COLONIZATION OF GRAPES BERRIES BY ALTERNARIA sp. AND THEIR ABILITY TO PRODUCE MYCOTOXINS

Our research focused on identify the Alternaria species from grapes (surface sterilized berries and non-surface sterilized berries) of Slovak origin and characterize their toxinogenic potential in in vitro conditions. We analyzed 47 samples of grapes, harvested in years 2011, 2012 and 2013 from various wine-growing regions. For the isolation of species, the method of direct plating berries and surface-sterilized berries (using 1 % freshly pre-pared chlorine) on DRBC (Dichloran Rose Bengal Chloramphenicol agar) was used. For each analysis was used 50 berries. Only undamaged berries have been used for analysis. The cultivation was carried at 25 ±1°C, for 5 to 7 days in dark. After incubation, the colonies of Alternaria were transferred on PCA potato-carrot agar and CYA Czapek-yeast extract agar and cultured for 7 days at room temperature and natural light. A total 4 species-groups of the genus Alternaria were isolated from grapes berries: Alternaria alternata (1369 isolates), Alternaria arborescens (734 isolates), Alternaria infectoria (143 isolates), and Alternaria tenuissima (3579 isolates). According to European Union legislation mycotoxins produced by species genus Alternaria are not monitored in foods and food commodities. Mycotoxins such as alternariol and alternariol monomethylether are mutagenic and genotoxic in various in vitro systems. Selected strains were tested for production of altenuene, alternariol monomethylether and alternariol. In neither case of A. infectoria species-group isolates was confirmed the production of tested mycotoxins in in vitro conditions by TLC method. The ability to produce altenuene, alternariol monomethylether and alternariol in in vitro conditions was detected in isolates of Alternaria alternata, Alternaria arborescens and Alternaria tenuissima species-groups. Isolates of Alternaria alternata species-group (44 tested isolates) were able to produce altenuene (24 isolates), alternariol monomethyleter (42 isolates) and alternariol (43 isolates). Only one isolate did not produce any mycotoxins. Isolates of Alternaria arborescens species-group (38 tested isolates) were able to produce altenuene (24 isolates), alternariol monomethyleter (33 isolates) and alternariol (36 isolates). Only two isolates did not produce any mycotoxins. Isolates of Alternaria tenuissima species-group (87 tested isolates) were able to produce altenuene (42 isolates), alternariol monomethyleter (41 isolates) and alternariol (73 isolates). Thirteen isolates did not produce any mycotoxins.


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). The black mould genus Alternaria Ness is ubiquitously distributed and includes various saprophytic, endophytic and pathogenic species. Many of the genus Alternaria Ness commonly cause spoilage of various food crops in the field or post-harvest decay (Ostrý, 2008;Logrieco et al., 2009). Alternaria species are pathogenic and saprophytic fungi widely distributed in soil. They are widespread in both humid and semiarid regions and can infect growing plants in the field. They are the principal contaminating fungi in wheat, sorgum and barley. In addition to cereal crops, Alternaria species have been reported to occur in oilseeds such as sunflower and rapeseed, tomato, apples, citrus fruits, olives and several other fruits and vegetables. Alternaria species grow at low temperature; hence they are generally associated with extensive spoilage during refrigerated transport and storage (Ostrý, 2008). Alternaria genus is the main component of the wine grape mycobiota at harvest time (Serra et al., 2005;Prendes et al., 2015;Tančinová et al., 2015). The most common fungi spoiling grapes were Alternaria, Botrytis cinerea and Cladosporium (Tournas, et al., 2005). Moreover, several Alternaria species are known to produce toxic secondary metabolites, Alternaria mycotoxins (Rotem, 1994;Prendes et al., 2015). Mycotoxins are secondary metabolites produced by filamentous fungi that have been detected in food commodities, including grapes and wine (Serra et al., 2005). Alternaria species have the ability to produce a variety of secondary metabolites, which plays important roles in food safety (Andresen, et al., 2015). The major Alternaria mycotoxins belong to three structural classes: tetramic acid derivate, tenuazonic acid; the dibenzopyrone derivates, alternariol, anternariol methylether and altenuene; and the perylene derivates, the altertoxins (Andersen et al., 2002). Food relevant Alternaria species are able to produce many more metabolites (Ostrý, 2008;Logrieco et al., 2009). Alternaria toxins occurred regularly in cereals, tomato sauces, figs, wine and sunflower seeds. Only incidental occurrence of the Alternaria toxins was observed in fresh apples, fresh citrus, fresh tomatoes and olives (López et al., 2016).
Our research focused on the identify the Alternaria species from grapes of Slovak origin and characterize their toxinogenic potential in in vitro.

