THE DETERMINATION OF MICROSCOPIC FUNGI FROM CHESTNUT (Castanea sativa Mill.) FRUITS, LEAVES, CRUST AND POLLEN

KAČÁNIOVÁ, M., SUDZINOVÁ, J., KADÁSI-HORÁKOVÁ, M., VALŠÍKOVÁ, M., KRÁČMAR, S.: The determination of microscopic fungi from Chestnut (Castanea sativa Mill.) fruits, leaves, crust and pollen. Acta univ. agric. et silvic. Mendel. Brun., 2010, LVIII, No. 4, pp. 73–78 The plant-microbial interactive relations with respect to determination of the mycofl ora of the Castanea sativa Mill. nuts, crust, leaves and pollen and their eff ect on the host organism in four Slovak regions were studied. In the experiments were isolated 7 genera and 10 species of microscopic fungi from the nut, crust and leaves. It was found, that isolates from the Castanea sativa Mill. pollen were represented by 8 genera and 11 species of microscopic fungi. Alternaria, Cladosporium, Mucor and Rhizopus appeared to be the most frequently occurring genera of nuts, leaves and crust. Acremonium, Alternaria, Cladosporium, Fusarium, Penicillium and Trichoderma are the most frequently fungi of pollen. On the base of further taxonomic determination from the genera Aspergillus were isolated and identifi ed representatives of species A. fl avus, A. fumigatus, A. ochraceus, A. terreus and A. versicolor. From the genera Fusarium was isolated F. oxysporum and from Penicillium genera were isolated P. crustosum and P. glabrum. It is necessary to underline that the isolated genera Aspergillus, Fusarium and Penicillium are considered as the most important producers of mycotoxins. Castanea sativa Mill., nuts, leaves, crust, pollen, microscopic fungi The chestnut fruits produced in Slovakia are gene ral ly of small size and of lower quality because of less favorable climatic conditions and absence of breeding and selection work. The European chestnut trees spread all over the world and produce wood and chestnuts that have considerable economical value. Specifi c li tera tu re describes some utilization for chestnuts in Europe being emphasized the marrons glaces production; some references also relate their use in baking, mainly under economically diffi cult condition (Demiate et al., 2001). Also it is generally known, that the main component of chestnuts is starch, there are a little studies related to chemical composition of the chestnuts. Many studies are oriented to the agri cul tu ral features of the trees and especially to the phytopathology (Rutter et al., 1991). Moulds are distributed in nature ubiquitously and their spores can be found in the atmosphere even at high altitudes. They are drive and disseminate by wind and air currents, as well as can be spread by insects, rodents, and other animals. The metabolic activities accompanying the growth and mould development decomposes organic substrate ensuring the recycling of elements that comprise organic matters. Food products, being organic substances and containing essential nutrients, are very suitable substrates for the mould growth (Leoni et al., 2001; Sa lee mul lah et al., 2006; Carmo et al., 2008). Because of their hydrolytic enzymes powerful arsenal, moulds may cause a high degree of deterioration when they occur in/on foods and can be in charge of considerable economic losses (Overy et al., 2003; Kawashima and Valente Soares, 2006). Besides the possible food decaying caused by moulds and ultimate changes in it nutritional and organoleptic characters, the moldiness in foodstuff s is toxicologically signifi cant since the mould species growing on such products is known as potentially mycotoxicogenic (Battilani et al., 2006; Carmo et al., 2008). My co to74 M. Kačániová, J. Sudzinová, M. Kádasi-Horáková, M. Valšíková, S. Kráčmar xins are secondarily toxic metabolites produced by fi lamentous fungi that have been detected in several food commodities. Levels of mycotoxins and mycotoxicogenic moulds, which may cause risk to population, are refused by consumers and many countries have set regulations in various agricultural foods. The consumption of mouldy products may cause human or animal mycotoxicoses, and some of more important mycotoxins are strong carcinogens (Rasooli and Abyaneh, 2004; Wangikar et al., 2005; Kawashima and Valente Soares, 2006; Carmo et al., 2008). For the better understanding of ecological and practical interactions occurring on the nuts, crust, leaves and pollen of Castanea sativa Mill., some analy ses including the study of the diversity and exact species identifi cation are needs. Our study was intended on the survey of fungal community composition occurring on four diff erent parts of C. sativa Mill. species from four diff erent regions of Slovakia. MATERIALS AND METHODS The samples were taken from four diff erent parts of Castanea sativa Mill. in localities of Krupina, Mlyňany, Príbelce and Radošiná. The samples as leaves, crust and pollen were collected in June and July of year 2007. The samples of nut were collected in September and October of year 2007. 