Transcriptomic analysis of inhibition by eugenol of ochratoxin A biosynthesis and growth of Aspergillus carbonarius

Abstract

Aspergillus carbonarius is a saprophytic filamentous fungus that produces the mycotoxin ochratoxin A (OTA), a powerful hepatotoxin, nephrotoxin and teratogen, which causes global problems with food contamination during storage. Various methods were applied to assess the impact of eugenol on the OTA production and mycelial growth of A. carbonarius. The minimum inhibitory concentration (MIC) for eugenol treatment was 0.8 μL/mL. Eugenol treatment at 0.2 μL/mL (1/4 MIC) resulted in 87.7% decrease in OTA production in spite of only a 26.9% inhibition of mycelial growth, and actually caused marked morphological changes of spores and mycelia by Scanning electron microscopy (SEM). A total of 400 differentially expressed genes (DEGs) were detected by comparatively transcriptomic analysis of A. carbonarius in the presence or absence of eugenol, including 324 down-regulated and 76 up-regulated DEGs. The transcription of the clustered genes for OTA biosynthesis was significantly reduced under eugenol stress and further confirmed by RT-qPCR. LaeA, a global regulator for secondary metabolites biosynthesis, was markedly downregulated by eugenol. Eugenol disrupted cell integrity by the disturbance of the DEGs expression in relation with cellular structure, which was also proved by monitoring chitinase activity, malondialdehyde (MDA) and ergosterol content. Changes in superoxide dismutase (SOD) and catalase (CAT) activity, and glutathione (GSH) content showed that eugenol induced oxidative stress, which was also consistent with the changes in gene expression. These findings provide useful insights into the antifungal and antimycotoxigenic mechanisms of eugenol and improve understanding of the effects of eugenol on A. carbonarius.