Mycological analysis
For the isolation of Alternaria sp. was used the method of direct plating berries: surface-sterilized berries and nonsterilized berries on DRBC (Dichloran Rose Bengal Chloramphenicol agar) 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 (agar with dichloran, rose bengal and chloramphenicol) (Samson et al., 2002). Cultivation lasted from 5 to 7 days in darkness at 25 ±1 °C. For each analysis was used 50 berries. Only undamaged berries have been used for analysis. After incubation, the colonies of Alternaria were transferred onto appropriate identification media. Identification of Alternaria species-groups. Grown micromycetes were classified into the genera and then isolated by re-inoculation on the identification nutrient media and identified by accepted mycological keys and publications. Isolates of the genus Alternaria were reinoculated on PCA -potato-carrot agar and CYA -Czapekyeast extract agar (Samson et al., 2002) and cultured for 7 days at room temperature and natural light.
In order to improve study of sporulation pattern we proceeded as follows. The colonized agar (piece of approx. size 0.5 x 1.0 cm) was cut and transferred to the agar surface, outside the colony. The growth was observed as early as one to two days of cultivation on the edge of the removed part. Main used identification keys were These values were calculated according to González et al., (1996) as follows: where ns = number of samples with a species; N = total number of samples.

Toxinogenity analysis
Toxinogenity of selected isolates was analysed by means of thin layer chromatography (TLC) by Samson et al., (2002). This method was performed with modifications according to Labuda and Tančinová (2006). Testing was focused on determination of the ability to produce mycotoxins altenuene (ALT), alternariol (AOH) and alternariol monomethylether (AME).
The colonies grown on yeast extract sucrose agar (YES) (7, respectively 14 days, in darkness at 25 ±1°C) were cut into squares of approximate size 2 cm x 2 cm and placed in an Eppendorf tube with 0.5 mL of extraction solution (chloroform: methanol -2:1; Reachem, SR). The content of the tubes was stirred for 5 minutes by Vortex Genie ® 2 (MO BIO Laboratories, Inc. -Carlsbad, CA). The obtained extracts were applied to silica gel chromatography plate (Alugram ® SIL G, Macherey -Nagel, Germany) and plates were put into the TEF solvent (toluene: ethyl acetate: formic acid -5 :4 :1; toluene -Mikrochem, SR; ethyl acetate and formic acid -Slavus, SR). After elution and drying, the mycotoxins identity was confirmed by visual comparison with the standards of mycotoxins (AME, ALT and AOH -Merck, Germany) under UV light with a wavelength of 254 nm and 366 nm.

