Intraspecific variability of growth and patulin production of 79 Penicillium expansum isolates at two temperatures

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Abstract

Penicillium expansum is the main species responsible for patulin production in apples and pears. Generally, fruit is stored at suboptimal conditions for mould growth and this situation could influence on the intra-species variability in both capability for growth and mycotoxin production. The aim of this research was to assess the impact of suboptimal environmental conditions on the intra-specific variability of P. expansum growth and patulin production using seventy nine isolates of this mould. Petri dishes with Apple Concentrate Agar Medium (ACAM) were inoculated centrally and incubated at two temperatures, one near optimal (20 °C) and the other representative of suboptimal cold storage (1 °C). For each condition, 10 Petri dishes were inoculated, and colony growth and patulin production was measured over time. The Kruskal–Wallis test revealed significant differences among growth rate (μ) and lag phase (λ) within the seventy nine assayed isolates. Coefficients of variation revealed a wider dispersion of μ (mm/day) and λ (days) at 1 °C compared with 20 °C. There were significant differences (p < 0.05) among patulin levels (ng/mm2) for the different conditions, values being lower at the lower temperature. Coefficients of variation revealed a wider dispersion of mycotoxin production at 1 °C. In order to address the strain variability in growth initiation and prove the well-established notion of reducing patulin production in foods by preventing fungal growth, a greater number of strains should be included.

Highlights

► Effect of marginal conditions on intraspecific variability on growth of Penicillium expansum. ► Effect of marginal conditions on patulin production by this mould. ► Ecophysiology of P. expansum and intraspecific variability among 79 isolates.

Introduction

Growth of moulds in foods is an important quality problem and may lead to significant economic losses for the food industry (Andersen and Thrane, 2006). Mycotoxin contamination of food products is an important health hazard of growing interest globally. Penicillium expansum is the most important fungal species causing spoilage in cold stored fruit and is the causative agent of “blue mould” spoilage in pears and apples. This mould is a psychrophile and can grow at very low temperatures (− 2 to − 6 °C) (Brooks and Hansford, 1923, Pansenko, 1967). However, the optimum temperature is near 25 °C and the maximum near 35 °C (Pansenko, 1967). This mould can also produce patulin, a mycotoxin with carcinogenic and teratogenic effects which can cause acute and chronic effects if ingested (McKinley et al., 1982, Lee and Röschenthalen, 1987, Pfeiffer et al., 1998, Liu et al., 2003). Some authors have reported that 100% isolates of P. expansum are patulin producers (Andersen et al., 2004). Moreover, this toxin is both stable in acidic conditions and resistant to thermal inactivation (Lovett and Peeler, 1973), making it difficult to remove from food products.

Both fungal growth and mycotoxin production are influenced by different factors such as temperature, substrate aeration, water activity, inoculum concentration, microbial interactions, physiological state of mould, genetic information, isolate age, etc., however the effects of these factors on mycotoxin production might be different from those on growth (Garcia et al., 2009). Many studies have been published on the effects of some of these factors on growth of mycotoxigenic strains. Some reports have shown high variability in growth rates and mycotoxin production when several strains were included in the studies (Bellí et al., 2004, Parra and Magan, 2004, Pardo et al., 2004, Pardo et al., 2005a, Arroyo et al., 2005, Astoreca et al., 2007, Astoreca et al., 2010, Romero et al., 2007, Tassou et al., 2009). By contrast, others found few differences among isolates of the same species (Pardo et al., 2005b, Bellí et al., 2004). However, a recent study on Aspergillus carbonarius growth and ochratoxin A (OTA) production was made with thirty isolates; in this study a wide dispersion was observed in both growth rate and mycotoxin production, especially under marginal growth conditions (Garcia et al., 2011).

The use of cocktail inocula of different isolates in ecophysiological studies to minimize the variation that might be expected among different isolates of the same species has been proposed by different authors (e.g. Hocking and Miscamble, 1995, Patriarca et al., 2001, Pose et al., 2009, Romero et al., 2007, Romero et al., 2010). This concept was introduced for physiological studies on foodborne bacterial pathogens, particularly in acquisition of data for predictive modelling studies, as a way of determining the extremes of growth limits for particular species (Gibson et al., 1987, Buchanan et al., 1993).

Generally, foods and feeds are stored under conditions that are marginal for mould growth. Different environmental factors could influence the variability of responses of different isolates of the same species in their growth patterns and mycotoxin production, and this might be increased under marginal conditions. This point requires clarification for further development of predictive mycology. The aim of this study was to assess the impact of a non-optimal temperature (1 °C, representative of apple storage conditions) and one near to optimal (20 °C), on the intraspecific variability of P. expansum growth and patulin production.

Section snippets

Fungal isolates and preparation of inoculum

This work was carried out on seventy nine isolates of P. expansum previously isolated from apples in Lleida (Spain) during 2004 and 2005 seasons. All isolates are maintained in the culture collection of the Food Technology Department of Lleida University. The isolates were sub-cultured on malt extract agar (MEA) plates and incubated at 25 °C for 7 days to enable significant sporulation. After incubation, a sterile inoculation loop was used to remove the conidia from MEA plates. Conidia were

Effect of marginal conditions on intraspecific growth differences

All isolates of P. expansum grew under both conditions tested. Analysis of variance revealed significant differences in growth among incubation conditions; moreover, significant differences among isolates were found and these differences were dependent on environmental conditions (p < 0.05). Table 1 shows median values of growth rate (μ, mm/day) and lag phase (λ, day) of all isolates for the conditions studied and their dispersion. Both parameters calculated for the 79 isolates were not normally

Discussion

Several isolates of P. expansum could contaminate the same food. However, individual strains exhibit differences in their growth and in the amount of mycotoxin produced (Romero et al., 2010).

In our work, we compared growth parameters (μ and λ) and patulin production of seventy nine isolates of P. expansum incubated at two different temperatures. All isolates grew at 1 and 20 °C. Nevertheless, some isolates showed an exceptional response with a minor adaptation compared to the rest of the

Acknowledgement

The authors are grateful to the European (MYCOREDKBBE-2007-2-5-05 project), Spanish (AGL2010-22182-C04-04 project), Comissionat per a Universitats i Recerca d'Innovació, Universitats I Empresa de la Generalitat de Catalunya (AGAUR) and European Social Fund for the financial support.

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