The grapevine (Vitis vinifera) phyllosphere is colonized by bacteria and fungi that substantially modulate grapevine health, fruit development and ripening, as well as the quality of grapes and wine (Barata et al., 2012; Martiniuk et al., 2023). The microbiome of a grapevine plant has direct and indirect relationships with its host and specific microbial biodiversity linked to a particular vineyard location is reported to be a crucial aspect, in conjunction with edaphic, climatic and human factors, in the concept of wine terroir (Gilbert et al., 2014). The concept of terroir is essential in viticulture since it links the sensory qualities of wine to the environmental conditions in which the grapes are grown (Van Leeuwen and Seguin, 2006).

Several scientific studies have shown that non-random biogeographic distribution patterns of microbial communities of grape surface in vineyards can be modulated by a combination of numerous factors, including geographical location, farming system, soil, cultivar, vintage and climate at various levels (Bokulich et al., 2014; Martins et al., 2014; Knight et al., 2015; Singh et al., 2018). These factors also affect the biology of the vines and the composition of the grapes, which in turn affect the microbiota, and, consequently, the qualitative properties of grapes and wine (Belda et al., 2017). This cycle has given rise to the concept of microbial-wine-terroir, intended as the close correlation between microbes, wine and territories (Gilbert et al., 2014; Knight et al., 2015).

To know the biogeographical distribution of vineyard microbial communities in specific regions, it is important to understand whether the vines themselves select different microorganisms according to their physiological responses, different environmental conditions and viticultural practices (Gilbert et al., 2014; Zarraonaindia et al., 2015; Pacifico et al., 2019; Vitulo et al., 2019). Therefore, in recent years, there has been a surge in the search for regional microbial signatures and microbial biogeography of wine and we now have a greater understanding of microbial diversity among wine-producing regions to begin to understand how this biodiversity can contribute to wine quality and regional characterization (Bokulich et al., 2014, 2016). Native yeasts, residing in a niche site, represent an important component of the microbiota of a vineyard, as they can influence the chemical and sensory profile and the so-called typicality of wine in a unique, reproducible, and identifiable way (Belda et al., 2017). Several studies have shown that the microbiota involved during the early fermentation stages, which is partially determined by endophytic plant-borne yeasts and bacteria, can comply with a well-delineated biogeography reflecting the signatures of different winegrowing regions with a minor influence from the grape variety and vintage year (Barata et al., 2012; Bokulich et al., 2014; Pretorius, 2020; Kamilari et al., 2021).

The diversity of the grape microbiota has long been studied using culture-dependent techniques (Barata et al., 2012; La Hens et al., 2014; Takahashi et al., 2014; Testa et al., 2014; Camilo et al., 2022), which have the disadvantage of being limited by the cultivability of microorganisms (Cocolin et al., 2013). Indeed, although these techniques are widely used for rapid and sensitive profiling of microbial communities, they are unable to detect a significant fraction of the microbial species present on grapes as rare taxa, low-abundance taxa and living but non-viable microorganisms (Bodor et al., 2020). The limitations of the conventional methods have led to the development of high-throughput sequencing (HTS)-based approaches to study the structure of microbial communities (Bleidorn, 2015). These innovative techniques allowed the scientific community to retrieve novel information about microbial communities in all different kinds of environments. As a result, the application of HTS methods for in-depth assessment of the grape and wine microbiome has increased in recent years (Salvetti et al., 2016; Morgan et al., 2017; Dissanayake et al., 2018; Kamilari et al., 2021). In a previous study we already assessed the impact of pedoclimatic conditions on the enological performance of two red cultivars grown throughout Southern Italy (Iorizzo et al., 2023). In this study, we obtained new information by adding other wine-grape samples of two cultivars (Cabernet and Aglianico) to determine relationships between spatial proximity and fungal composition through amplicons sequencing of the internal transcribed spacer (ITS2) region using a HTS approach combined with bioinformatics.

