Viticulture is an important activity in many countries (Einloft et al., 2017). Vine growing and viticulture have a very long tradition in Slovakia and are parts of the country's cultural and historical heritage. Hundreds of years of viticulture and viniculture have created a specific type of landscape (Bezák et al., 2010), with unique cultural and aesthetic values (Salašová and Štefunová, 2009). In total there exist six viticultural regions in Slovakia with forty areas and wine-growing villages (ÚKSÚP, 2019a). Slovakia features almost 660 producers growing around 13.500 ha of vines (of a potential 15.300 ha) (ÚKSÚP, 2019b) for a production of about 300.000 hL annually, which is primarily sold within the national market.

The microflora of the grapes is highly variable, mostly due to the influence of external factors as environmental parameters, geographical location, grape cultivars, and application of phytochemicals on the vineyards (Pretorius, 2000; Pinto et al., 2014). A variety of fungal genera, mainly Botrytis, Alternaria, Aspergillus, Penicillium, and Cladosporium, can contribute to grape spoilage before harvest (Bellí et al., 2006; Magnoli et al., 2003; Medina et al., 2005). Filamentous fungi impact negatively in the production, sensory quality, and safety characteristics of the wine in several ways. Their development in wine grapes brings significant yield losses for winemaking, alters the chemical composition of wine grapes, and produces secondary fungal metabolites and enzymes that together adversely affect wine flavor and color as well as yeast and lactic acid bacteria growth during vinification (Fleet, 2003). Among them, it is of great concern the presence of toxicogenic fungi in wine grapes capable of producing mycotoxins that could persist during the winemaking process up to wine, being a high risk for consumer's health (Paterson et al., 2018; Prendes et al., 2015).

The genus Alternaria is ubiquitously distributed and includes both saprophytic and opportunistic plant-pathogenic species, which may affect crops in the field or cause harvest and postharvest decay of plant products. Moreover, several Alternaria species are known to produce toxic secondary metabolites, Alternaria mycotoxins. The major Alternaria mycotoxins are the tetramic acid derivate, tenuazonic acid, and the dibenzopyrone derivates, alternariol (AOH), and alternariol monomethyl ether (AME) (Prendes et al., 2015). Despite the toxic effects of the Alternaria toxins and their documented occurrence, they have not yet received the same attention as others mycotoxins and up to now, there is no regulation about them (EFSA, 2011). As an opportunistic pathogen, it has the potential to cause a grape berry rot in the field under high disease pressure situations. Strikingly, Alternaria has not been extensively studied in wine grapes as a hazardous genus.

Penicillium has gained attention as grapevine pathogens. Penicillium expansum can cause rot in grapes, but does not usually attack grapes before harvest. Aside from losses in fruit, this species is regarded as the major producer of patulin, although this species produces many other toxic metabolites such as citrinin, roquefortine C or chaetoglobosins among others (Andersen, Smedsgaard and Frisvad, 2004).

Fifteen fungal genera were identified from the grape samples: Alternaria, Aspergillus, Aureobasidium, Botrytis, Cladosporium, Epicoccum, Fusarium, Mucor, Penicillium, Phoma, Rhizopus, Syncephalastrum, Trichoderma, Trichothecium, and Ulocladium. About 2 % of the isolated fungi did not produce conidiophores or conidia on the tested conditions and were nominated as non-sporulated fungi Mycelia sterilia. The number of isolates within the several genera found on grapes from different varieties are shown in Table 1. The highest number of isolates (from 101 to 199) with 7, 8, or 9 genera were isolated from varieties Müller Thurgau (13), Irsai Oliver (12), Blaufränkisch (3), Palava (4), and Alibernet (1). The lower number of isolates (26, 28, respectively) were isolated from the white variety Rheinriesling (8) with the number of genera 6 and Pinot Blanc (11) with the number of genera 4. It is interesting to note the absence of isolates belonging to microscopic filamentous fungi in one sample Green Veltliner (5). This wine grape was colonized only by yeasts. All samples (except sample 5) were colonized by genera Alternaria and Rhizopus. Genus Alternaria was dominated in samles Palava (4), Cabernet Sauvignon (2) and Welschriesling (9), genus Rhizopus in sample Alibernet (1), genus Penicillium in samples Müller Thurgau (13), Irsai Oliver (12), Blaufränkisch (3) and Welschriesling (9) and genus Cladosporium in samples Irsai Oliver (12) and Sauvignon (10).

