Mycotoxin-producing Penicillium spp. and other fungi isolated from grapes for wine production in Small Carpathians wine region

The diversity of mycobiota associated with grapevine in Vrbove, Slovakia at the harvest time in 2018 was evaluated. Fourteen samples of grapes were analyzed by plating methods and by plating methods with surface disinfection. The identification of fungi was performed using the morphological and microscopical characteristics. From the 1001 strains detected and identified from exogenous mycobiota, the most frequent genera were Alternaria, Rhizopus and Sordaria. Their relative density was low, except Alternaria. The most frequently encountered moulds and with the highest relative density from endogenous mycobiota were Alternaria, Cladosporium and Penicillium. Most of all genera had relative density less than 1%. Penicillium contributed small proportion in both sources. Penicillium citrinum was the most dominant species in exogenous and endogenous mycobiota. Penicillium expansum and P. glabrum were recorded in exogenous source and P. hordei, P. chrysogenum and P. griseofulvum in endogenous. Potentially toxigenic Penicillium species were tested for their toxigenic ability by thin layer chromatography method. Out of 15 tested isolates representing five potentially toxigenic species 11 produced at least one mycotoxin. Positive toxinogenity was detected in all tested strains of Penicillim citrinum (9/9).


INTRODUCTION
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.

Scientific hypothesis
During maturation of grapes, the spoilage agents, Aspergillus, Penicillium and Rhizopus, increase their incidence.When the temperature is higher then 37 °C, species in Aspergillus section Nigri become predominant (Valero et al., 2007).At harvest time, the conditions are optimal for fungal invasion, especially if physical damage had occurred on grape.Certainly the Aspergillus species are present worldwide, in all the grape products and under all environmental conditions.

Study area
The grapes (Vitis vinifera) used in this study originated from a small family winery founded in 2002.Their goal is to produce quality wines mainly from grapes from their own production.The farm is situated in Vrbovsky subregion, in the village of Vrbove, which is part of the Small Carpathians wine region.In the years 2014 -2016, 10 ha of their own vineyards were planted in Vrbove.Young vineyards are great advantage because they had the opportunity to choose many varieties according to the climate, soil type, but also their own experience so that they could obtain a high-quality raw material for the production of unique wines.They planted old varieties that were grown in Vrbove in the past, for example Green Veltliner, Müller Thurgau, Sauvignon, but also modern varieties such as Pálava, Cabernet Sauvignon and Alibernet.
The Small Carpathians wine region has a medium climate and abundant moisture.The spring of 2018 was an extremely abnormal temperature in Slovakia.April and May were two recordable warm months in Hurbanovo.The average monthly air temperature reached 16.2 °C in April and 19.7 °C in May.Summer was extremely hot with the absence of longer, cooler days.Intense rainfall occurred at the beginning of September (Siman, 2019).

Grape samples
Wine company provided the grapes grown for commercial winemaking.A total of 14 samples were taken: ten from white varieties (Green Veltliner, Seteasca Regala, Chardonnay, Rheinriesling, Welschriesling, Sauvignon, Pálava, Pinot Blanc, Irsai Oliver, Müller Thurgau and the remaining 4 from red varieties: Dornfelder, Blaufränkisch, Alibernet, Cabernet Sauvignon, during the period from the end of August to the beginning of September 2018, in the maturation stages corresponding to harvest.The samples comprised 10 bunches of grapes collected across two diagonal transects.Grape samples were put directly each into a sterile plastic bag.Samples were brought into the laboratory and kept at 5 °C till fungal analysis.

Isolation and identification of fungi
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 detection of fungi in grape samples was also made by plating methods with surface disinfection.The whole 50 grapes were surfacedisinfected in 1% NaClO for 1 min according methods of Magnoli et al. ( 2003).After thorough washing with sterile distilled water (3 times, total amount 1L), and drying were inserted on the agar surface DRBC as a whole fruit and incubated at 25 ±1 °C in the dark for 5 -7 days.
The identification of fungal taxa based on macroscopic and microscopic features, with guidelines by Pitt and Hocking (2009).
Media used for isolation of fungi were: a) Dichloran Rose Bengal Chloramphenicol agar medium (DRBC, MERCK, Germany) to which rose bengal (25 µg.mL -1 ) was used to inhibit rapidly growing fungi and chloramphenicol (100 µg.mL -1 ) was used as bacteriostatic agents.
Penicillium strains were isolated and cultivated on these Extracellular metabolitescitrinin, griseofulvin and patulin were carried out on YES agar and intracellular roquefortine C and cyclopiazonic acid 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, -Carlsbad, CA, USA).

Mycotoxins detection
Mycotoxins extracted from fungal isolates cultures were determined by thin layer chromatographic technique on pre-coated silica gel plate (Alugram ® SIL G, Macherey -Nagel, Germany).The volume 30 µL of liquid phase of extracts along with 10 µL standards (Sigma, Germany) was applied on TLC plate.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.Mycotoxins were identified by comparison with appropriate reference standards of mycotoxins.
Roquefortine C was visible after spraying with Ce(SO4)2 x 4 H2O as an orange spot.Cyclopiazonic acid was visible directly in daylight after spraying with the Ehrlich reagent as a violet-tailed 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 were visualized citrinin as a yellow-green-tailed spot and griseofulvin as a blue spot.

Statisic 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 et al., 2009).These values were calculated according to González et al. (1999) as follows: RD (%) = (ni / Ni) x 100 where ninumber of isolates of a species or genus; Nitotal number of isolated fungi.The isolation frequency (%) is defined as the percentage of samples within which the species or genus occurred at least once.These values were calculated according to González et al. (1999) as follows: Fr (%) = (ns / N) x 100 where: nsnumber of samples with a species or genus, Ntotal number of samples.

RESULTS AND DISCUSSION
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 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 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.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.
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.

CONCLUSION
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.
medias: b) Malt extract agar (MEA), c) Czapek yeast agar (CYA), d) Creatine-Sucrose agar (CREA), e) Yeast Extract agar (YES) (Samson et al., 2010).Genus Penicillium was identified to species level based on morphological characters according to special mycological literature of Pitt and Hocking (2009), Samson and Frisvad (2004) and Samson et al. (2002a, 2010).Mycotoxins analysis A total of 15 different isolates of filamentous fungi from different samples belonging to Penicillium spp.were examined for their ability on mycotoxins production by thin layer chromatography (TLC) according to Samson et al. (2002b), modified by Labuda and Tančinová (2006).

Table 1
Fungi identified in Slovak wine grapes from 2018 by the direct plating method.

Table 2
Fungi identified in Slovak wine grapes from 2018 by the direct plating method with surface disinfection.

Table 3
Toxinogenity of selected Penicillium strains, isolated from exogenous and endogenous mycobiota of wine grapes.
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 frequentPenicillium 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 (

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 (Table1).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.