SPECIES OF GENERA BOTRYTIS , FUSARIUM AND RHIZOPUS ON GRAPES OF THE SLOVAK ORIGIN

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.


INTRODUCTION
Grapes have a complex microbial ecology including filamentous fungi, yeasts and bacteria with different physiological characteristics and effects upon wine production ( Barata et al., 2012).Moulds commonly isolated from grapes are Alternaria, Cladosporium and Botrytis cinerea, the latter causing bunch rot.Pathogenic and opportunistic species of Fusarium, Penicillium and Aspergillus can also colonize inducing grape disease (Oliveri and Catara, 2011).
The concern about filamentous fungi in the vineyasrd has traditionally been linked to spoilage of grapes due to fungal growth.The main fungus responsible for grape rot is Botrytis cinerea, a pathogen that damages the berries and had a detrimental effect on the organoleptic properties (Serra et al., 2006).Botrytis is a common genus in the temperate zones, where it occurs mainly as a pathogen on a variety of plant crops.Vegetable and small berry fruits are particularly susceptible.Invasion may occur before maturity or postharvest, both in transport and in storage.Onions and other allium species and grapes are the most susceptible crops.In the latter, it is notable that the disease is sometimes encouraged.Grapes affected by Botrytis, in this ciccumstance called "the noble rot", are used int the production of certain high quality sweet wines in France, Germatny, Australia and other countries (Pitt and Hocking, 2009).
Rhizopus stolonifer is one of the most common and fastest-growing species in the Zygomycota phylum.Disease caused by this fungus is known as soft rot, black mould and Rhizopus rot (Bautista-Baños et al., 2014).Rhizopus rot is common on soft fruits, more abundant in warm, humid climates than in cool climate viticulture.In several fruits and crops such as strawberry, tomato, cucumber and table grapes Rhizopus rot causes soft rot during transport and storage (Kassemeyer and Berkelmann-Löhnertz, 2009).
Fusarium species are renowned for their role as plant pathogens, causing a wide renage of diseases such as vascular wilts, root and stem rots, pre-and post-emergence blight and many others (Pitt and Hocking, 2009).
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 identify toxinogenic potential of chosen strains of species Fusarium.

MATERIAL AND METHODOLOGY Samples
Fifty samples of wine grapes, harvested in years 2011, 2012 and 2013 from various wine-growing regions of Slovakia, from small and medium-sized vineyards were analysed.
Slovak wine region is characterized as a territory of the Slovak Republic, where grapes are grown.It is divided into 6 viticulturally regions, which are divided into 40 wine area and 690 wine-growing villages.The samples which were analysed were taken from all viticulturally regions (Table 1).

Mycological analysis
For the isolation of Botrytis sp., Fusarium sp. and Rhizopus sp. was used the method of direct plating berries: surface-sterilized berries, non-sterilized and damaged berries on DRBC (Dichloran Rose Bengal Chloramphenicol agar) (Biolife, Italia) according

Samson et al. (2002).
The endogenous mycobiota was determined by the method of direct placing of superficially sterilized berries on agar plates (Samson et al., 2002).More than 50 pieces of undamaged berries from each sample were superficially sterilized (using 1% freshly pre-pared chlorine).Sterilization was carried out for 2 minutes.Berries were rinsed 3 times with sterile distilled water and dried on sterile filter paper.Exactly 50 berries from each sample were placed on DRBC plates (Samson et al., 2002).The total mycobiota (non-sterilized berries) was determined by the method of direct placing of grape berries on DBRC plates, also.Only the undamaged berries were used for analysis.For determination fungal colonization of damaged berries all berries (from 7 to 15 berries from sample) with some evident defect were used.Berries from each sample were placed on DRBC plates.
Cultivation lasted from 5 to 7 days in darkness at 25 ±1 °C (in the all cases).
Grown micromycetes were classified into the genera and then isolated by reinoculation on the identification nutrient media and identified through macroscopic and microscopic observation in accordance with accepted mycological keys and publications.

Identification of Fusarium species
Potato   Obtained results were evaluated and expressed in isolation frequency (Fr) at the species level.The isolation frequency (%) was defined as the percentage of samples within which the species occurred at least once.These values were calculated according to González et al. (1996) as follows: Fr (%) = (ns / N) x 100 Where: ns = number of samples with a species; N = total number of samples.

