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.
Keywords: grape; filamentous fungi; Penicillium;
mycotoxin; Slovakian vineyard
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., 2006;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 plant-pathogenic 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 METHODOLOGYStudy 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.
Mycological analysis
Fifty berries were selected randomly from each sample (totaling 650 berries) and
placed in Dichloran Rose Bengal Chloramphenicol agar (DRBC) (Samson et al., 2002). Plates were incubated
at 25 ±1 °C for 7 days in darkness. Genera identification was
conducted according to microscopic and macroscopic criteria using the key of
Pitt and Hocking (2009).
Aspergillus strains were isolated and cultivated on MEA
(Malt extract agar) (Samson et al.,
2010), CYA (Czapek yeast extract agar) (Samson et al., 2010), and CY20S (Czapek yeast extract with 20%
sucrose) (Pitt and Hocking, 2009). The
Aspergillus colonies were identified to species level
according to micro and macroscopic criteria, using the keys of Klich (2002) and Pitt and Hocking (2009). Penicillium
strains were isolated and cultivated on MEA, CYA, Creatine-Sucrose agar (CREA)
(Samson et al., 2010) and Yeast
Extract agar (YES) (Samson et al., 2010).
The Penicillium colonies were identified to species level
according to Pitt and Hocking (2009) and
Samson and Frisvad (2004).
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% AlCl3 in 60% ethanol and heating at
130 °C for 8 min as a dark blue spot. Roquefortin C was visible after
spraying with Ce(SO4)2 x 4 H2O 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 González et al. (1999) as follows:
RD (%) = (ni/Ni) x 100
where ni – number of isolates of a species or genus; Ni – total 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 ns – number of samples with a species or genus; N –
total number of samples.
RESULTS AND DISCUSSION
Fifteen fungal genera were identified from the grape samples:
Alternaria, Aspergillus,
Aureobasidium, Botrytis,
Cladosporium, Epicoccum,
Fusarium, Mucor, Penicillium,
Phoma, Rhizopus,
Syncephalastrum, Trichoderma,
Trichothecium, and Ulocladium. About 2 % of
the isolated fungi did not produce conidiophores or conidia on the tested conditions
and were nominated as non-sporulated fungi Mycelia sterilia. The
number of isolates within the several genera found on grapes from different
varieties are shown in Table 1. The highest
number of isolates (from 101 to 199) with 7, 8, or 9 genera were isolated from
varieties Müller Thurgau (13), Irsai Oliver (12), Blaufränkisch (3),
Palava (4), and Alibernet (1). The lower number of isolates (26, 28, respectively)
were isolated from the white variety Rheinriesling (8) with the number of genera 6
and Pinot Blanc (11) with the number of genera 4. It is interesting to note the
absence of isolates belonging to microscopic filamentous fungi in one sample Green
Veltliner (5). This wine grape was colonized only by yeasts. All samples (except
sample 5) were colonized by genera Alternaria and
Rhizopus. Genus Alternaria was dominated in
samles Palava (4), Cabernet Sauvignon (2) and Welschriesling (9), genus
Rhizopus in sample Alibernet (1), genus
Penicillium in samples Müller Thurgau (13), Irsai Oliver
(12), Blaufränkisch (3) and Welschriesling (9) and genus
Cladosporium in samples Irsai Oliver (12) and Sauvignon
(10).
Fungi identified in Slovak wine grapes from exogenous mycobiota in 2019 by
direct plating method.
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 (Ponsone et al., 2010;Visconti et al., 2008). A low occurrence of
this fungus was previously reported in Argentina (Chiotta et al., 2009;Ponsone et al., 2010), Brazilia (Einloft et al., 2017) and Lebanon (El Khoury et al., 2006), and the absence of this
fungus was observed in cold regions, like Germany, North Hungary, Czech Republic and
Portugal (Abrunhosa et al., 2001;Ostrý et al., 2007;Varga et al., 2005).
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.
The data in Table 2 obtained from the
cultivation of the berry samples revealed a high diversity of fungal species (a
total of 1044 isolates were obtained). Alternaria and
Rhizopus were the most frequently occurring genera (92%, each),
followed by Cladosporium (85%), Penicillium (77%),
Botrytis, and Epicoccum (54%, each).
Penicillium spp. was predominant in terms of relative abundance
(25%), followed by Alternaria (24%), Cladosporium
(20%), Rhizopus (12%), and Botrytis (6%). Besides,
a minor portion (<5%) of Aspergillus and other genera was
found.
The occurrence, isolation frequency and relative density of filamentous
microscopic fungi in surface mycobiota of grapes (n = 13) harvested in Small
Carpathian region.
Fungal taxa
No.
