The V. vinifera phyllosphere is colonized by bacteria and fungi which modulate health, development and quality of grape and the produced wine characteristics (Barata, Malfeito-Ferreira andLoureiro, 2012). The grape surface inhabiting microorganisms are sensitive to environmental changes during wine fermentation and cannot survive in low pH, ethanolic and anaerobic conditions. At the same their metabolic activity on the grape surface can have consequences for wine quality, e.g. metabolic changes produced by phytopathogenic fungi (Hong et al., 2011).

The grape microbiota more often demonstrates the beneficial effect, and the participation of microbiota in wine fermentations can improve the sensory characteristics of wines (Ciani et al., 2010). Studies of microbiota involved in wine fermentations allowed a discovery of microbial species, which show positive enological properties. The application of those microorganisms with Saccharomyces yeasts was commercialized in winemaking (Ciani et al., 2010). With improvement of detection and identification methods in winemaking, a growing number of microorganisms were recognized as active contributors in wine fermentations with significant improvement of sensory qualities of wine (Ciani et al., 2010).

Regional wines characteristics potentially are influenced by microbial biogeography, which is another important factor for winemaking. Traditional winemaking relies mostly on native grape microbiota for fermentations. This practice thought to enhance the regional typicity. The role of microorganisms is well described for grape health, fruit and wine quality (Barata, Malfeito-Ferreira and Loureiro, 2012), but the effect of grape microbiota on regional characteristics of wines is still of limited knowledge. The effect of geographic region, grape variety, and climatic factors influence the bacterial and fungal communities of wine grapes was shown through the growing years (Bokulich et al., 2014). The regional fungal biodiversity of grapes was demonstrated also globally (Taylor et al., 2014; Gayevskiy and Goddard, 2012; Pinto et al., 2015). The local Saccharomyces cerevisiae strains, the principal yeast species for wine fermentations purposes, resulting in distinct wine chemical compositions, thus the role of regional microbiota is important for winemaking (Knight et al., 2015).

Study of microbiota of grape berries was conducted in Slovakia (Kačániová et al., 2018). A total of 33 species of 8 Gram-negative (G^-, 20.72%) and 10 Gram-positive (G⁺, 31.53%) bacteria and 10 yeasts species of 8 genera (47.74%) were identified with MALDI-TOF Mass Spectrometry. These results show that the yeasts were the most common group pf microorganisms isolated from grapes, but the yeasts and bacteria were isolated from each grape variety. Bacteria counts were higher than yeast. The highest counts of yeast species were identified in Irsai Oliver (10.06%), Pálava, Pinot Blanc and Rheinriesling (9.43%) grape varieties (Kačániová et al., 2018).

In our study, the total bacteria counts isolated from different wine grape berries ranged from 2.57 ±0.09 in Chardonnay to 4.39 ±0.21 log cfu.g^-1 in Pálava (Table 1). Microscopic filamentous fungi count ranged from 1.18 ±0.03 in Blue Frankish to 2.60 ±0.17 log cfu.g^-1 in Welschriesling. Kántor et al. (2015) found the bacteria counts on Acetobacter agar (AA) from 1.76 to 2.80 log cfu.mL^-1. The highest counts of acetic acid bacteria on AA agar was found in grape variety Blaufränkisch (2.80 log cfu.mL^-1). Lactic acid bacteria (LAB) counts on MRS agar ranged from 0.48 to 2.06 log cfu.mL^-1, but the LAB was not isolated from white grape varieties.

The highest LAB counts were found in grape variety Cabernet sauvignon of 2.06 log cfu.mL^-1, and the highest counts of LAB were detected in Blaufränkisch grape variety. The yeasts count on Sabouraud dextrose agar (SDA) ranged from 2.47 log cfu.mL^-1 to 2.76 log cfu.mL^-1, and the highest counts were on Blaufränkisch grape variety surface. In generally, limited yeast diversity and low bacteria counts (10 – 10³ CFU.mL^-1) were detected on immature grape berries, but the yeasts count increased to 10⁴ – 10⁶ CFU.mL^-1 as the grapes were ripe enough to harvest. During ripening, the sugars diffuse from the inner tissues of the grape to the surface, that facilitating yeast growth. Unripe grapes mostly harbour Rhodotorula, Cryptococcus and Candida species. These species could be isolated from mature, ripe grapes, however, the apiculate yeasts as Hanseniaspora (anamorph Kloeckera) and Metschnikowia, were mostly distributed. Hanseniaspora (Kloeckera), Candida and Metschnikowia species, as well as species of Saccharomyces and Zygosaccharomyces has increased incidence on the damaged grapes (Fleet, 2003).

