SURVEY OF MYCOBIOTA ON SLOVAKIAN WINE GRAPES FROM SMALL CARPATHIANS WINE REGION

A total of 13 samples of grapes (bunches) without apparent fungal contamination were analyzed. The samples were collected during the 2019 harvest from Vrbové village in the Small Carpathian region of Slovakia. For the isolation of fungi were used the direct plating technique on DRBC plates. The plates were incubated aerobically at 25 ±1 °C for one week in the dark. The data obtained from the cultivation of the grape berry samples revealed a high diversity of fungal species (a total of 1044 isolates were obtained). Alternaria and Rhizopus were the main components of the wine grape mycobiota of the Vrbovský subregion at harvest time (92%, each), followed by Cladosporium (85%), Penicillium (77%), Botrytis and Epicoccum (54%, each). The most abundant genera found by descending order were Penicillium (25%), Alternaria (24%), Cladosporium (20%), and Rhizopus (12%) and only in minor percentage by Aspergillus (3%) among others. The main fungal species isolated from genera Penicillium and Aspergillus were Penicillium expansum (57% RD) and A. section Nigri (97% RD). Of 17 analyzed Penicillium strains, 65% were able to produce at least one of the six mycotoxins analyzed in in vitro conditions by means of thin-layer chromatography method: citrinin, griseofulvin, patulin, cyclopiazonic acid, penitrem A, and roquefortin C.


INTRODUCTION
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., 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 plantpathogenic 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).

Scientific hypothesis
Some of the fungal species occurring on grapes and grape products can produce mycotoxins, so species identification is critical to predicting the potential mycotoxin contamination of grapes and wine.

MATERIAL AND METHODOLOGY Study area
Village Vrbové is located in the Vrbovský subregion in the Small Carpathian wine region. The Small Carpathian wine region is the most extensive of the six wine regions in Slovakia (vineyards are covering 4175 hectares) and is located in the southwestern part of Slovakia (ÚKSÚP, 2019b). Vines have been grown on the south-facing slopes of the Small Carpathian mountains in locality Záhorie for more than three thousand years. This region has a medium climate and abundant moisture.
Last year, as a whole, was extremely warm. The year 2019 had the same average annual temperature in Hurbanovo 12.42 °C as in 2018. This value is a record high for Hurbanovo since the record began. During the whole year, 2019 was only one month of the territory temperature below normal. It was May (Beránek and Faško, 2020).

Grape sampling
A total of 13 samples were taken: 3 from red varieties (Alibernet, Cabernet Sauvignon, and Blaufränkisch) and 10 from white varieties (Palava, Green Veltliner, Seteasca Regala, Chardonnay, Rheinriesling, Welschriesling, Sauvignon, Pinot Blanc, Irsai Oliver, and Müller Thurgau). The sampling was conducted at the 2019 vintage, at the end of September. Two diagonals crossing the vineyards were delimited, and five healthy and undamaged bunches from each diagonal were obtained. Each bunch was collected in a sterilized plastic bag and sent to the laboratory chilled on ice.

Mycotoxin production
Toxinogenity of selected isolates was screened in in vitro conditions by means of thin-layer chromatography (TLC) according to Samson et al. (2002), modified by Labuda and Tančinová (2006). Extracellular metabolites -citrinin griseofulvin and patulin were carried out on YES agar and intracellular cyclopiazonic acid, penitrem A, and roquefortin C on CYA agar. At 14 days of incubation, five agar plugs (4 mm diameter) were cut from the edge of a colony (extracellular metabolites) or cut from a colony (intracellular metabolites) from each Petri plate and placed in an Eppendorf tube. The plags were extracted in 500 µL of chloroform-methanol (2:1, v/v) (Reachem, Slovak Republic). The content of the tubes was stirred for 5 min by Vortex Genie ® 2 (MO BIO Laboratories, Inc. -Carlsbad, CA, USA). The extract of liquid phase 30 µL along with 10 µL of standards (Sigma, Germany) was transferred to the TLC plate (Alugram ® SIL G, Macherey -Nagel, Germany). The plate was put into TEF solvent (toluene:ethyl acetate:formic acid -5:4:1, toluene -Mikrochem, Slovak Republic; ethyl acetate and formic acid -Slavus, Slovak Republic). After elution, the plate was air-dried. The identification of the metabolites was done by comparison with metabolite standards. Cyclopiazonic acid was visible directly in daylight after spraying with the Ehrlich reagent as a violet-tailed spot. Penitrem A was visible after spraying with 20% AlCl 3 in 60% ethanol and heating at 130 °C for 8 min as a dark blue spot. Roquefortin C was visible after spraying with Ce(SO 4 ) 2 x 4 H 2 O as an orange spot. Patulin detection was achieved by spraying with 0.5% methylbenzothiazolone hydrochloride (MBTH) (Merck, Germany) in methanol and heating at 130 °C for 8 min and then detected as a yellow-orange spot under visible light. Citrinin was detected directly as an intense yellow-green streak under ultraviolet light (365 nm) as well as griseofulvin, which was visible as a blue spot.

Statistical analysis
The obtained results were evaluated and expressed according to relative density (RD) and isolation frequency (Fr). The relative density (%) is defined as the percentage of isolates of the species or genus, occurring in the analyzed sample (Guatam, Sharma and Bhadauria, 2009). These values were calculated according to Fr (%) = (ns/N) x 100; where ns -number of samples with a species or genus; N -total number of samples.
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 ( 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.
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 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).
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%  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).

CONCLUSION
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.   Note: * -number of isolates with ability to produce mycotoxin, ** -number of tested isolates, C -citrinin, G -griseofulvin, P -patulin, CA -cyclopiazonic acid, PA -penitrem A, RC -roquefortin C.