The aim of study was to detect the microscopic filamentous fungi from wine surface of sterilized grapes berries of Slovak origin. We analyzed 21 samples of grapes, harvested in the year 2012 of various wine-growing regions. For the isolation of species we used the method of direct plating surface-sterilized berries (using 0.4% freshly pre-pared chlorine) on DRBC (Dichloran Rose Bengal Chloramphenicol agar). The cultivation was carried at 25±1°C, for 5 to 7 days. A total number of 2541 fungal isolates pertaining to 18 genera including Mycelia sterilia were recovered. Isolates of genus Alternaria were found in all of tested samples with the highest relative density 56.4%. The second highest isolation frequency we detected for genus Fusarium (90.48% positive samples), but with low relative density (31 isolates and 2.99% RD). Another genera with higher isolation frequency were Cladosporium (Fr 85.71%, RD 14.6%), Mycelia sterilia (Fr 85.71%, RD 4.25%), Penicillium (Fr 80.95%, RD 13.42%), Botrytis (Fr 71.43%, RD 2.95%) Rhizopus (Fr 66.66%, RD 1.34%), Aspergillus (Fr 57.14%, RD 0.87%), Epicoccum (Fr 47.62%, RD 1.22%), Trichoderma (Fr 42.86%, RD 1.26%). Isolation frequency of another eight genera (Arthrinium, Dichotomophtora, Geotrichum, Harzia, Chaetomium, Mucor, Nigrospora and Phoma) was less than 10% and relative density less than 0.5%. Chosen isolates of potential producers of mycotoxin (species of Alternaria, Aspergillus, Fusarium and Penicillium) were tested for the ability to produce relevant mycotoxins in in vitro conditions using TLC method. None isolate of Aspergillus niger aggregate (13 tested) did not produce ochratoxin A – mycotoxin monitored in wine and another products from grapes berries. Isolates of potentially toxigenic species recovered from the samples were found to produce another mycotoxins: aflatoxin B1, altenuene, alternariol, alternariol monomethylether, citrinin, diacetoxyscirpenol, deoxynivalenol, HT-2 patulin, penitrem A and T-2 toxin in in vitro conditions. In conclusion, another research should be performed to detect the occurrence of these mycotoxins in grapes, must, wine and another products from grape.
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
The current study investigated an endogenous mycobiota of grapes in Eastern wine region, Slovakia and detection a potentially pathogenic isolates to produce selected mycotoxins. Intact berries from four wine grape cultivars were tested. Seven/eight berries superficially sterilized from each samples were placed on a Dichloran Rose Bengal Chloramphenicol agar in a total of 50 and incubated at 25 °C, 5 - 7 days. A total of 582 isolates were obtained that belonged to ten genera: Alternaria, Aspergillus, Botrytis, Cladosporium, Epicoccum, Fusarium, Mucor, Penicillium, Rhizopus, Trichoderma and one unidentified genus Mycelium sterillium without creation fruiting bodies. The most frequent were genera Alternaria, Aspergillus, Botrytis and Penicillium with 100 % frequention. The most frequent Aspergillus species was Aspergillus section Nigri (100 %) and Penicillium species was Penicillium chrysogenum (50 %). The largest number of isolates belonged to Alternaria (275 isolates), Cladosporium (114 isolates) and Penicillium (92 isolates). For that reason the relative density of both genera were the highest 42 %, 19.6 % and 15.8 %, respectively. The selected isolates – Aspergillus flavus, Aspergillus section Nigri, Penicillium citrinum, Penicillium expansum and Penicillium chrysogenum were tested for patulin, citrinin, penitrem A, roquefortin C, ochratoxin A, aflatoxin B1, G1 and cyclopiazonic acid producing ability. Out of 11 strains 54 % produced at least one mycotoxin. In our research ochratoxigenic microfungi (some species of Aspergillus section Nigri) were found in grape samples but without production of OTA.
The aim of this study was to investigate a surface mycobiota of grapes and detection a potentially pathogenic isolates to produce selected mycotoxins. Three samples of wine grapes Furmint, Lipovina and Yellow muscat were collected in the Tokaj wine region in Viničky during the November harvest in 2012. Seven/eight berries from each sample were placed on a Dichloran Rose Bengal Chloramphenicol agar in a total of 50 and incubated at 25 °C for 5 - 7 days. After incubation from the samples were isolated following filamentous fungi: Alternaria, Aspergillus, Botrytis, Cladosporium, Mucor, Penicillium, Phoma, Rhizopus, Trichoderma and Mycelium sterillium. The most frequent were genera Aspergillus, Botrytis and Penicillium with 100 % frequention. The largest number of isolates belonged to Penicillium (368 isolates) and Botrytis (334 isolates). For that reason the relative density of both genera were the highest 47 % and 43 %, respectively. The selected isolates - Aspergillus section Nigri, Penicillium crustosum, Penicillium expansum and Penicillium chrysogenum were tested for patulin, citrinin, penitrem A, roquefortin C and ochratoxin A producing ability. A higher percentage of Penicillium crustosum isolates (79 %) were positive for penitrem A and all were positive for roquefortin C. A higher percentage of Penicillium expansum (83 %) were positive for patulin, 72 % for citrinin and they were all positive for roquefortin C. All isolates of Penicillium chrysogenum produced roquefortin C. As would be expected, ochratoxin A was not detected from Aspergillus section Nigri.
The Southern Slovak wine growing region is warmest part of Slovakia and is suitable for cultivating the grapes for production of wines at high quality. From the eight vineyards were collected 8 samples of wine grapes (white 7, blue 1) during harvesting 2011, 2012 and 2013. The aim of this work was to gain more knowledge about mycobiota on grapes originating from Slovakia, to identify Aspergillus and Penicillium species according to their morphopogy and evaluate the presence of secondary metabolites (also including intracellular and extracellular mycotoxins) produced in in vitro conditions by thin layer chromatography method from fresh grape berries. 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 17 genera. One hundred percent of samples were colonies by the genus Penicillium and 75% by the genus Aspergillus. During the survey, 135 isolates belonging to 9 Penicillium species (P. aurantiogriseum, P. canescens, P. citrinum, P. crustosum, P. decumbens, P. expansum, P. funiculosum, P. chrysogenum and P. purpurogenum) and 26 isolates belonging to 3 Aspergillus species (A. clavatus, A. flavus and A. section Nigri) were isolated and identified from exogenous contamination. The main occurring penicillium species of the samples were P. expansum (37.5% Fr), followed P. citrinum, P. chrysogenum and P. crustosum (25% Fr). The main occurring aspergillus species of the samples were A. section Nigri (62.5%). Eight potentially toxigenic species were tested for their toxigenic ability. It was confirmed the production of various mycotoxins such as aflatoxin B 1 , citrinin, patulin, cyclopiazonic acid, penitrem A and roquefortin C. Out of 34 strains, 56% produced at least one mycotoxin.
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