Abstract 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.

BACKGROUND:Fungal endophytes are important members of the holobiont of all plants, including that of Scots pine (Pinus sylvestris), potentially affecting host performance. One of the most important pathogens of Scots pine in Europe is Diplodia sapinea, which causes necrotic lesions and is becoming increasingly prevalent in northern regions. Although endophytes are known to affect plant performance, it remains unclear whether naturally established fungal communities in Scots pine shoots can modulate D. sapinea-induced necrosis. Using a field experiment, we tested the hypothesis that exclusion of airborne fungal inoculum shapes the endophytic community in shoots of pine seedlings, and that such alterations in this community influence the necrosis-inducing capacity of D. sapinea. RESULTS:In the field site, airborne fungal inoculum was reduced in half of the saplings by covering shoots with mesh bags. Covered (bagged) and free (unbagged) shoots were transported to the laboratory and inoculated with D. sapinea. The morphology and physiological status of the shoots were monitored using a multispectral 3D scanner, and the necrotic lesion development was assessed. The propagule exclusion resulted in endophytic communities with slightly lower richness, while shoots showed no detectable morphological or physiological differences prior to inoculation. Shoots inoculated with D. sapinea developed clear necrotic lesions, which were significantly larger in covered shoots than in the free ones. Long-read Oxford Nanopore metabarcoding revealed that community shifts following inoculation were more pronounced in covered shoots. Community composition clearly separated necrotic and healthy tissues. CONCLUSIONS:Our findings suggest that the structure of the resident fungal endophytic community may influence the extent of necrotic lesions caused by D. sapinea in Scots pine shoots. A more established, diverse fungal community was associated with smaller lesion sizes, whereas shoots exposed to lower propagule pressure developed larger lesions following inoculation. These results highlight the functional role of fungal community assembly in shaping disease outcomes and suggest that endophyte-based approaches may provide new opportunities for improving disease resistance in forest tree species. The results also suggest that endophytic status may need to be considered when lesion size is used to evaluate resistance to pathogens in tree breeding programs.

Abstract Ectomycorrhizal (ECM) fungi are well-known for their crucial roles in forest health and productivity, yet their responses to various forest management practices are understudied, particularly in oak-dominated forests. The purpose of this study was to better understand the effects of silvicultural treatments on the diversity and community composition of ECM fungi in an oak-hornbeam forest in northern Hungary. We analyzed ITS2 rDNA metabarcoding data of soil-borne fungi to compare richness and community composition of ECM fungi among forest treatment types (clear-cutting, gap-cutting, preparation-cutting, tree retention in clear-cut areas, and control) and between sampling years (2020 and 2021). We found 268 ECM fungal genotypes, with the most diverse phylogenetic clades being /russula-lactarius (52), /tomentella-thelephora (47), /inocybe (40), /sebacina (27), and /cortinarius (20). We found significant compositional difference of ECM fungi among silvicultural treatments in both years, with some variations in richness. There were also small, but still significant compositional differences between the two years. Treatment effect was partly explained by altered environmental variables, such as relative humidity and soil temperature. These results highlight the importance of forest structure and the abiotic environment in driving community dynamics of plant-symbiotic fungi, with potential implications for forest health and productivity.

Dry hopping, a popular technique in modern craft brewing, introduces non-sterile hop material that may act as a source of microorganisms. Although beer is generally considered microbiologically stable, recent findings indicate that hops can harbour viable fungal and bacterial strains with potential effects on beer quality and hop creep enzymes. We investigated the fungal DNA reservoir of commercial hop pellets and its transfer into beer during dry hopping. Using ITS2 metabarcoding, we characterized fungal communities in hop pellets, pre-hopping beer, and dry-hopped beer, complemented by untargeted volatile profiling (HS-SPME-GCMS). Hop pellets contained diverse fungal assemblages dominated by common foliar endophytes. Several yeast genera of fermentative or spoilage relevance were also detected, including Saccharomyces, Wickerhamomyces, Rhodotorula, and Debaryomyces. While most taxa were found only in hops, four genera (Wickerhamomyces, Vishniacozyma, Bipolaris, and Curvularia) were additionally found in dry-hopped beer but absent from pre-hopping samples, indicating transfer from hops. Metabolomic screening revealed that, besides enrichment of hop-derived metabolites, dry hopping induced shifts in volatile profiles through increases in ethyl esters, higher alcohols, and short-chain fatty acids. Our results demonstrate that commercial hop pellets carry diverse fungal assemblages and that their DNA is detectable in beer after dry hopping, together with aroma shifts consistent with microbial or enzymatic activity.

