Menadione (2-methyl-1,4-naphthoquinone) induces oxidative stress to regulate growth and ochratoxin A biosynthesis in Aspergillus westerdijkiae fc-1.

Abstract

Aspergillus westerdijkiae fc-1 is a foodborne fungus that produces ochratoxin A (OTA), a toxic and potentially carcinogenic mycotoxin posing serious food safety and health risks. This study investigated the impact of the oxidative stress-inducing agent menadione (2-methyl-1,4-naphthoquinone) on A. westerdijkiae fc-1 growth and OTA biosynthesis. Wild-type (WT) and ΔOTAbZIP (OTAbZIP knockout) mutant strains of A. westerdijkiae fc-1 were exposed to varying concentrations of menadione (0, 0.05, 0.25, 0.50, and 0.75 mmol/L) to evaluate their oxidative stress responses. High-performance liquid chromatography (HPLC) was used to quantify OTA production, and quantitative real-time polymerase chain reaction (qRT-PCR) was performed to determine the relative expression levels of the OTA biosynthetic genes (otaA-D) and the HOG1 gene. Increasing menadione concentrations significantly inhibited both fungal growth and OTA production. At 0.75 mmol/L, fungal growth inhibition reached 88.91% (WT) and 88.89% (ΔOTAbZIP) at 6 dpi. OTA production in the WT strain decreased significantly with increasing menadione concentrations and was undetectable at ≥ 0.50 mmol/L. qRT-PCR analysis showed up-regulation of the HOG1 gene at higher menadione levels. In the WT strain, HOG1 expression increased at 0.25 mmol/L, whereas in the ΔOTAbZIP mutant it was elevated at both 0.25 and 0.50 mmol/L, indicating activation of the HOG-MAPK (high-osmolarity glycerol-mitogen-activated protein kinase) pathway under oxidative stress. These results demonstrate that menadione inhibits both fungal growth and OTA biosynthesis in A. westerdijkiae fc-1 and implicate oxidative stress signaling in the regulation of OTA production. The ΔOTAbZIP mutant of A. westerdijkiae fc-1, which lacks the OTAbZIP transcription factor, showed restricted growth and failed to produce OTA. Compared with the wild type, the mutant exhibited higher HOG1 expression, whereas the OTA biosynthetic genes (otaA-D) were significantly down-regulated. This study advances understanding of OTA regulation and provides a basis for controlling its contamination in agriculture and food systems.