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Potravinarstvo Slovak Journal of Food Sciences Volume 11 403 No. 1 Potravinarstvo Slovak Journal of Food Sciences Potravinarstvo Slovak Journal of Food Sciences vol. 11, 2017, no. 1, p. 403-409 doi: https://dx.doi.org/10.5219/763 Received: 20 February 2017. Accepted: 15 May 2017. Available online: 29 June 2017 at www.potravinarstvo.com © 2017 Potravinarstvo Slovak Journal of Food Sciences, License: CC BY 3.0 ISSN 1337-0960 (online) SPECIES OF GENERA BOTRYTIS, FUSARIUM AND RHIZOPUS ON GRAPES OF THE SLOVAK ORIGIN Dana Tančinová, Zuzana Mašková, Ľubormír Rybárik, Viera Michalová ABSTRACT Our research was focused to identify the Botrytis, Fusarium and Rhizopus species from grapes of the Slovak origin. A further goal of the project was to characterized toxinogenic potential of chosen strains of species Fusarium. 50 samples of grapes, harvested in years 2011, 2012 and 2013 from various wine-growing regions were analyzed in this study. For the isolation of species the of direct plating method was used: a) surface-sterilized berries (using 1% freshly pre-pared chlorine) b) berries and c) damaged berries on DRBC (Dichloran Rose Bengal Chloramphenicol agar). For each analysis were used 50 berries (or all damaged berries from sample). The cultivation was carried at 25 ±1°C, for 5 to 7 days in dark. After incubation, the colonies of Botrytis, Fusarium and Rhizopus were transferred to identification media and after incubation strains were identified to species level. Thirteen species of fusaria (F. acuminatum, F. avenaceum, F. culmorum, F. equiseti, F. graminearum, F. oxysporum, F. proliferatum, F. semitectum, F. solani, F. sporotrichioides, F. subglutinans F. tricinctum and F. verticilioides) were identified. Frequency of fusaria isolation was 92 %. Botrytis cinerea was determined from 86% samples and Rhizopus from 94%. Chosen strains of species of genus Fusarium were able to produce following mycotoxins: deoxynivalenol, T-2 toxin, HT-2 toxin and diacetoxyscirpenol in in vitro conditions as determinated by thin-layer chromatography. Thirty-two (68%) of tested isolates of Fusarium species were able to produce at least one mycotoxin. Keywords: Botrytris; Fusarium; Rhizopus; grapes; trichothecenes INTRODUCTION this ciccumstance called “the noble rot”, are used int the Grapes have a complex microbial ecology including production of certain high quality sweet wines in France, filamentous fungi, yeasts and bacteria with different Germatny, Australia and other countries (Pitt and physiological characteristics and effects upon wine Hocking, 2009). production (Barata et al., 2012). Moulds commonly Rhizopus stolonifer is one of the most common and isolated from grapes are Alternaria, Cladosporium and fastest-growing species in the Zygomycota phylum. Botrytis cinerea, the latter causing bunch rot. Pathogenic Disease caused by this fungus is known as soft rot, black and opportunistic species of Fusarium, Penicillium and mould and Rhizopus rot (Bautista-Baños et al., 2014). Aspergillus can also colonize inducing grape disease Rhizopus rot is common on soft fruits, more abundant in (Oliveri and Catara, 2011). warm, humid climates than in cool climate viticulture. In The concern about filamentous fungi in the vineyasrd has several fruits and crops such as strawberry, tomato, traditionally been linked to spoilage of grapes due to cucumber and table grapes Rhizopus rot causes soft rot fungal growth. The main fungus responsible for grape rot during transport and storage (Kassemeyer and is Botrytis cinerea, a pathogen that damages the berries Berkelmann-Löhnertz, 2009). and had a detrimental effect on the organoleptic properties Fusarium species are renowned for their role as plant (Serra et al., 2006). Botrytis is a common genus in the pathogens, causing a wide renage of diseases such as temperate zones, where it occurs mainly as a pathogen on vascular wilts, root and stem rots, pre- and post-emergence a variety of plant crops. Vegetable and small berry fruits blight and many others (Pitt and Hocking, 2009). are particularly susceptible. Invasion may occur before Our research was focused to identify the Botrytis, maturity or postharvest, both in transport and in storage. Fusarium and Rhizopus species from grapes of the Slovak Onions and other allium species and grapes are the most origin. A further goal of the project was to identify susceptible crops. In the latter, it is notable that the disease toxinogenic potential of chosen strains of species is sometimes encouraged. Grapes affected by Botrytis, in Fusarium. Volume 11 403 No. 1/2017 Potravinarstvo Slovak Journal of Food Sciences MATERIAL AND METHODOLOGY into 6 viticulturally regions, which are divided into 40 Samples wine area and 690 wine-growing villages. The samples Fifty samples of wine grapes, harvested in years 