Investigation of the mode of action and cytotoxicity of prothioconazole on Fusarium phytopathogens

Abstract

Prothioconazole's effects on Fusarium graminearum PH-1 and F. culmorum FcUK99 reference strains were investigated at the cellular level and via genomic, epigenomic, transcriptomic, and chromatographic approaches. Prothioconazole's minimum inhibitory concentrations (MIC) on them were determined as 1.2 μg/mL and 1 μg/mL, respectively. Its MIC₂₅ and MIC₅₀ doses (0.3 μg/mL and 0.6 μg/mL for PH-1; 0.25 μg/mL, and 0.5 μg/mL for FcUK99, respectively) reduced linear growth rates and cell viabilities in both strains. Additionally, MIC₅₀ stimulated the macroconidia production of FcUK99 and swelled them. Oxidative stress effect of prothioconazole was demonstrated by 2′,7′-dichlorodihydrofluorescein diacetate staining, monitoring of lipid peroxidation levels, and detection of alterations of antioxidant enzyme activities and expression levels of their transcripts. Apoptosis-like cell death was shown by acridine orange/ethidium bromide staining, determination of specific gene expression changes, and monitoring of caspase-3 activity. The correlation analysis revealed that apoptosis-like cell death could have resulted from prothioconazole-induced oxidative stress. Coupled restriction enzyme digestion-random amplification and enzyme-linked immunosorbent assay revealed that prothioconazole reduced the 5-mC content and the genomic template stability. The thin layer chromatography indicated that prothioconazole did not affect deoxynivalenol production in both strains while reducing 3-acetyl deoxynivalenol production in FcUK99. Real-time PCR findings demonstrated that prothioconazole upregulated tri5 expression in PH-1, downregulated it in FcUK99. Moreover, this compound was not toxic to HEK293 and L929 cells. The study showed that prothioconazole was a suitable antifungal compound for controlling Fusarium spp. because of its strong, complex, and species-specific effects on these two phytopathogens while maintaining non-toxicity towards mammalian cells.