Elsevier

Food Microbiology

Volume 46, April 2015, Pages 521-527
Food Microbiology

Effect of interaction between Aspergillus carbonarius and non-ochratoxigenic grape-associated fungal isolates on growth and ochratoxin A production at different water activities and temperatures

https://doi.org/10.1016/j.fm.2014.09.014Get rights and content

Highlights

  • The interaction between Aspergillus carbonarius and grape associated mycobiota was studied.

  • Interactions on growth and OTA production were assayed in various aw and temperatures.

  • A. carbonarius presented mutual antagonism with A. section Nigri strains.

  • Penicillium spinulosum, Cladosporium spp. and Botrytis cinerea were competitive against A. carbonarius.

  • OTA level was decreased depending on fungal competitor and environmental conditions.

Abstract

The effect of water activity (0.90, 0.94, and 0.98 aw) and temperature (15, 20, and 25 °C) on the in vitro interactions between three ochratoxigenic strains of Aspergillus carbonarius (Ac-28, Ac-29, and Ac-33) and eleven non ochratoxigenic grape-associated fungal strains was assessed in this study. Fungal strains were allowed to grow in dual cultures on Synthetic Grape-juice Medium (SGM) for 15 days and fungal interactions were given a numerical score to obtain an Index of Dominance (ID) for each fungus. Results showed that in most cases A. carbonarius toxigenic strains were dominant against other fungal species. However, A. carbonarius presented mutual antagonism with A. section Nigri strains regardless of water activity (aw) and temperature. Moreover, interactions with Penicillium spinulosum and Cladosporium spp. at 15 °C, as well as Botrytis cinerea at 20 °C, showed that the antagonists were more competitive against A. carbonarius. In some cases, growth rates of A. carbonarius strains were either slightly stimulated or inhibited after interaction in dual cultures, depending on temperature, aw and competing species. Regarding OTA production, the presence of other species sometimes decreased the production or slightly enhanced it, depending on fungal competitor and environmental conditions. Overall, OTA production was higher at 15 °C/0.98 aw and 20 °C/0.98 aw for all target strains and at 20 °C/0.94 aw for Ac-33 strain only, but decreased at higher temperatures regardless of aw and interacting species.

Introduction

Wine grapes can be contaminated by several fungal species at preharvest period and during harvesting or grape processing (Valero et al., 2007). Isolates from wine and table grapes belonging to the genera Alternaria, Aspergillus, Botrytis, Cladosporium, Epicoccum, Eurotium, Fusarium and Rhizopus have been found to be responsible for grape infection (Abrunhosa et al., 2001, Bellí et al., 2004, Kizis et al., 2014). Some species belonging in Aspergillus and Penicillium genera have been found as the main source of ochratoxin A (OTA) and in particular Aspergillus carbonarius as the major ΟΤΑ producer in grapes and their derivatives (Bellí et al., 2002, Cabañes et al., 2002, Ostry et al., 2002, Soufleros et al., 2003, Stefanaki et al., 2003). OTA in wine was first detected by Zimmerli and Dick several years ago (1995). It is a fungal secondary metabolite that has been reported as a mycotoxin with nephrotoxic, hepatotoxic, genotoxic, teratogenic and immunotoxic impact to humans and animals (IARC, 1993, Castegnaro et al., 1998). Additionally, it has been reported that OTA is a disease determinant of Balkan endemic nephropathy (Krogh, 1978). According to the annual report of Rapid Alert System for Food and Feed (European Commission, 2012), OTA is the second more frequent mycotoxin found in food products after aflatoxins.

Although, numerous studies have examined the ecophysiology of A. carbonarius at different environmental conditions (Bellí et al., 2004, Tassou et al., 2009, Spadaro et al., 2010), only few focused on the interactions between A. carbonarius and other fungal species (Valero et al., 2007, Magan et al., 2010). Magan and Lacey, 1984, Magan and Lacey, 1985 investigated the competitiveness of different fungal species under diverse environmental conditions and classified these interactions by giving a numerical score to each species. Thus, for mutual intermingling of hyphae between two species the score 1-1 was assigned, for mutual antagonism on contact or distance the score 2-2 and 3-3 was given, respectively, and for dominance by one species over another the score 4-0 was given when species were in contact, and 5-0 when in distance. The second number in the score always indicates the antagonist strain. The score for each species can be added to obtain an overall Index of Dominance (ID). The ID has been reported to change significantly with water availability (aw), temperature and nutritional status and it has been employed as an index to compare the variations under different environmental conditions in food-based ecosystems (Marin et al., 1998, Lee and Magan, 1999; Magan and Aldred, 2007; Magan et al., 2010). Moreover, the interaction and competition between A. carbonarius and other fungal species has an influence on OTA production (Lee and Magan, 2000). It is thus necessary to extend our knowledge on the outcome of interactions between A. carbonarius and other grape-related species under diverse environmental conditions, in an attempt to elucidate the potential of growth and OTA production in these products.

The aim of this study was to examine the effect of different environmental factors (aw and temperature) and fungal interactions on the (a) growth rates, (b) OTA production, and (c) profile of hyphae intermingling, between three A. carbonarius strains and eleven grape colonizing related fungi.

Section snippets

Fungal strains and media preparation

The black aspergilli strains used in this study were three A. carbonarius OTA-positive producers (Ac-28, Ac-29, Ac-33), an A. carbonarius OTA-negative (Ac-27), Aspergillus niger (An-1), Aspergillus tubingensis (At-4), Aspergillus japonicus (Aj-1) and Aspergillus ibericus (Ai-1). Other grape-associated fungal strains used were Alternaria alternata, Fusarium oxysporum, Cladosporium spp., Botrytis cinerea, Penicillium spinulosum and Aspergillus flavus. All the strains have been previously isolated

Effect of aw and temperature on Index of Dominance (ID)

In this study, the effect of the competitiveness of grapes' mycobiota against three high OTA-producing strains of A. carbonarius at marginal, sub-optimum and optimum conditions has been examined. Until now, only few studies focused on interactions between A. carbonarius strains and other fungal species which are associated with grape mycobiota and particularly interactions with black aspergilli species (Valero et al., 2007, Magan et al., 2010). The numerical interaction scores and indices of

Acknowledgements

This work has been supported by the project ‘Design and development of innovative tools for the detection of ochratoxigenic fungi in wine and table grapes FungalPrognosis_242’ co-financed by the European Union (European Social Fund – ESF) and Greek national funds through the Operational Program “Education and Lifelong Learning” of the National Strategic Reference Framework (NSRF) – Research Funding Program: ARISTEIA-I.

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