RESEARCH ARTICLE The e¡ect of fungicides on yeast communities associated with grape berries Nez ˇaC ˇ adez ˇ 1 , Jure Zupan 1 & Peter Raspor 1 1 Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia Correspondence: Nez ˇaC ˇ adez ˇ , Department of Food Science and Technology, Biotechnical faculty, University of Ljubljana, Jamnikarjeva, Ljubljana 1000, Slovenia. Tel.: 1386 1 423 11 61; fax: 1386 1 257 40 92; e-mail: neza.cadez@bf.uni-lj.si Received 2 November 2009; revised 7 April 2010; accepted 7 April 2010. Final version published online 12 May 2010. DOI:10.1111/j.1567-1364.2010.00635.x Editor: Teun Boekhout Keywords fungicide; grape berry; wine yeast; basidiomycetes; polyphasic identification; halo assay. Abstract The influence of three commonly used fungicides (iprodione, pyrimethanil and fludioxonil plus cyprodinil) on the density and diversity of yeast populations present on grape berries was evaluated. At the time of harvest, the fungicide residues on grapes were below the maximum permitted levels. In general, larger yeast counts were found on the treated grapes than on the control samples. Among 23 species identified, Cryptococcus magnus, Rhodotorula glutinis and Sporidiobolus pararoseus dominated on sound grape berries. The results showed that the tested fungicides had only a minor impact on the composition of grape berry commu- nities in comparison with the effect of weather conditions and the mode of grape berry sampling. Halo assays using filter discs loaded with fungicides were used as in vitro tests of the sensitivity of grape berry isolates. The fungicide containing pyrimethanil suppressed the growth of all basidiomycetous yeast species, while the sporadically occurring fermentative yeasts were unaffected. Fungicides with fludioxonil plus cyprodinil and iprodione as active substances showed specificity for certain species. Our results suggest that after the safety interval, the presence of fungicides has a minor impact on the composition of grape berry communities, although at the time of fungicide applications, the yeast species composition changes. Introduction Grapes are a primary source of indigenous yeast microbial communities that plays an important role in alcoholic fermentation. Although Saccharomyces cerevisiae is the main organism responsible for the conversion of grape juice into wine, indigenous yeasts were found to be important con- tributors to the chemosensory properties of the wine. They can either contribute to a more complex and better aroma or they can cause wine spoilage (Heard & Fleet, 1985; Granchi et al., 2002). There are two types of detrimental effects of natural yeast flora on wine fermentation: by the production of off-flavours or by the prevention of the normal predomi- nance of S. cerevisiae (Bisson, 1999). For these reasons, the ecology of natural yeast flora is an important factor influen- cing wine quality. Grape berry surfaces provide a physical environment suitable for the growth of microbial communities that depend on the grape vine for nutrients, water and protection (Schreiber et al., 2004). Yeast populations are spatially distributed over the grape berries (Belin, 1972) and grape bunches (Rosini et al., 1982), and are dynamic during the course of grape berry development (Rosini et al., 1982; Prakitchaiwattana et al., 2004). The community dynamics is also influenced by external factors such as geographical location, climatic conditions, grape cultivar, vine canopy and the use of agrichemicals (reviewed by Fleet et al., 2002). The application of chemical fungicides to prevent the growth of the fungus Botrytis cinerea (teleomorph: Botryoti- nia fuckeliana), a causal agent of grey mould, is a routine viticultural practice in areas where weather conditions favour the disease (Rosslenbroich & Stuebler, 2000). Although the application of fungicides is an effective control against B. cinerea, their mode of action might be nonspecific and might act on organisms other than the target fungus (Calhelha et al., 2006). Based on these predictions, we examined the influence of three commonly used fungicides (iprodione, pyrimethanil and fludioxonil1cyprodinil) on the density and diversity of indigenous yeast populations present on grape berries. Because we cannot avoid the effect FEMS Yeast Res 10 (2010) 619–630 c  2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved YEAST RESEARCH