In our samples, different bacterial, yeasts and fungal species were found (Table 2). The most abundant microscopic filemanous fungi were: Alternaria sp., Aspergillus niger, Botrytis cnerea, Cladosporium sp. and Penicillium expansum. Alternaria sp. was found in 8 (66.7%) wine grape berries samples. Altogether nine yeast species were isolated: Candida magnoliae, Hanseniospora uvarum, Ignatzschineria indica, Issatchenkia orientalis, Kazachstania exigua, Kluyveromyces marxianus, Metschnikowia pulcherrima, Rhodotorula glutinis and Yarrowia lipolityca. Issatchenkia orientalis was the most abundant yeast, which was found in 10 grapes varieties (83.33%). In total, 14 bacterial species were isolated: Arthrobacter koreeniss, Bacillus endophyticus, B. cereus, B. licheniformis, Escherichia coli, Enterobacter cloacae, Lactobacillus acidophilus, L. paracasei, L. feremntum, Leuconostoc mesenteroides subs. mesenteroides, Micrococcus luteus, Pantotea agglomerans, Staphylococcus epidermidis and Stenotrophomonas maltophilia. The most distributed bacterial species was Leuconostoc mesenteroides subs. mesenteroides, which was isolated from 10 grape berry varieties (83.33%). Figure 4

The microorganisms can contaminate from different environmental sources. The origin of microorganisms could be the vineyard, can be residents of the winery flora, or can be transmitted with insects such as fruit flies and, bees (Fleet et al., 2002). Over twenty yeast species have been identified in wines (Renouf et al., 2007). Hanseniaspora uvarum (anamorph: Kloeckera apiculata), Metschnikowia pulcherrima (anamorph: Candida pulcherrima), and Candida stellata are thought to be the principal yeasts of grapes. In some studies, Hanseniaspora was reported to be the dominant species (Beltran et al., 2002; Combina et al., 2005; Hierro et al., 2006), while Candida assumed to be widespread as well (Clemente-Jimenez et al., 2004). Majority of Candida stellata isolates from wine are actually Candida zemplinina (Csoma and Sipiczki, 2008).

Kačániová et al. (2018) isolated from the surface of grape berries different species of microorganisms. The most abundant G^- bacteria were Stenotrophomonas maltophilia and Ignatzschineria indica. Same results were found in the present study with wine grape berries. Within 22 different species of G⁺ bacteria, Bacillus endophyticus, Paenibacillus glucanolyticus, Paenibacillus lautus and Staphylococcus succinus were the most isolated among bacteria Rhodotorula mucilaginosa was the most abundant among yeasts.

Kunová et al. (2018) found fungal counts ranged from 2.85 log cfu.g^-1 in Cabernet Sauvignon to 4.83 log cfu.g^-1 in Feteasca regala. After identification of 627 isolates of microscopic fungi, moulds belonged to genera Alternaria and Penicillium were the most widespread and were isolated from 100% of samples. Alternaria sp. was the most abundant fungal species in our study also. The high prevalence of Aspergillus (76.92%) and Cladosporium (76.92%) was found (Table 2).

Alternaria, Cladosporium and Penicillium were the most abundant moulds after identification of 1377 cultures of microscopic fungi isolates Felsöciová et al. (2017). The identified prevalence found was similar to our results (100%). The higher prevalence was detected for Fusarium (100%), Epicoccum, Rhizopus (87.5%), Botrytis, Aspergillus (75%) and Mucor (62.5%). Different fungal genera with higher prevalence in comparison with our study were identified. Kántor et al. (2017) found 11 genera of G^- (11%), 11 of G⁺ (27%) bacteria and nine genera of yeasts (62%) among 200 isolates of 19 Slovak grape samples. The most frequently isolated G^- bacteria were Acinetobacter (22%), Pseudomonas (22%) and Sphingomonas (13%). The most common genera of G⁺ bacteria were Bacillus (20%), Lactobacillus (19%), Leuconostoc and Staphylococcus (11%). The most common yeasts genera were Hanseniaspora (37%), Metschnikowia (31%), and Rhodotorula (10%). Our results on diversity of microbial species in grape samples corresponded to Kántor et al. (2017) results.

Similar results were described in Kántor et al. (2016) study, who studied similar grape wine varieties as sampled in our study. The most dominant species was Saccharomyces cerevisiae isolated from all 15 new wine samples, that was a very good wine quality indicator. Altogether, seven different S. cerevisiae strains were identified with mass spectrometry, the second most common species was Kloeckera apiculata (Hanseniaspora uvarum) found in seven new wine samples. Also, other non – Saccharomyces yeasts such as Metschnikowia pulcherrima, Pichia occidentalis and Pichia kluyveri were identified.

Natural microflora of grape berries is very diverse. In our study, the bacteria were the most distributed in comparison with other groups of microorganisms. The highest bacterial counts were found in Palava grape variety, followed by Welschriesling. In our study, 5 different yeast, 9 moulds and 14 bacteria species on grape berries were identified.