Fresh grapes are prone to fungal contamination in the fields, during harvesting, transporting, marketing and during storage under domestic conditions. If the spoiling fungi are toxigenic or pathogenic, they could pose a health risk for the consumer. Grape fruit contain high levels of sugars and other nutrients, and they possess and ideal water activity for microbial growth, their low pH makes them particularly susceptible to fungal spoilage (Tournas and Katsoudas, 2005).

The most frequent filamentous fungi found in grapes were species of Cladosporium, Penicillium, Botrytis, Alternaria and Aspergillus (Serra et al., 2005; Fredj et al., 2007). The grape mycrobiota may change in response to various factors such as: the climate, grape variety and geographical region (Raspor et al., 2006).

Penicillium species are one of the most common fungi occurring in a diverse range of habitats, from soil to vegetation, air, indoor environments and various food products (Visagie et al., 2014). Species of Penicillium are economically, ecologically and medically important microorganisms playing vital roles in natural ecosystems, agriculture and biotechnology (Visagie, 2008). Many species of the Penicillium are among the common postharvest pathogens on a wide range of fruits and vegetables. Penicillium expansum, the main cause of green (blue) mold of apple, pear and fruits of other deciduous trees, is an example of destructive pathogens that causes much of post-harvest economic losses on food during storage and marketing stages (Barkai-Golan, 2008). Two main genera are responsible for mycotoxin production in grapes: Aspergillus and Penicillium. The mycotoxin production is characteristic of the species and therefore by identifying the species one can predict potential mycotoxin hazards (Serra et al., 2006). Green mould produces mycotoxins for example patuline which is however degraded during fermentation and by sulphurization. Berries affected by green mould have an off-flavour and even a small amount of infected berries add a mouldy taste to the wine (Kassemeyer and Berkelmann-Löhnertz, 2009). Ochratoxin A (OTA) producing fungi are members of the genera Aspergillus and Penicillium. Natural occurrence of OTA has been reported from temperate to tropical climates mainly on cereals and their products, beverages such as bread, beer, coffee, dried fruits, grape juice and wine among others. Only some OTA producing species are known to be a potential source of OTA contamination of these commodities, including A. niger aggregate, A. carbonarius, A. ochraceus, A, westerdijkiae, A. steynii, Penicillium verrucosum and P. nordicum (Cabañes et al., 2010).

In our work the goal was to assess the fungi present on the surface and inside of healthy grapes destined for commercial winemaking at harvest time. Emphasis was given to Penicillium species due to their relevance for mycotoxin production.

A survey study was conducted on 14 samples of fresh grape which collected from Vrbove, Slovakia. Isolation of fungi contaminated fresh grape resulted in collecting of 1001 fungal isolates. Data in Table 1 show that, eleven fungal genera namely Alternaria, Aspergillus, Aureobasidium, Botrytis, Cladosporium, Epicoccum, Mucor, Penicillium, Rhizopus, Sordaria, Trichoderma and Mycelia sterilia (unidentified fungus without creation fruiting bodies) were identified from fresh grape samples.

Data in the same Table 1 showed that, Alternaria was the most frequently occurring genus (100 %) which record 868 isolates, with the highest relative density 87%. Rhizopus was the second predominant genus which recorded frequency of 93%, but low relative density (5%), followed by Sordaria (71% Fr, 1% RD). Botrytis, Epicoccum, Penicillium, Cladosporium, Aspergillus, Mucor Aureobasidium and Trichoderma were less fungal frequency occurred in all grape samples.