Grapevine is one of the most valuable crops globally, and environmental factors have long been found to have significant effects on them. Studies on the effects of environmental change on grapevine rootstock have focused on either physiology and physical properties or its influence on microbial communities. Despite growing research, the relationship between scion physiology and leaf microbiome is still largely unexplored. Environmental aspects that alter plant physiology may also influence the plant-associated microbiome, including pathogenic, symbiotic, and commensal fungi that could directly affect plant health. The main objective of this study was to explore the influence of rootstock genotype on the scion berry and leaf mycobiome, nutrient content, and physiological activities. To achieve this, we conducted fungal DNA metabarcoding of berry and leaf samples collected in August and September 2020 from the Nagy-Eged Hill terroir in Northern Hungary from grapevines of the same scion cultivar grafted on three different rootstocks. We statistically compared the composition, richness, and abundance of taxonomic and fungal functional groups among the rootstocks and sampling months. The results illustrated significant differences in the measured physiological parameters, nutrient content, and fungal community composition among rootstocks and months. Of the total of 1,474 fungal amplicon sequence variants identified at the genus level, Alternaria and Aureobasidium were the most prolific. Temporal variation was larger than variation among rootstocks. The interaction between rootstock and month explained the highest proportion of variance for the composition of the fungal community. Regression analyses showed positive relationships between leaf mycobiome composition and physiological parameters, such as transpiration rate and stomatal conductance.Copyright (c) 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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.

The composition, diversity and dynamics of microbial communities associated with grapevines may be influenced by various environmental factors, including terroir, vintage, and season. Among these factors, terroir stands out as a unique possible determinant of the pathobiome, the community of plant-associated pathogens. This study employed high-throughput molecular techniques, including metabarcoding and network analysis, to investigate the compositional dynamics of grapevine fungal pathobiome across three microhabitats (soil, woody tissue, and bark) using the Furmint cultivar. Samples were collected during late winter and late summer in 2020 and 2021, across three distinct terroirs in Hungary’s Tokaj wine region. Of the 123 plant pathogenic genera found, Diplodia, Phaeomoniella, and Fusarium displayed the highest richness in bark, wood, and soil, respectively. Both richness and abundance exhibited significant disparities across microhabitats, with plant pathogenic fungi known to cause grapevine trunk diseases (GTDs) demonstrating highest richness and abundance in wood and bark samples, and non-GTD pathogens prevailed soil. Abundance and richness, however, followed distinct patterns Terroir accounted for a substantial portion of the variance in fungal community composition, ranging from 14.46 to 24.67%. Season and vintage also contributed to the variation, explaining 1.84 to 2.98% and 3.67 to 6.39% of the variance, respectively. Notably, significant compositional differences in fungi between healthy and diseased grapevines were only identified in wood and bark samples. Cooccurrence networks analysis, using both unweighted and weighted metrics, revealed intricate relationships among pathogenic fungal genera. This involved mostly positive associations, potentially suggesting synergism, and a few negative relationships, potentially suggesting antagonistic interactions. In essence, the observed differences among terroirs may stem from environmental filtering due to varied edaphic and mesoclimatic conditions. Temporal weather and vine management practices could explain seasonal and vintage fungal dynamics. This study provides insights into the compositional dynamics of grapevine fungal pathobiome across different microhabitats, terroirs, seasons, and health statuses. The findings emphasize the importance of considering network-based approaches in studying microbial communities and have implications for developing improved viticultural plant health strategies.

This research was started in 2014, led by the Pilis Forestry Systems Experiment (PFSE), a long-term ecological study established in the Pilis Mountains that investigaties the effects of the forestry treatments on forest site, regeneration and multi-taxon biodiversity. We compared the effects of different treatments of rotation and selection

Ectomycorrhizal (EM) fungi are one of the most ecologically and culturally important fungi in temperate forest ecosystems, as root contacts with EM fungi are essential for the survival of most forest tree species. The Pilis Forestry Systems Experiment, ongoing since 2016, compares five different forest management methods (four treatments and control) in terms of their effects on abiotic environmental variables, vegetation and mesofauna. The project presented here provides the first insight in our country on the impact of forest management on the composition of EM fungal communities based on DNA data from soil. A total of 4480 fungal genotype DNA sequences were determined in 30 plots sampled in October 2020, more than half of which could be identified at the genus level or higher. Of these, EM fungi were represented by 534 genotypes from 38 genera. Silvicultural practices affected both diversity and composition of fungal communities. Diversity of EM fungi was reduced in the clear-cut and 20 m gap

The Pilis Forestry Systems Experiment,

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.

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.

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.