Fr (%)
RD (%)
Alternaria
252
92
24
Aspergillus
32
46
3
Aureobasidium
1
8
<1
Botrytis
64
54
6
Cladosporium
206
85
20
Epicoccum
24
54
2
Fusarium
17
31
2
Mucor
17
46
2
Penicillium
264
77
25
Phoma
3
23
<1
Rhizopus
123
92
12
Syncephalastrum
1
8
<1
Trichoderma
11
38
1
Trichothecium
1
8
<1
Ulocladium
3
15
<1
Mycelia sterilia
25
54
2
Total isolates
1044
Note: No – number of isolated micromycetes, Fr – isolation
frequency, RD – relative density.
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 (Garmendia and
Vero, 2016), Argentina (Magnoli et al.,
2003;Prendes et al., 2015), Spain (Medina et al., 2005), Slovakia (Felšöciová et al., 2015c;Felšöciová, Mašková and
Kačániová, 2018;Felšöciová and
Kačániová, 2019a;Felšöciová and
Kačániová, 2019b).
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).
Data in Table 3 show that, 32
Aspergillus species were identified from grape samples. The
section Nigri was predominant within the Aspergillus genus,
representing 94% of species isolated from this genus with 38% frequency. Certainly,
the Aspergillus species are present worldwide, in all the grape
products and under all environmental conditions (Somma, Perrone and Logrieco, 2012). From the 12 vineyards in the Small
Carpathian area (14 samples), 79% of the samples were coloniezed by the genus
Aspergillus (Felšöciová et al., 2015c). During the 3 years survey
(2011, 2012, and 2013), 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. The main occurring
Aspergillus species of the samples were A.
section Nigri (64%), as in our research. On the other hand, the
most species were not been isolated from any of the samples analyzed in the present
study.
The occurrence, isolation frequency and relative density of
Aspergillus species in surface mycobiota of grapes (n =
13) harvested in Small Carpathian region.
Aspergillus species
No.
Fr (%)
RD (%)
A. ochraceus
1
8
3
A. section
Nigri
30
38
94
A. sp.
1
8
3
Total isolates
32
Note: No. – number of isolates, Fr – isolation frequency,
RD – relative density.
Data in Table 4 show 3 different
Penicillium species from the 264 fungal strains.
Penicillium expansum was predominant within the
Penicillium species, representing 57% of the isolates and 54%
frequency, which agrees with previous publications by Felšöciová and Kačániová
(2019a), Felšöciová and
Kačániová (2019b). The predominant species of
Penicillium isolated from grapes differs between vineyards. For
example, Penicillium expansum is the species most frequently
isolated in South Slovak wine region (28% RD) (Felšöciová et al., 2017), P.
aurantiogriseum in East Slovak wine region (34% RD) (Felšöciová et al., 2015a),
P. chrysogenum in Small Carpathian wine region (64% RD) (Felšöciová et al., 2015c), in
Nitra wine region (28% RD) (Felšöciová et al., 2013), in Central Slovak wine region
(53% RD) (Felšöciová et al.,
2014) and Tokaj (39% RD) (Felšöciová et al., 2015b). Penicillium
expansum was found frequently in botrytized grapes (Morales-Valle et al., 2011). This species was
the second frequent (after P. chrysogenum) in Tokaj (33% RD).
The occurrence, isolation frequency and relative density of
Penicillium species in surface mycobiota of grapes (n =
13) harvested in Small Carpathian region.
Penicillium species
No.
Fr (%)
RD (%)
P. crustosum
1
8
<1
P. expansum
150
54
57
P. griseofulvum
10
8
4
P. sp.
103
54
39
Total isolates
264
Note: No. – number of isolates, Fr – isolation frequency,
RD – relative density.
The toxicogenic profile of the 17 Penicillium isolates representing
P. crustosum, P. expansum and P.
griseofulvum from the Slovak grapes is shown in Table 5.
Toxinogenity of selected Penicillium strains, isolated from
exogenous mycobiota of wine grapes.
Species
C
G
P
CA
PA
RC
P. crustosum
1/1
0*/1**
P. expansum
13/14
11/14
9/14
P. griseofulvum
2/2
2/2
1/2
1/2
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.
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% of Penicillium expansum strains are patulin producers
(Andersen, Smedsgaard and Frisvad, 2004;Morales et al., 2008), which does not fully
correspond to our results. Penicillium expansum is commonly
associated with apple rot, production of geosmin − a well‐known compound with
a strong earthy smell, and patulin contamination in apple derivatives (Morales-Valle et al., 2011). However, patulin
has been reported in grapes (Moake, Padilla‐Zakour
and Worobo, 2005), processed grape juice (Scott, Fuleki and Harwig, 1977), and fermenting wine (Majerus, Hain and Kölb, 2008;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.
Acknowledgments:
This work has been supported by grants of the European Community of project No.
26220220180: Building Research Centre “AgroBioTech” and of KEGA
015SPU-4/2018.
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