MYCOBIOTA OF SLOVAK WINE GRAPES WITH EMPHASIS ON ASPERGILLUS AND PENICILLIUM SPECIES IN THE SMALL CARPATHIAN AREA

The Slovak wine-growing region is divided into six viticulture areas. The largest in size and the most important over the centuries has been the Small Carpathian area (around 5800 ha of vineyards) spreads in the western of Slovakia. The objectives of this study were: to gain more knowledge about mycobiota on grapes originating from Slovakia, with a focus on genera Aspergillus and Penicillium and their ability to produce mycotoxins in in vitro conditions by thin layer chromatography method. From the twelve vineyards were collected 14 samples of wine grapes (white 6, blue 8) during harvesting 2011, 2012 and 2013. Fifty wine grapes per bunch (approximately 7-8 berries per plate) that showed no symptoms were randomly selected on Dichloran Rose Bengal Chloramphenicol agar medium. The plates were then incubated aerobically at 25 ±1 °C for 5 to 7 days in the dark. Of these samples were identified 22 genera. Ninety-three percent of samples were colonies by the genus Penicillium and 79% by the genus Aspergillus. During the survey, 251 isolates belonging to 14 Penicillium species (P. aurantiogriseum, P. citrinum, P. coprophylum, P. crustosum, P. expansum, P. funiculosum, P. glabrum, P. griseofulvum, P. chrysogenum, P. oxalicum, P. polonicum, P. purpurogenum, P. roqueforti and P. thomii) and 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 and identified from exogenous contamination. The main occurring penicillium species of the samples were P. chrysogenum (36% Fr), followed P. crustosum (29% Fr), P. griseofulvum (21% Fr) and P. expansum (21% Fr). The main occurring aspergillus species of the samples were A. section Nigri (64%). Thirteen potentially toxigenic species were tested for their toxigenic ability. It was confirmed the production of various mycotoxins such as aflatoxin B1, G1, citrinin, griseofulvin, patulin, cyclopiazonic acid, penitrem A, roquefortin C and sterigmatocystin. Out of 124 strains, 84% produced at least one mycotoxin.


INTRODUCTION
Grapevine can be attacked by a number of fungi and fungus-like organisms which affect the berries and cause loss of quality and influence the taste of the wine (Pitt and Hocking, 2009).Several fungi are pathogenic to grapevines, infecting the roots, trunk, canes, leaves and berries (Pearson and Goheen, 1988).Fungi which commonly infect berries include the mildew pathogens Erysiphe necator and Plasmopara viticola, as well as Alternaria spp., Aspergillus spp., Botrytis cinerea, Cladosporium spp., Penicillium spp., Epicoccum spp.and Rhizopus spp.(Bellí et al., 2004;Sage et al., 2002).During maturation, the spoilage agents, Aspergillus, Botrytis, Penicillium and Rhizopus, increase their incidence.When the temperature is higher than 37 °C, species in Aspergillus section Nigri, usually called "black aspergilli", predominate (Valero et al., 2005).At harvest time the conditions are optimal for fungal invasion, especially if physical damage has occurred on berries.From single infected berries the whole cluster may be affected causing mummified clusters covered with green mould Penicillium expansum.Green mould produce mycotoxins (Abrunhosa et al., 2001;Serra et al., 2006) for example patuline which is however degraded during fermentation and by sulphurization.Berries affected by green mould have an off-flavor and even a small amount of infected berries add a mouldy taste to the wine (Kassemeyer and Berkelmann-Löhnertz, 2009).Samson et al., (2004) considered 15 species provisionally accepted in Aspergillus section Nigri, four of those producing ochratoxin A (OTA) and only two occurring on grapes, raisins and in wine -Aspergillus carbonarius and to a lesser extent A. niger.Ochratoxin A is the main mycotoxin of concern in grape products.OTA is produced primarily when Aspergillus carbonarius infects berries before harvest.The relatively few toxigenic strains of the related species, Aspergillus niger is by far the most common species of Aspergillus present on grapes (Leong et al., 2007).The aflatoxigenic species, Aspergillus flavus and Aspergillus parasiticus, have occasionally been isolated from grapes (Sáez et al., 2004).Toxigenic isolates of Aspergillus ochraceus have also only occasionally been isolated from grapes.Generally, the colonisation of grape bunches by black aspergilli and other fungi occurs when berry skin damage allows the entry into fruit tissues, where the low pH and high sugar content under aerobic Volume 9 502 No. 1/2015 conditions provide a competitive advantage for moulds.However, fungal invasion may occur without visible symptoms (Bellí et al., 2007).
The aim of our study was to detect mycobiota on grapes and species of genera Aspergillus and Penicillium and potentially toxigenic producing species tested by thin layer chromatography for the ability to produce selected mycotoxins in in vitro conditions.