RESULTS AND DISCUSSION
In the current study from all samples were isolated Alternaria species (from superficially sterilized berries and berries without sterilization, too). The cosmopolitan fungal genus Alternaria consists of multiple saprophytic and pathogenic species. Based on phylogenetic and morphological studies, the genus is currently divided into 26 sections. Alternaria section Alternaria contains most of the small-spored species with concatenated conidia, including important plant, human and postharvest pathogens (Woundenberg et al., 2015). A total of 4 species-groups (Table 1) of the genus Alternaria (Alternaria section Alternaria) were isolated from grapes berries, namely Alternaria alternata group, Alternaria arborescens group, Alternaria infectoria group, and Alternaria tenuissima group. Isolates, which could not be closer specified or contaminated another species were specified as Alternaria sp., Sporulation patterns of Alternaria species-group are listed according to Simmons, (2007). The typical sporulation pattern of Alternaria alternata group (Figure 1) comprises a single suberect conidiophore and an apical cluster of branching chains of small conidia separated by short secondary donidiophores. Long, well-defined primary conidiophores of Alternaria arborescens group (Figure 2) characteristically bear a few terminal and subterminal branches. Each conidiophore branch bears a branching chain of conidia, giving a relatively tall, three-dimensionally arborescent apperarance to the suberect system.      Mycotoxins are abiotic hazards produced by certain fungi that can grow on a variety of crops (Marin et al., 20013). According to European Union legislation mycotoxins produced by species genus Alternaria are not monitored in foods and food commodities. Mycotoxins such as alternariol and alternariol monomethylether are mutagenic and genotoxic in various in vitro systems. In addition, it has been suggested that in certain areas in China Alternaria toxins in grains might be responsible for oesophageal cancer. Hence, due to their possible harmful effects, Alternaria toxins are of concern for public health (EFSA, 2011). According to Prendes et al., (2015), Alternaria, one of the most mycotoxigenic genus commonly found in wine grapes, could represent a high risk for the wine consumer´s health. Representative isolates were selected for analysis to produce mycotoxins in in vitro conditions randomly from all obtained isolates. The results are presented in Table 2 (Table 2). In neither case of the 15 tested isolates of Alternaria infectoria species-group we confirmed the production of mycotoxins ALT, AOH and AME. Although, the reputation of "nontoxigenic" strains of the Alternaria infectoria species-group has been undermined in recent years by isolation unknown metabolites (Mašková et al., 2012).
Conversely, isolates of other tested species-groups proved to be highly toxigenic (Table 2). Only one isolates of Alternaria alternata species-group and two isolates of Alternaria arborescens species-group did not produce tested mycotoxins in in vitro conditions detectable by TLC method. Robiglio and Lopez were tested eleven Alternaria alternata strains, isolated from Red Delicious apples in cold storage in Argentina, for alternariol and alternariol methyl ether production in laboratory media and in whole fresh fruits. Most of them were able to produce both toxins in all media. They were detected also in mycelium free filtrates from liquid cultures and in asymptomatic tissues from inoculated fruit. Thus, in the evaluation of mouldy core incidence in apples, the presence of Alternaria alternata toxins in tissues should be considered even in the absence of mycelia (Robiglio and Lopez, 1995).
Small-spored Alternaria, such as Alternaria alternata group, Alternaria arboresces group, Alternaria infectoria group and Alternaria tenuissima group are important producers of mycotoxins, or other unknown metabolites but they were dominant fungal consortium in grapes berries in our samples. Considering that literature reported about the effectiveness of Alternaria endophytes against important grapevine pathogens, it should be interesting to elucidate the chemical structure of Alternaria unknown metabolites and to evaluate them as new biological method in the control of grapevine diseases (Polizzotto et al., 2012).

CONCLUSION
From the 2350 surface-sterilized (47 samples) grape berries have been isolated 2964 strains of genus Alternaria and from the same number of non-sterilized berries 3099 isolates of this genus. Isolates were identified acording to sporulation patterns to four species groups: namely Alternaria alternata (1369 isolates), Alternaria arborescens (734), Alternaria infectoria (143), and Alternaria tenuissima (3579) and 238 isolates were not identified to species group. There were found out the ability to produce following mycotoxis: altenuene, alternariol and alternariol monomethylether in in vitro conditions by TLC method of chosen strains of genus Alternaria. In another research would be advisable to follow occurrence of these mycotoxins in grapes, must, wine and another grape products.