Khashaba et al. (2018) reported that prevalence of Alternaria was moderate fungal frequency (50%) and Aspergillus was the most frequently occurring genus (100%) in Egypt. Penicillium was the second predominant genus which recorded frequency of 92.5%. In our study Aspergillus and Penicillium were less fungal frequency occurred in all grape samples. Similar results that Aspergillus is the main fungal genus were obtained by several studies (Alisa et al., 2007; Fredj et al., 2007). Alternaria, Botrytis and Cladosporium were three of the most frequent genera in all four winemaking regions in Portuguese (Serra et al., 2006), representing 16, 17 and 24% of the total identified strains, respectively. According to the region considered, other frequent fungi were Aureobasidium pullulans, Aspergillus niger, Epicoccum nigrum, Penicillium brevicompactum, P. thomii and Rhizopus. Aspergillus and Penicillium were also an important part of the mycobiota representing 15 and 24%, respectively, of all the fungi found in the regions. Aspergillus was more frequent than Penicillium in almost all regions. In our samples Botrytis represented 2% of the total identified strains and Cladosporium less than 1%, but Alternaria was the most frequent genus, too. Serra et al. (2006) described that the most frequent Penicillium species were P. brevicompactum, P. thomii and P. glabrum which together accounted for approximately 71% of the strains identified in the genus. Five species of Penicillium including P. brevicompactum, P. citrinum, P. echinulatum, P. expansum and P. solitum reported from grapes in Korea (Won et al., 2007). Serra and Peterson (2007) described two new species of Penicillium, namely, P. astrolabium and P. neocrassum from contaminated grapes in Portugal. The reports on Penicillium species occurring on grape and raisin from Iran are very rare (Rahmani et al., 2012; Maulani et al., 2012), mainly P. expansum, P. brevicompactum and P. glabrum (Houbraken et al., 2014). Five species represent new records for the mycobiota of Iran are Penicillium crocicola, P. olsonii, P. sumatrense, Talaromyces atroroseus and T. minioluteus (Khodaei et al., 2016). During our survey, five isolates belonging to three Penicillium species (P. citrinum, P. expansum and P. glabrum) were isolated and identified from exogenous colonisation (Table 1). Their occurrence was very sporadically. On the other hand, Penicillium were between the most common species identified in grape from Small Carpathian area during harvesting 2011 to 2013 (Felšöciová et al., 2015). From 13 different Penicillium species with high frequency (93%) were 4 main species: P. chrysogenum, P. crustosum, P. expansum and P. griseofulvum. Penicillium citrinum was less common species identified in grape samples.

By the endogenous (surface-disinfected) plating method were identified nine different genera from the 509 fungal strains (Table 2): Alternaria, Aspergillus, Botrytis, Cladosporium, Epicoccum, Fusarium, Penicillium, Rhizopus, Trichoderma and Mycelia sterilia. The three most abundant genera found by descending order and with the highest relative density were Alternaria, Cladosporium and Penicillium. Most of all genera had relative density less than 1%.

Most of the fungi found are ubiquitously distributed, such as the field fungi Alternaria, Cladosporium and Epicoccum, which occur commonly in the air, plant surfaces, debris and soil (Serra et al., 2006). Fungal species capable of causing rot in grapes (e.g. Aspergillus niger, Botrytis cinerea, Rhizopus) were not common inhabitants of the berry surface.

Four Penicillium species were found: Penicillium citrinum, P. griseofulvum, P. hordei and P. chrysogenum (Table 2). According Felšöciová et al. (2013) some similar species but in lower number of abundances were isolated from the Nitra wine growing region in 10 analysed samples, namely P. citrinum, P. corylophilum, P. crustosum, P. decumbens, P. expansum, P. chrysogenum and Penicillium spp. The occurrence of most was also very sporadically, except P. crustosum, P. chrysogenum and P. expansum. Nine Penicillium species (P. canescens, P. citrinum, P. crustosum, P. expansum, P. funiculosum, P. glabrum, P. griseofulvum, P. chrysogenum and P. variabile) were found from grapes grown in the Central Slovak wine region, but there occurrence was also very sporadically except two P. expansum and P. chrysogenum (Felšöciová et al., 2015).

As shown in Table 3, thin layer chromatographic analysis of 15 tested isolates representing 5 potentially toxigenic species showed that 11/15 (73%) produced at least one mycotoxin. Positive toxigenity was detected in P. citrinum (9 out of 9 strains screened). Penicillium expansum (Figure 1) produced only citrinin, did not produce patulin and roquefortin C, Penicillium griseofulvum produced griseofulvin and patulin, the production of cyclopiazonic acid and roquefortin C was not confirmed. Negative toxigenity were detected in Penicillium hordei and P. chrysogenum (Figure 2) on roquefortin C.

Penicillium spp. in our samples was generally low (3% RD). On the other hand, Penicillium was a common component of the grapes mycobiota from 2011 to 2013 in the Small Carpathian area (Felšöciová et al., 2015). Ninety three percent of samples were colonies by the genus Penicillium. During the survey, 251 isolates belonged to 14 Penicillium species. Out of 124 strains, 84% produced at least one mycotoxin by TLC method. The most frequent was P. chrysogenum. Interesting was, that almost all tested strains on roquefortin C were producted (100 out of 102). No mycotoxin (RC) was formed by the examined four species (P. expansum, P. griseofulvum, P. hordei and P. chrysogenum) isolated from our grape samples.

The present work indicated that the examined grape fruits were contaminated with several fungi especially members of Alternaria. Members of Aspergillus and Penicillium, fungi capable of producing mycotoxins such as aflatoxins and ochratoxin A, were very rare. These findings indicate that strict hygiene microbiological must be applied during different stages of harvest, transport, storage and handling to avoid the harmful effects on human health.