Microorganisms have a key role in the winemaking process as they drive fermentation and are able to influence the aroma and quality of wine (Verginer et al., 2010; Belda et al., 2017; Sherman et al., 2020). Grape berries are one of the main sources of microbial communities that affect the winemaking process, therefore the study of the microbial diversity of wine grapes is of particular interest (Morrison-Whittle and Goddard, 2018). The structure of microbial communities could be, in turn, influenced by several factors such as geographical location, climatic conditions, grape variety, viticultural practices (e.g., fungicides, herbicides, fertilizers) and many others (Bokulich et al., 2014; Pinto et al., 2014; Setati et al., 2015; Grangeteau et al., 2017; Vitulo et al., 2019). As far as wine is concerned, individual vineyards are the smallest scale through which wine diversity can be studied (Knight et al., 2020) and understanding the scale at which microbial communities differ could be important in clarifying the concept of terroir.

Due to the complexity of the microbial population found on grape berries, it is evident that traditional culture-dependent methods have limitations in studying microbial diversity. On the contrary, high-throughput sequencing (HTS), coupled with multivariate data analysis, represents a valid method for studying microorganisms in a variety of contexts. In the present study, we used both HTS and multivariate data analysis to investigate the fungi community structure of grape samples collected in three different vineyards located in Campania, Molise and Sicily regions (Southern Italy) and belonging to two red grape varieties (Aglianico and Cabernet). In general, the descriptive approach through relative abundance analysis confirmed what we already found in our previous study (Iorizzo et al., 2023), but in the present research, new information is derived from the addition of new samples from the Campanian vineyard. Moreover, the statistical approach used furnished new important information on the structure of fungal communities as related to vineyard location and grape variety.

In detail, we found that the main phyla identified on grape berries were represented by Ascomycota, Basidiomycota and Chytridiomycota. In particular, the most abundant phylum in our samples was Ascomycota (∼ 88%), followed by Basidiomycota (∼ 12%). Similarly, Gao et al. (2019) reported that Ascomycota, Basidiomycota and Zygomycota were the main phyla found on the surface of grape berries from six wine regions in Xinjiang - China. In addition, other authors have also found Ascomycota and Basidiomycota as the dominant fungal phyla on grape berries (Morrison-Whittle et al., 2017). Recently, Xu et al. (2022) conducted a study in which they reported that Ascomycota and Basidiomycota were the dominant fungi present on wine-grape surfaces.

Regarding the fungal community structure at the family level, we showed that Cladosporiaceae, Saccotheciaceae, Pleosporaceae, Saccharomycodaceae and Didymellaceae were the most representative families within the phylum Ascomycota, while Sporidiobolaceae, Filobasidiaceae and Bulleribasidiaceae were the most abundant ones among the phylum Basidiomycota. Similarly, in previous studies, other authors reported that Pleosporaceae and several members of Saccotheciaceae and Cladosporiaceae were the dominant fungi in Riesling grape berries (Kecskeméti et al., 2016). Interestingly, stratifying the dataset according to the variable VL, we observed that Cladosporiaceae, Saccotheciaceae, Pleosporaceae, and Filobasidiaceae were present in all samples (n = 12), suggesting that these 4 families represent the core taxa. Similar results were also obtained by Gao et al. (2019) in their work regarding the study of the fungal community structure of grape surfaces. In addition, Wei et al. (2022) reported that the core taxa of Cabernet Sauvignon grapes are retained at the genus level, although the fungi diversity changes during the berry development.