Thirty of the 32 Aspergillus isolates were identified as A. section Nigri and 1 isolate as A. ochraceus. Sixteen black aspergilli were isolated from the Blaufränkisch variety (3), 6 from Cabernet Sauvignon (2), 5 from Irsai Oliver (12), 2 from Palava (4), and 1 from Chardonnay (7). Among section Nigri, A. carbonarius is considered the predominant species responsible for the occurrence of OTA in wine grapes and derivatives (Ponsone et al., 2010; Visconti et al., 2008). A low occurrence of this fungus was previously reported in Argentina (Chiotta et al., 2009; Ponsone et al., 2010), Brazilia (Einloft et al., 2017) and Lebanon (El Khoury et al., 2006), and the absence of this fungus was observed in cold regions, like Germany, North Hungary, Czech Republic and Portugal (Abrunhosa et al., 2001; Ostrý et al., 2007; Varga et al., 2005).

Three species of Penicillium were isolated from grapes. Species Penicillium expansum were dominated from the Müller Thurgau variety (13), Blaufränkisch (3), Seteasca Regala (6), Rheinriesling (8), and Palava (4). Penicillium expansum has a high incidence in certain wine regions such as bordering regions of North Portugal and Galiza (Spain) (Serra et al., 2006). The incidence of P. expansum in some wine regions is high, but the attack of this fungus to vineyards, is rare, being B. cinerea the most common disease. Morales et al. (2013) observed that, in vitro, the presence of P. expansum spores enhanced B. cinerea growth, while the latter avoided patulin accumulation.

The data in Table 2 obtained from the cultivation of the berry samples revealed a high diversity of fungal species (a total of 1044 isolates were obtained). Alternaria and Rhizopus were the most frequently occurring genera (92%, each), followed by Cladosporium (85%), Penicillium (77%), Botrytis, and Epicoccum (54%, each). Penicillium spp. was predominant in terms of relative abundance (25%), followed by Alternaria (24%), Cladosporium (20%), Rhizopus (12%), and Botrytis (6%). Besides, a minor portion (<5%) of Aspergillus and other genera was found.

Alternaria genus was the main component of the wine grape mycobiota of the Vrbovský subregion (Small Carpathian wine-growing region) at harvest time, which is in agreement with previous studies carried out in several winemaking regions worldwide, e.g. from Uruguay (Garmendia and Vero, 2016), Argentina (Magnoli et al., 2003; Prendes et al., 2015), Spain (Medina et al., 2005), Slovakia (Felšöciová et al., 2015c; Felšöciová, Mašková and Kačániová, 2018; Felšöciová and Kačániová, 2019a; Felšöciová and Kačániová, 2019b).

It was followed by Penicillium, which recorded a frequency of 77% and a high relative density of 25%. From the previous study by Felšöciová and Kačániová (2019a), Penicillium contributed a small proportion (21% Fr, <1% RD) from mycobiota associated with grapevine in Vrbové. The Botrytis genus, which is regarded as the main spoilage cause in wine grapes, was isolated in this study, but the absence of this genus has already been reported by Magnoli et al. (2003) in Argentina, and Medina et al. (2005) in Spain. Grey mold Botrytis cinerea is responsible for severe economic loss. Musts obtained from botrytized grapes are more liable to oxidation because of the polyphenol oxidizing activity of B. cinerea laccase and are not suitable for wine production (Morales et al., 2013).

Aspergillus was one the less common genera (46% Fr, 3 % of all fungi). These results differ from those obtained by other authors, who reported a much higher frequency from this genus, ranging from 70% to 95% (El Khoury et al., 2008; Magnoli et al., 2003; Medina et al., 2005).