Study area
Twelve vineyards were studied (Modra, Zeleneč, Svätý Martin, Doľany, Dolné Orešany, Dvorníky, Pezinok, Moravany n.Váhom, Gajary, Skalica, Bratislava -Rača) during a 3-year period (2011 -2013) in Small Carpathian wine-growing region.Slovak republic has 6 distinct winegrowing zones (the Small Carpathians, the Southern Slovak, the Nitra, the Central Slovak, the Eastern Slovak and the Tokaj wine regions).They spread from the west to the east of the country along its southern and south-western borders.The largest in size and the most important over the centuries has been the Small Carpathian area (around 5800 ha of vineyards) spreads in the western of Slovakia.The Small Carpathian wine region is divided to 12 subregions.The subregion is the area with the same soil and climate conditions.Wine-growing zones are defined as geographic regions with distinct climatic conditions for grape cultivation.The Small Carpathian wine-growing region has medium climates and abundant moisture.

Samples
Samples were collected from early September to late October, in the maturation stage harvest.Fourteen samples: 6 of white grape varieties (Sauvignon, 2 x Pinot Blanc, 2 x Green Veltliner, Riesling) and 8 of blue grape varieties (2 x Cabernet Sauvignon, 2 x André, 4 x Blue Frankish) were mycologically analyzed.Three kilograms of samples were collected at the time of technological ripeness.Picked grapes were stored at 4 ±1 °C and analyzed within 24 h after harvest.

Mycological analysis of grapes
A total of 50 berries (7 -8 berries per bunch) from each sample were plated in Dichloran Rose Bengal Chloramphenicol agar medium (DRBC) and incubated at 25 ±1 °C in the dark for one week.The spore-producing filamentous fungi detected were identified to genus level based on morphological characters according to the manual of Pitt and Hocking (2009).Different media were used for the taxonomic identification of obtained fungi according to that used for standard strains.Specifically, Penicillium and Aspergillus strains were identified down to the species level first using Malt extract agar (MEA)  (2009).The berries from the vineyards sampled were generally in good condition without visible damage.
The obtained results were evaluated and expressed according to isolation frequency (Fr) and relative density (RD).The isolation frequency (%) is defined as the percentage of samples within which the species or genus occurred at least once.The relative density (%) is defined as the percentage of isolates of the species or genus, occurring in the analyzed sample (Guatam et al., 2009).These values were calculated according to González et al. (1999) as follows: Fr (%) = (ns / N) x 100 ; RD (%) = (ni / Ni) x 100 Where: nsnumber of samples with a species or genus; Ntotal number of samples; ninumber of isolates of a species or genus; Nitotal number of isolated fungi.