Studying the fungal community structure of grape berries at genus level, on a dataset stratified in accordance with the variable VL, we found that the main genera (about 71% of OTUs) were represented by Cladosporium, Aureobasidium, Alternaria, Stemphylium, and Filobasidium. Similarly, when the fungal community structure of grape berries was studied on a dataset stratified by the variable GV, it was observed that, apart from the genus Stemphylium, all other genera were conserved, indicating that Cladosporium, Aureobasidium, Alternaria, and Filobasidium constitute the core taxon. Some of these genera were also reported as the most common fungal organisms isolated from grapes in other parts of the world. For example, in a study conducted in different regions of Portugal, the authors reported that Cladosporium, Alternaria, and Aureobasidium were the main genera of Alvarinho grapes (Fernandes et al., 2023). In another recent work conducted in the Xinjiang region of China, the authors found that the dominant fungi on nine grape skins variety were represented by Alternaria, Filobasidium, Aureobasidium, Erysiphe and Naganishia (Xu et al., 2022). In Italy, Perpetuini et al. (2022) reported that the common genera of fungi detected in grape samples of Montepulciano d’Abruzzo cultivar (Abruzzo Region, Central Italy) were represented by Hanseniaspora, Aureobasidium, Botrytis, Pichia, Cladosporium and Alternaria. In another work conducted by Perpetuini et al. (2023), it was found that Aureobasidium, Metschnikowia, Hanseniaspora, Botrytis, Cladosporium, Pichia, Alternaria, Epicoccum, Vishniacozyma and Candida were the main genera detected on the minor grapevine cultivar cv. Nero Antico from Chieti (Abruzzo Region, Central Italy). Similarly, Rossetti et al. (2023) studied the fungal community associated with conventional and organic Trebbiano Abruzzese grapes from vineyards of Chieti (Abruzzo Region, Central Italy), and a core taxon of 8 genera was detected (Zygosaccharomyces spp., Cladosporium spp., Botrytis spp., Hanseniaspora spp., Pichia spp., Alternaria spp., Candida spp., Aureobasidium spp.) in all samples investigated.

For a better understanding of the fungal community structure of grape samples and to assess the hypothesis that the fungal population of grape berries differs between samples depending on vineyard location(VL) and/or grape variety (GV), we used several statistical tools based on both univariate and multivariate approaches.

In detail, the alpha diversity values indicated that there were no significant differences (p > 0.05) in terms of richness (Chao1) between the analyzed samples, highlighting that the number of species present in the samples was essentially the same, regardless the VL and the GV (Figure 4). On the other hand, alpha diversity as measured with the Shannon index, evidenced that the VL may have a significant effect on the fungal community composition, as grapes from Molisian vineyard have a greater diversity than grapes from Sicilian one (Figure 4). This result indicates that there is a greater presence of rare species in grape samples from Molisian vineyard than those observed in Sicilian samples.

The effect of VL on the diversity of fungal communities among samples was also supported by the beta diversity measurement. In fact, both PCoA and sPLS-DA analyses showed that samples are mainly grouped according to the VL (Figure 5). These results are consistent with those reported in other studies where it was demonstrated that the diversity of the microbial community is influenced by geographical location of vineyards (Bokulich et al., 2014; Mezzasalma et al., 2017; Kioroglou et al., 2019; Papadopoulou et al., 2022; Kazou et al., 2023). Similar results were also obtained by other authors who have reported that VL has a greater impact on the fungal community than the state of ripeness (Kioroglou et al., 2019).

In the present study, a strong association between some families genera or species and their VL was confirmed by the results obtained using both sPLS-DA and LEfSe. For instance, both sPLS-DA and LEfSe analyses evidenced that C. herbarum, S. roseus and A. methacromatica were associated to Molisian vineyard. Similarly, the Campanian vineyard showed a strong association with the Phaeosphaeriaceae family, while they were weakly associated with Pichia terricola, Zygoascus meyerae and Papiliotrema flavescens. Regarding the Sicilian vineyard, a weak association with Hanseniaspora genus or with Leotiomycetes was observed since the results were not confirmed by both analyses (sPLS-DA and LEfSe).