Data in Table 3 show that, 32 Aspergillus species were identified from grape samples. The section Nigri was predominant within the Aspergillus genus, representing 94% of species isolated from this genus with 38% frequency. Certainly, the Aspergillus species are present worldwide, in all the grape products and under all environmental conditions (Somma, Perrone and Logrieco, 2012). From the 12 vineyards in the Small Carpathian area (14 samples), 79% of the samples were coloniezed by the genus Aspergillus (Felšöciová et al., 2015c). During the 3 years survey (2011, 2012, and 2013), 37 isolates belonging to 7 Aspergillus species (A. clavatus, A. flavus, A. section Nigri, A. ostianus, A. parasiticus, A. versicolor and A. westerdijkiae) were isolated. The main occurring Aspergillus species of the samples were A. section Nigri (64%), as in our research. On the other hand, the most species were not been isolated from any of the samples analyzed in the present study.

Data in Table 4 show 3 different Penicillium species from the 264 fungal strains. Penicillium expansum was predominant within the Penicillium species, representing 57% of the isolates and 54% frequency, which agrees with previous publications by Felšöciová and Kačániová (2019a), Felšöciová and Kačániová (2019b). The predominant species of Penicillium isolated from grapes differs between vineyards. For example, Penicillium expansum is the species most frequently isolated in South Slovak wine region (28% RD) (Felšöciová et al., 2017), P. aurantiogriseum in East Slovak wine region (34% RD) (Felšöciová et al., 2015a), P. chrysogenum in Small Carpathian wine region (64% RD) (Felšöciová et al., 2015c), in Nitra wine region (28% RD) (Felšöciová et al., 2013), in Central Slovak wine region (53% RD) (Felšöciová et al., 2014) and Tokaj (39% RD) (Felšöciová et al., 2015b). Penicillium expansum was found frequently in botrytized grapes (Morales-Valle et al., 2011). This species was the second frequent (after P. chrysogenum) in Tokaj (33% RD).

The toxicogenic profile of the 17 Penicillium isolates representing P. crustosum, P. expansum and P. griseofulvum from the Slovak grapes is shown in Table 5.

The 65% of the 17 analyzed Penicillium strains were able to produce at least one of the six mycotoxins tested (citrinin, griseofulvin, patulin, cyclopiazonic acid, penitrem A, and roquefortin C). Citrinin was the toxin produced by the majority of the strains P. expansum (93%). It was followed by patulin produced by 79% of the strains P. expansum, and roquefortin C produced by 64% of the strains. Penicillium crustosum produced only penitrem A, did not produce roquefortin C. Two strains of Penicillium griseofulvum produced griseofulvin and patulin, the production of cyclopiazonic acid and roquefortin C was confirmed by one isolate.

Almost 100% of Penicillium expansum strains are patulin producers (Andersen, Smedsgaard and Frisvad, 2004; Morales et al., 2008), which does not fully correspond to our results. Penicillium expansum is commonly associated with apple rot, production of geosmin − a well‐known compound with a strong earthy smell, and patulin contamination in apple derivatives (Morales-Valle et al., 2011). However, patulin has been reported in grapes (Moake, Padilla‐Zakour and Worobo, 2005), processed grape juice (Scott, Fuleki and Harwig, 1977), and fermenting wine (Majerus, Hain and Kölb, 2008; Bragulat, Abarca and Cabañes, 2008), although the occurrence in wine is low because it is well-known to be degraded partially by the fermentation process (Moss and Long, 2002). Patulin mainly induces gastrointestinal disorders including ulceration, distension, and bleeding. The compound provokes congestion and oedema of pulmonary, hepatic, and gastrointestinal blood vessels and tissues. Subcutaneous injection of patulin produced local sarcomas in rats and is classified in group 3 as not classifiable as to its carcinogenicity to human by IARC (Varga et al., 2015).

Our results indicate a high diversity of fungal species with a high incidence of Alternaria genus. Out of 17 potentially toxigenic Penicillium strains isolated from exogenous mycobiota, namely P. crustosum, P. expansum and P. griseofulvum, 65% produced at least one mycotoxin by thin-layer chromatography method. The occurrence of the potentially toxigenic fungus Aspergillus was overall very low what indicates the high quality of the wine grapes produced in Slovakia.