Toxinogenity analysis
Toxinogenity of selected isolates was screened in in vitro conditions by means of thin layer chromatography (TLC) according to Samson et al., (2002b), modified by Labuda and Tančinová (2006).Extracellular metabolitescitrinin, patulin, griseofulvin, ochratoxin A, aflatoxin B 1 , G 1 were carried out on YES agar and intracellular roquefortin C, penitrem A, cyclopiazonic acid and sterigmatocystin on CYA agar.A few pieces of mycelium with approximate size 5 x 5 mm were cut from colonies and placed in an Eppendorf tube with 500 µL of chloroform:methanol -2:1 (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 volume 30 µL of liquid phase of extracts along with 10 µL standards (Sigma, Germany) was applied on 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.Identification of the metabolites was done by comparison with metabolite standards.Roquefortin C was visible after spraying with Ce(SO 4 ) 2 x 4 H 2 O as an orange spot.Cyclopiazonic acid was visible directly in daylight after spraying with the Ehrlich reagent as a violet-tailed spot.Penitrem A after spraying with 20% AlCl 3 in 60% ethanol and heating at 130 °C for 8 min as a dark blue spot.Patulin by spraying with 0.5% methylbenzothiazolone hydrochloride (MBTH), (Merck, Germany) in methanol and heating at 130 °C for 8 min and then detectable as a yellow-orange spot.Directly under UV light with a wavelength of 365 nm was visualized citrinin as a yellow-green-tailed spot, griseofulvin as a blue spot, ochratoxin A as a blue-green spot, aflatoxin B 1 as a blue spot, aflatoxin G 1 as a green-blue spot and sterigmatocystin as a reddish spot.
The filamentous fungi identified from samples by the direct plating method are indicated in Table 1.Without surface disinfection, a total of 2774 strains belonging to 20 genera were identified.The three most abundant genera found by descending order were Alternaria (42%), Cladosporium (33%) and Penicillium (9%).Epicoccum, Botrytis, Fusarium, Rhizopus, Trichoderma were detected in more than 1% of the berries analyzed.The remaining 12 genera were detected in less than or equal to 1% of the berries.The genus Alternaria, Cladosporium, Fusarium, Rhizopus, Trichoderma colonised 100% of samples, followed Epicoccum, Penicillium (93%, each) and Aspergillus (79%).In our study was also unidentified Mycelia sterilia without creation fruiting bodies.

RESULTS
The Aspergillus and Penicillium strains were isolated and identified to species level.The isolation rates for Aspergillus from the berries were 79%.The relative densities were low (Table 1).Table 2 shows the number of isolates and isolation frequency (%) of Aspergillus spp.The species of Aspergillus section Nigri were the predominant in mycobiota.The species of A. clavatus and A. flavus were the other most important species recorded with high isolation frequency.
In total 124 isolates representing 13 potentially toxigenic species were tested for their toxigenic ability (Table 4).Out of 124 strains, 84 % produced at least one mycotoxin as revealed by the method used here.Positive toxigenity was detected in A. clavatus, A. parasiticus, P. crustosum and P. chrysogenum.Aspergillus flavus produced aflatoxin B 1 and cyclopiazonic acid (CPA, 2 out of 5 strains screened, each) but did not produce aflatoxin G 1 .Aspergillus ostianus produced sterigmatocystin and did not produce OTA and citrinin.Ochratoxin A production was tested also in 7 strains belonging to Aspergillus section Nigri.Among them, the production of ochratoxin A was not confirmed.Penicillium citrinum produced citrinin (1 out of 2), Penicillium expansum produced roquefortin C (RC), patulin (3 out of 5) and citrinin (two out of 5), P. griseofulvum produced CPA, RC, griseofulvin (12 out of 13) and patulin (10 out of 13).Negative toxigenity was detected in A. versicolor, P. coprophilum and P. roqueforti.