2011, which were analysed were taken from all viticulturally 2012 and 2013 from various wine-growing regions of regions (Table 1). Slovakia, from small and medium-sized vineyards were White and red grape varieties were analysed. White analysed. grape: Chardonnay (4), Grüner Veltliner (5), Müller Slovak wine region is characterized as a territory of the Thurgau (1), Riesling (3), Velsch Riesling (7), Pálava (1), Slovak Republic, where grapes are grown. It is divided Pinot blanc (2), Pinot gris (2), Sauvignon (2), Tramin (2), Zala gyöngye (1) and grape using for special Tokay vine Table 1 List of analysed samples of grapes and their origin. Number Town or willage Wine-growing area Vineyard region Variety of sample 1. Báb Šintavský Nitra Chardonnay 2. Nitra Nitriansky Nitra Velsch Riesling 3. Oponice Radošinský Nitra Chardonnay 4. Beladice Zlatomoravecký Nitra Velsch Riesling 5. Vinodol Vrábeľský Nitra Chardonnay 6. Komjatice Žitavský Nitra Riesling 7. Čaka Želiezovský Nitra Velsch Riesling 8. Nová Dedina Tekovský Nitra Grüner Veltliner 9. Brhlovce Pukanecký Nitra Pinot Blanc 10. Hontianske Moravce Hontiansky Central Slovak Konkordia 11. Gbelce Strekovský Southern Slovak Velsch Riesling 12. Mužla Štúrovský Southern Slovak Velsch Riesling 13. Pribeta Hurbanovský Southern Slovak Riesling 14. Veľký Krtíš Modrokamenský Central Slovak Pinot Gris 15. Veľký Krtíš Modrokamenský Central Slovak Pinot Noir 16. Veľký Krtíš Modrokamenský Central Slovak Sauvignon 17. Modra Modranský Small Carpathian Pinor Blanc 18. Zeleneč Trnavský Small Carpathian Cabernet Sauvignon 19. Báb Šintavský Nitra Tramin 20. Báb Šintavský Nitra Blaufrankise 21. Svätý Martin Senecký Small Carpathian André 22. Doľany Dolanský Small Carpathian Pinot Noir 23. Doľné Orešany Orešanský Small Carpathian Blaufrankise 24. Dvorníky Hlohovecký Small Carpathian Sauvignon 25. Pezinok Pezinský Small Carpathian Blaufrankise 26. Moravany nad Váhom Vrbovský Small Carpathian Grüner Veltliner 27. Vinica Vinický Central Slovak Blaufrankise 28. Šahy Ipeľský Central Slovak Zala gyöngye 29. Sebechleby Hontiansky Central Slovak Saint Laurent 30. Gajary Záhorský Small Carpathian André 31. Skalica Skalický Small Carpathian Blaufrankise 32. Zeleneč Trnavský Small Carpathian Cabernet Sauvignon 33. Nové Zámky Palárikovský Southern Slovak Grüner Veltliner 34. Abrahám Galanstký Southern Slovak Velsch Riesling 35. Čamovce Fiľakovský Central Slovak Pálava 36. Rimavská Sobota Gemerský Central Slovak Blaufrankise 37. Kráľ Tornaľský Central Slovak Müller Thurgau 38. Orechová Sobranecký Eastern Slovak Pinot Gris 39. Vinné Michalovský Eastern Slovak Grüner Veltliner 40. Streda na Bodrogom Kráľovsko-chlmecký Eastern Slovak Tramin 41. Hrušov Moldavský Eastern Slovak Alibernet 42. Viničky Tokajský Tokaj Furmint 43. Viničky Tokajský Tokaj Lipovina 44. Viničky Tokajský Tokaj White Frontignan 45. Bratislava - Rača Bratislavský Small Carpathian Riesling 46. Bratislava - Rača Bratislavský Small Carpathian Blaufrankise 47. Stupava Stupavský Small Carpathian Grüner Veltliner 48. Nesvady Komárňanský Southern Slovak Velsch Riesling 49. Veľký Meder Dunajsko-Stredský Southern Slovak Chardonnay 50. Šamorín Šamorínsky Southern Slovak Blaufrankise Volume 11 404 No. 1/2017 Potravinarstvo Slovak Journal of Food Sciences Table 2 Preparation of the chromatographic plates before visualisation of the diacetoxyscirpenol (DAS), deoxynivalenol (DON), HT-2 toxin (HT-2) and T-2 toxin (T-2) and the manifestation of visualization. Mycotoxin Chromatographic plate preparation Visualisation under UV light with a wavelenght of 366 nm DAS, DON application of 20% AlCl3 in 60% light blue fluorescet spot ethanol heating-up HT-2, T-2 application of 20% H2SO4 in water green-blue fluorescent spot heating-up for 8 min.at 130 oC Furmit (1), White Frontignan (1) and Lipovina (1). Red were placed on DRBC plates (Samson et al., 2002). The grape: Alibernet (1 sample), André (2 samples), total mycobiota (non-sterilized berries) was determined by Blaufrankise (8), Cabernet Sauvignon (2), Konkordia (1), the method of direct placing of grape berries on DBRC Pinot noir (2), Saint Laurent (1). Informations about plates, also. Only the undamaged berries were used for analysed samples are shown in Table 1. Samples (3 kg) analysis. For determination fungal colonization of were collected at the time of technological ripeness. damaged berries all berries (from 7 to 15 berries from Picked grapes were stored at 4 ±1 °C and analysed within sample) with some evident defect were used. Berries from 24 h after harvest. each sample were placed on DRBC plates. Cultivation lasted from 5 to 7 days in darkness at Mycological analysis 25 ±1 °C (in the all cases). For the isolation of Botrytis sp., Fusarium sp. and Grown micromycetes were classified into the genera and Rhizopus sp. was used
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