Mycotoxins screening by a modified agar plug method
For the determination of toxigenity a modified agar plag method using thin-layer chromatography according to the Samson et al. ( 2002), modified by Labuda and Tančinová (2006) was used.A total of 47 randomly selected strains of the F. acuminatum, F. culmorum, F. equiseti, F. graminearum, F. oxysporum, F. proliferatum, F. semitectum, F. sporotrichioides (the most important species according to their occurrence), F. tricinctum and F. verticillioides were re-inoculated on YES (yeasts extract agar) (Samson et al., 2002), cultured in the dark at a temperature of 25 ±1 °C from 7 to 14 days and then tested for the ability to produce mycotoxins deoxynivalenol (DON), diacetoxyscirpenol (DAS), HT-2 toxin (HT-2) and T-2 toxin (T-2).From the grown colonies squares of the approximate size 2 x 2 cm were cut and put them in small chunks to Eppendorf tube with 1 ml of extraction reagent chloroform : methanol, 2 : 1 (for DON) and acetonitrile (Fischer, Slovakia) : water, 50 : 50 (for DAS, HT-2, T-2).After a 5 minute mixing the extract was applied to the chromatographic plate (Alugram®SIL G, Macherey -Nagel, Germany).Subsequently, developing solution toluene : acetone : methanol (5 : 3 : 2) (Centralchem, Slovakia) were used.Before visualisation, chromatographic plates were processed as is given in Table 2. Mycotoxins were confirmed by comparison with standards (Merck, Germany) under UV light with a wavelength of 366 nm.

RESULTS AND DISCUSSION
Botrytis cinerea and Erysiphe necator are among the most relevant fungi in viticulture (Lopez Pinar et al., 2016).Botrytis cinerea (Figure 1) was identified in 86% of samples in our research (Table 3, 4).Significant difference was observed in the number of isolates from Tokaj viticulturally region.From 3 samples from this region were isolated 257 isolates and only 187 isolates from 47 samples from other regions.The grapes from Tokaj viticulturally region are used for production of typical Table 3 List of the isolated moulds of genera Botrytis, Fusarium and Rhizopus from wine grapes berries of the Slovak origin, isolated from berries, berries superficially sterilized and damaged berries.

Serra et al. (2005)
shown that Fusarium strains were primarily detected at the early maturation stages of grapes, with and without surface disinfection.Isolation frequency of Fusarium was very high -92%.Isolates were detected   2005) reported that species described as producers of mycotoxins represented 8.0% of the grape mycobiota, distributed as follows: potential produceres of aflatoxins (0.3%), ochratoxin A (6.0%), patulin (0.5%) and trichothecenes (1.2%).Selected isolates (47) of ten species were tested for their ability to produce relevant mycotoxins -trichothecenes in in vitro condition, by means of thin-layer chromatography.The results are presented in Table 5. Thirty-two (68%) of tested isolates were able produce at least one mycotoxin.All isolates of F. sporotrichioides were able to produce some mycotoxin in in vitro conditions.Isolates of potential producers of mycotoxins can produce more than one mycotoxin.11 isolates of F. sporotrichioides produced T-2 toxin and HT-2 toxin, 4 isolates T-2 toxin, HT-2 toxin and diacetoxyscirpenol.F. graminearum (2 tested isolates) produced T-2 toxin, HT-2 toxin and diacetoxyscirpenol.

CONCLUSION
From 2500 surface-sterilized berries, 2500 berries without sterilization and 550 damaged berries (50 samples) wine grape berries were isolated 440 strains of Botrytis cinerea, 504 strains of Fusarium spp.and 373 strains of Rhizopus stolonifer.Significant difference was observed in the number of strains of Botrytis cinerea from Tokaj viticulturally region (3 samples -257 strains) to another samples (47 samples -183 strains).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 by means of thinlayer chromatography.In another research would be advisable to follow occurrence of these mycotoxins in grapes, must, wine and another products from grapes.
Dextrose agar (PDA) (Samson et al., 2002) was used for observation of colony characteristics."Synthetischer nährstoffarmer agar" (SNA) (Samson et al., 2002) was used for micromorphological features.Cultures were incubated at the room temperature and natural light.Species identification was done after 10 days according to Leslie and Summerell (2006), Nelson et al.

Table 1
List of analysed samples of grapes and their origin.

Table 4
Species of Botrytis, Fusarium and Rhizopus isolated from berries of the Slovak origin determinated by using plate direct method on DRBC agar from 50 samples.Fusarium, * -isolates from Tokaj viticulturally region.

Table 5
Potential ability of Fusarium species isolates to produce mycotoxins in in vitro conditions, tested by TLC method.Ochratoxin A is a secondary metabolite produced by filamentous fungi of the two genera Aspergillus and Penicillium present in a wide variety of foodstuffs (

Amézqueta et al., 2012; Vega et al., 2012).
These two genera are main genera responsible for mycotoxin production in grapes (

Serra et al., 2006).
Species of genus Fusarium are important producers of mycotoxins, too.Serra et al. (