KOG1058 were also included in the phylogenetic analyses. Identification of secondary metabolites and metabolite profiling of the isolates were performed using high-performance liquid chromatography (HPLC)–high-resolution tandem mass spectrometry (HR-MS/MS). The multilocus phylogeny results revealed two distinct groups in grapevine, namely A. alternata and the A. arborescens species complex (AASC). Eight main metabolites were identified in all collected Alternaria isolates, regardless of their affiliation to the species and lineages. Multivariate analyses of untargeted metabolites found no clear separations; however, a partial least squares-discriminant analysis model was able to successfully discriminate between the metabolic datasets from isolates belonging to the AASC and A. alternata. By conducting univariate analysis based on the discriminant ability of the metabolites, we also identified several features exhibiting large and significant variation between A. alternata and the AASC. The separation of these groups may suggest functional differences, which may also play a role in the functioning of the plant microbiome.

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.