DISCUSSION
The native mycobiota of seven grape varieties (14 samples) grown in Small Carpathian wine-growing region (Slovak Republic) has been studied.We used plating methods without surface disinfection to detect the sporulating fungi colonizing the grape surface.Most of the fungi found are ubiquitously distributed, such as the field fungi Alternaria and Cladosporium, which occur commonly in the air, plant surfaces, debris and soil.Alternaria, Cladosporium and Penicillium occured with high isolation frequency and relative density.Magnoli et al., (2003) showed that Alternaria (80% of the samples), Aspergillus (70%), Cladosporium (40%) and Penicillium Certainly the Aspergillus species are present worldwide, in all the grape products and under all environmental conditions, most frequent in warmer regions and heatgenerating substrates (Somma et al., 2012).Our results agree with this because this genus represented 1% of all the fungi found in the region so the occurrence of Aspergillus spp. in our samples was generally low.Aspergillus section Nigri were the most prevalent, followed by A. clavatus and A. flavus.The isolation frequency of Aspergillus section Nigri in our contaminated samples was 64% and relative density 40% in nondisinfected grapes.From the thirteen samples of wine grapes in Czech Republic, a Slovak neighbouring country, Ostrý et al., (2007) were not found ochratoxigenic microfungi, e. g.Aspergillus carbonarius, and other species of section Nigri, A. ochraceus, Penicillium  Penicillium is a common component of the grapes mycobiota.This genus is ubiquitous saprophyt whose conidia are easily distributed in the atmosphere (Serra et al., 2006).Penicillium is described as being frequent in soils and temperate regions.Penicillium was more frequent than Aspergillus in all our samples.Isolation frequency among Penicillium species was maximum for P. chrysogenum (36%), followed by P. crustosum (29%), P. expansum and P. griseofulvum (21%, each).Cabañes et al., (2002) found Penicillium purpurogenum in all samples of the white Garnacha grape variety that they studied.Their samples were from Tarragona, Spain.Penicillium purpurogenum was isolated in our samples, however in low relative density (1%).Magnoli et al., (2003) found among Penicillium spp.P. chrysogenum, as the most frequent species isolated in 22% of the samples what correspond with our results.The most frequent Penicillium species in grape berries from Portuguese vineyards in four winemaking regions were P. brevicompactum, P. thomii and P. glabrum/spinulosum which together accounted for approximately 71% of the strains identified in the genus (Serra et al., 2006).In our study we isolated them by only in a low density.Penicillium thomii represented 1% of the isolates and P. glabrum 0,4%.The genera Penicillium (present in the range 27 -54%) was predominant in harvest time in grapes from Slovakia, and it was represented by P. brevicompactum, P. chrysogenum, P. crustosum, P. expansum, P. palitans, P. polonicum, P. verrucosum, P. citrinum and P. glabrum (Mikušová et al., 2012).Most of them also were isolated from our samples.It should be noted that despite the differences in geographic location, the varieties studied by the different authors were different as well, which could explain the disagreement of the results found among the samples.
Grapes that are heavily infected with moulds alter in chemical composition and secondary metabolities such as mycotoxins.These mycotoxins of greatest significance in grapes and grape products produced by Aspergillus and Penicillium spp., include ochratoxin A, aflatoxins, patulin and citrinin (Magnoli et al., 2003).Mycotoxins such as aflatoxin, patulin and citrinin are less common than ochratoxin A in grape and grape products.Ochratoxin A is a mycotoxin with nephrotoxic, nephrocarcinogenic, teratogenic and immunosuppressive properties, which has received growing interest from the scientific community and food committees in the last few years (Chulze et al., 2006).Ochratoxin A is a kidney toxin and probable carcinogen (Varga and Kozakiewicz, 2006)  Patulin can occur in many moldy fruits including grapes.Patulin causes gastrointestinal problems, skin rashes, and is known to be mutagenic (Abrunhosa et al., 2001).Patulin has been demonstrated to be acutely toxic (Dailey et al., 1977) The potential producers of patulin were isolated from 23 samples berries, 19 samples of surface-sterilized berries and 6 samples of grape juice.Overall, the representatives of producers of patulin were detected in 32 (68.1%) samples (75 isolates).The ability to produce patulin in in vitro condition was detected in 82% of isolates of Penicillium expansum, 65% of Penicillium griseofuvum and 100% of Aspergillus clavatus.
Penicillium chrysogenum may produce a very wide range of toxic compoundsroquefortine C, meleagrin and penicillin.These metabolites could be considered as a potential hazard to human health (Samson et al., 2002a).We tested 68 strains on roquefortine C from exogenous mycobiota which all were positive.