1. Nut sample number: N1–N13. 2. Leaves sample number: L1–L13. 3. Crust sample number: C1–C13. 4. Pollen sample number: P1–P43. The samples of nuts, leaves and crust numbered 1–4 were taken from locality Krupina, 5–9 were taken from Radošiná and 10–13 were taken from locality Príbelce. Sample of pollen numbered 1–14 were taken from Radošiná, 15–22 were taken from Krupina, 23–32 were taken from Príbelce and 33-43 were taken from Mlyňany. All samples were taken with scalpel previously cleaned with 70% ethylalcohol, transferred to sterile test-tubes and directly transported to the laboratory for further microbiological processing. Isolation and morphological characterization of fungi One gram of samples was transferred to 10 ml 0.01% Tween 80 solution and shaken for 30 min. Coarse-gained material was allowed to precipitate and 50 μl were homogeneously dispersed onto selective agar media in Petri dishes and the plates were incubated at room temperature. The isolates were purifi ed in two or three steps by transfer to fresh medium; purity was checked and maintained by re peti ti ve light microscopic observation. The fungi were grown in Petri dishes and slide cultures on 2% malt extract agar (MEA) and Czapek – Dox agar (CDA) (Imuna Šarišské Michaľany, Slovakia) for the morphological studies. A er 14–20 days of cultivation in darkness at room temperature, slide cultures and preparations were made in solution of lactic acid. The pure cultures were identifi ed according to method for diagnosis of fungi (Hoog et al., 2000; Klich, 2002; Samson et al., 2002). RESULTS AND DISCUSSION The study of mould spores have major importance because many fungi may cause considerable economic losses worldwide acting as plant pathogens or triggering respiratory diseases and allergenic processes in humans (Rodriguez-Rajo et al., 2005). The contamination of food products with micromycetes of the Penicillium, Aspergillus, Fusarium, Alternaria, Paecilomyces, Trichotecium, genera should receive particular attention. Some of the strains growing on a particular type of vegetable or fruit could synthesize and excrete diff erent toxic secondarily metabolites (Lugauskas et al., 2005; Piecková and Jesenská, 1999). The most frequently fungi found in nuts of Castanea sativa Mill. were Alternaria alternata, Cla do spo ridium cladosporoides, Fusarium oxysporium, Mucor mucedo and Rhizopus stolonifer. The less abundance was determined by Aspergillus fumigatus, Aspergillus terreus, Mucor racemosus, Penicillium crustosum and Penicillium glabrum (Tab. I). Cladosporium cladosporoides was the most common spoiler of nut samples contaminating 43.7%. The nationwide survey was carried out to assess mould spoilage of Castanea sativa nuts sold in Canadian grocery stores. Three mycotoxigenic fungi were dominated (Penicillium crustosum, Penicillium glabrum/spinulosum and Penicillium discolor) and isolated at frequencies of 67.1%, 18.6% and 17.7%, respectively, from a total sample size of 350 nuts. Aspergillus ochraceus was also isolated, but at a much lower frequency (Overy et al., 2003). Alternaria alternata aff ect diff erent cultivated and wild plants. Inhibitor from buckwheat also suppressed spore germination and mycelium growth of phytopathogenic fungi Alternaria alternata and Fusarium oxysporium (Valueva and Mosolov, 2004). The most frequently fungi found in leaves of Castanea sativa Mill. were Alternaria alternata, Cla do spori dium cladosporoides, Mucor mucedo, Mucor racemosus and Rhizopus stolonifer. The less abundance was determined by Aspergillus fumigatus, Aspergillus terreus, Fusarium oxysporum, Penicillium crustosum and Penicillium glabrum (Tab. I). Alternaria alternata was the most common spoiler of leave samples contaminating 49.7%. The leaf litter decomposition in streams is an important ecosystem level process, which depends on the activity of invertebrates and microorganisms. Both fungi and bacteria convert leaf carbon into microbial biomass, enhancing leaf pala ta bi li ty for shredding invertebrates (Pascoal and Cássio, 2004). Alternaria alternata (100%), Cladosporium cla do sporioi des (100%), Mycelia sterilia (67%) and Penicilium spp. (67%) were the most frequently isolated species of fungi encountered on the surface of the Sorbus doThe determination of microscopic fungi from Chestnut (Castanea sativa Mill.) fruits, leaves, crust and pollen 75 I: Frequency of fungi species of Castanea sativa Mill. Species of fungi Contaminated samples % Number of isolates (range)