Patulin

Penicillium expansum

Gene cluster

Mycotoxins

Apples

Gene expression

Patulin is a polyketide-derived mycotoxin, the risk of exposure to which has emerged as an important topic given the high number of toxic effects that have assigned to it. Patulin produces acute and chronic toxicity mainly genotoxicity, cytotoxicity, mutagenicity as well as immunotoxicity (Puel et al., 2010). It is currently one of the few mycotoxins whose levels in food are regulated in many countries around the world because of its perceived harmful effects in humans, particularly children, who are most susceptible to patulin toxicity (Brandon et al., 2012).

Patulin is produced by a number of species belonging to the Aspergillus, Byssochlamys and Penicillium genera (Varga et al., 2007, Houbraken et al., 2006). Of these, Penicillium is the most prolific with 14 species identified as patulin producers (Frisvad et al., 2004, Vansteelandt et al., 2012). Within this genus, Penicillium expansum is considered as the main source of patulin in food and consequently the species of greatest concern regarding public health and economic risk (Morales et al., 2008). P. expansum is the causal agent of a common post-harvest disease known as blue mold rot (Baert et al., 2007). Fruit products in general and apples in particular, are one of the sectors most affected by this pathogen, and are considered by far the main route of entry of patulin into the food chain (Chen et al., 2004, Baert et al., 2012). The Europe…

2.1. Fungal growth and DNA isolation

A filamentous fungal strain of P. expansum NRRL 35695 isolated from grapes in the Languedoc Roussillon region of France was used in this study. The isolate was previously shown to belong to this Penicillium species according to the DNA sequencing of the ITS gene region. Furthermore, its ability to produce patulin was confirmed. The fungal strain was freshly cultivated on PDA media (Fluka, Saint-Quentin Fallavier, France) and allowed to grow for 7 days at 25 °C to enhance sporulation. This culture was then used to inoculate a 250 mL Erlenmeyer flask containing 100 mL of yeast extract-sucrose (YES) broth with 105 spores and it was then incubated at room temperature under constant agitation for 4 days. The liquid culture was filtered through Whatman No. 42 filter paper and the mycelium was ground up under liquid nitrogen. DNA was extracted according to the method described by Gardes and Bruns (1993) with some modifications. In this method, the powdered mycelium was collected in microfuge tubes, homogenized with 700 μL of lysis buffer [2% CTAB (Fluka, Saint-Quentin Fallavier, France), 1.4 M NaCl (Euromedex, Souffelweyersheim, France), 20 mM EDTA pH 8 (Sigma-Aldrich, Saint-Quentin Fallavier, France), 100 mM Tris–HCl pH 8 (Euromedex, Souffelweyersheim, France)] and incubated at 50 °C for 10 min then cooled on ice for 1 h. The sample was late…