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Int J Food Microbiol. 2016 Mar 2;220:50-7. doi: 10.1016/j.ijfoodmicro.2016.01.004. Epub 2016 Jan 9.

Isoepoxydon dehydrogenase (idh) gene expression in relation to patulin production by Penicillium expansum under different temperature and atmosphere.

Author information

1
Institute for Agricultural and Fisheries Research (ILVO), member of Food2Know, Brusselsesteenweg 370, 9090 Melle, Belgium.
2
Ghent University, Faculty of Bioscience Engineering, Department of Food Safety and Food Quality, Laboratory of Food Microbiology and Food Preservation, member of Food2Know, Coupure Links 653, 9000 Gent, Belgium.
3
Ghent University, Faculty of Bioscience Engineering, Department of Food Safety and Food Quality, Laboratory of Food Chemistry and Human Nutrition (nutriFOODchem), member of Food2Know, Coupure Links 653, 9000 Gent, Belgium.

Abstract

Penicillium expansum growth and patulin production occur mainly at post-harvest stage during the long-term storage of apples. Low temperature in combination with reduced oxygen concentrations is commonly applied as a control strategy to extend apple shelf life and supply the market throughout the year. Our in vitro study investigated the effect of temperature and atmosphere on expression of the idh gene in relation to the patulin production by P. expansum. The idh gene encodes the isoepoxydon dehydrogenase enzyme, a key enzyme in the patulin biosynthesis pathway. First, a reverse transcription real-time PCR (RT-qPCR) method was optimized to measure accurately the P. expansum idh mRNA levels relative to the mRNA levels of three reference genes (18S, β-tubulin, calmodulin), taking into account important parameters such as PCR inhibition and multiple reference gene stability. Subsequently, two P. expansum field isolates and one reference strain were grown on apple puree agar medium (APAM) under three conditions of temperature and atmosphere: 20 °C - air, 4 °C - air and 4 °C - controlled atmosphere (CA; 3% O2). When P. expansum strains reached a 0.5 and 2.0 cm colony diameter, idh expression and patulin concentrations were determined by means of the developed RT-qPCR and an HPLC-UV method, respectively. The in vitro study showed a clear reduction in patulin production and down-regulation of the idh gene expression when P. expansum was grown under 4 °C - CA. The results suggest that stress (low temperature and oxygen level) caused a delay of the fungal metabolism rather than a complete inhibition of toxin biosynthesis. A good correlation was found between the idh expression and patulin production, corroborating that temperature and atmosphere affected patulin production by acting at the transcriptional level of the idh gene. Finally, a reliable RT-qPCR can be considered as an alternative tool to investigate the effect of control strategies on the toxin formation in food.

KEYWORDS:

Atmosphere; P. expansum; Patulin; RT-qPCR; Temperature; idh gene expression

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