CONCLUSION
Grapes were analyzed by plating methods from Small Carpathian wine-growing region at the harvest time between 2011 and 2013.From the 4463 strains detected and identified from exogenous mycobiota, the most frequent genera were Alternaria, Cladosporium and Penicillium.Potentially toxigenic Aspergillus and Penicillium species were tested for their toxigenic ability by thin layer chromatography.Out of 124 exogenous strains representing 7 potentially toxigenic species, 84% produced at least one mycotoxin.Potential producers of ochratoxin A Aspergillus section Nigri and roquefortin C Penicillium chrysogenum were the most frequent mycotoxigenic species isolated from grapes.In line with the results on OTA content of Slovak grapes, it appears that the mycotoxin does not present a significant hazard to consumers.

Table 1
Fungi identified in Slovak wine grapes from 2011 to 2013 by the direct plating method.Penicillium were four of the most frequent genera in all the regions.Botrytis, Cladosporium and Penicillium were also reported as the predominant mycobiota by Abrunhosa et al., (2001) in Portugal.In 41 samples of grape fruits grown in Eastern Spain the most infected samples were Cladosporium, Alternaria and Aspergillus section Nigri ( Note:No.number of isolates, Frisolation frequency, RDrelative density.

Table 2
Aspergillus species identified in Slovak wine grapes from 2011 to 2013 by the direct plating method.
Note:No.number of isolates, Frisolation frequency.

Table 3
Penicillium species identified in Slovak wine grapes from 2011 to 2013 by the direct plating method.
Note:No.number of isolates, Frisolation frequency.verrucosum and P. nordicum.Occurrence of Aspergillus spp. in grapes from Slovakia was surveyed during 2 years 2008 and 2009 by Mikušová et al., (2012).A large number of Aspergillus spp., including A. flavus, A. japonicus, A, niger, A. carbonarius and A. ibericus were identified.

.
According to studies ochratoxin A producing strains from the group Aspergillus section Nigri (A. carbonarius and A. niger aggregate) are the source of ochratoxin A in wines, grapes and dried vine fruits (

Hocking et al., 2007). Ochratoxin
A is produced primarily when A. carbonarius infects berries before harvest.Aspergillus niger may also contribute to ochratoxin A contamination.It is by far the most common species of Aspergillus present on grapes (

Chulze et al., 2006).
Toxigenic isolates of A. ochraceus have only occasionally been isolated from grapes (

Abrunhosa et al., 2001).
Penicillium verrucosum and P. nordicum, the only confirmed Penicillium species that are able to produce OTA, were not isolated.Ochratoxin A producers in grapes were isolated in 24.74% in the year 2008 and only one species Aspergillus niger (7.01%) in the year 2009 in South Slovak region by Mikušová et al., (2012).The higher presence of Aspergillus carbonarius (9.68%) was observed only in year 2008.However, their results confirmed a low production of the OTA, what indicates that there is low risk of OTA contamination of Slovak wine.This conclusion agrees with reported survey data, where OTA has been detected in less than 50% of analysed

Table 4
Toxinogenity of selected strains, isolated from exogenous mycobiota of wine grapes.So far, aflatoxin contamination in the grape and wine product chains does not seem to be a real risk for human and animal health.Aflatoxins may occur as common contaminants of dried vine fruits in some countries, i.e.Iran (

Feizy et al., 2012), Egypt (Youssef et al., 2000) and Greece (Kollia et al., 2013) at
very high levels.The strain of A. parasiticus isolate from our study was able to produce in vitro aflatoxins B 1 and G 1 .Other toxigenic species A. flavus produced AFG 1 (2 out of 5) but not produced AFB 1 .Citrinin, a hepato-nephrotoxic compound, also has been detected in grapes before storage (

Bragulat et al., 2008).
It is produced by different species of Penicillium, Aspergillus and Monascus.Citrinin producing strains A. ostianus, P. citrinum and P. expansum were isolated from our Slovak samples.