Apart from the well-known role of Hanseniaspora spp. on the quality of some wines (Lombardi et al., 2018; Testa et al., 2020), the exact functions of core taxa fungi or potential biomarker fungi in wine and winemaking are unknown or poorly investigated for most of the taxa identified in the present work, while some more information, even if sometimes in contrast, is available regarding their role on grapevines. For example, some studies reported that Filobasidium spp. was detected on undamaged Tempranillo grapes, but it was not involved in spontaneous must fermentation (Bougreau et al., 2019). Similarly, Aureobasidium would appear to have no impact on the fermentation process and the wine quality (Barata et al., 2012; Setati et al., 2015; Grangeteau et al., 2017). Also, Alternaria spp. is generally not associated with neither fermentation or spoilage of wine (Barata et al., 2012; Setati et al., 2015; Grangeteau et al., 2017), while Cladosporium genus is considered a spoilage of the winery environment (Barata et al., 2012; Grangeteau et al., 2017). Both Cladosporium spp. and Alternaria spp. are endophytic fungi detected in plants showing symptoms of grapevine trunk diseases (GTDs) although their direct role in the disease was not confirmed (Patanita et al., 2022). In other studies, these genera were detected on grapevines, but no visible symptoms of plant diseases were observed (Varanda et al., 2016). Some authors reported that the presence of Cladosporium genus, in particular C. herbarum, on grape berries, is associated with the Cladosporium rot (Barata et al., 2012). In our study, despite the association of C. herbarum with grape samples from Molise vineyard, no disease symptoms were visible on the grapes or grape plants. This could be attributed to the fact that the induction of disease may depend on many factors ascribable to the concurrent presence of this species with other microorganisms or with specific plant factors (Liu and Howell, 2021). Similar contrasting results were also reported regarding Alternaria spp., as some authors reported the potential role of Alternaria spp. as biological control agent against GTDs-associated fungi and other grapevine pathogens (Musetti et al., 2006; Bruez et al., 2014).

Sporobolomyces spp. have been detected in grape musts as well as at the beginning of wine fermentation (Li et al., 2018). These yeasts are not involved in the alcoholic fermentation, but they could contribute to the production of some metabolites that affect the aroma of wines. For instance, it is known that Sporobolomyces roseus can produce volatile compounds such as higher alcohols, acetate esters and thiols (Verginer et al., 2010). Moreover, there are evidence of its potential role as post-harvest biocontrol agent against Penicillium expansum (Sanzani et al., 2021).

Microbial terroir involves multiple interacting aspects, such as soil and plant-associated microbes and plant-microbe interactions. The present metataxonomic study explored the distribution of fungal communities at vineyard scale considering different vineyards located in Southern Italian regions dedicated to the production of Aglianico and Cabernet wines. Our results, although worthy of further investigation, highlighted the relevance of the vineyard location in the definition of grape-associated fungal communities. Moreover, from the results obtained in the present study, it appeared that the fungal community present on grape berries is more associated with vineyard location than with the grapevine cultivar, allowing to consider some fungal groups as territorial biomarkers regardless of the cultivar analyzed. For instance, in our study, the Phaeosphaeriaceae family was associated to the Campanian vineyard, while Cladosporium herbarum, Sporobolomyces roseus, and Alternaria metachromatica resulted significantly correlated to samples from Molisian vineyard and Leotiomycetes to the Sicilian one, and this for both varieties analyzed. Of course, further studies are needed, as numerous variables such as growing season, soil properties, plant status and other factors may contribute to the definition of fungal communities typical of vineyards located in near geographical areas.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://data.mendeley.com/datasets/tp2rrdz8wp/2, doi: 10.17632/tp2rrdz8wp. https://data.mendeley.com/drafts/6jcyrwj2gh, doi: 10.17632/6jcyrwj2gh.

MI: Conceptualization, Validation, Writing – original draft, Writing – review and editing. DB: Formal analysis, Writing – review and editing. FV: Formal analysis, Writing – review and editing. BT: Formal analysis, Writing – review and editing. PT: Conceptualization, Writing – review and editing. MS: Conceptualization, Writing – original draft, Writing – review and editing. GP: Data curation, Software, Writing – original draft, Writing – review and editing. FL: Formal analysis, Writing – review and editing. GA: Formal analysis, Writing – review and editing. SJL: Formal analysis, Writing – review and editing. RC: Conceptualization, Funding acquisition, Supervision, Validation, Writing – review and editing.