Antifungal activity, physiological disruption, and toxicity mechanisms of difenoconazole in Sclerotinia sclerotiorum

Abstract

In this study, we evaluated the baseline sensitivity and toxicity mechanisms of difenoconazole against Sclerotinia sclerotiorum. The mean EC₅₀ value for 97 isolates collected in 2022 was 0.2305 ± 0.1924 μg/mL with individual EC₅₀ values ranging from 0.0280 to 1.0989 μg/mL. The logarithms of EC₅₀ values fitted the normal distribution and with a relatively flat peak. Light microscopic observations revealed that difenoconazole treatment resulted in shorter, more contorted hyphal of S. sclerotiorum with increased offshoots at the tips. Transmission electron microscope observations showed that the cell wall of the difenoconazole-treated hyphae became thicker and some organelles were destroyed. The number of sclerotia significantly decreased with difenoconazole at 1.6 μg/mL (P < 0.05). When difenoconazole was combined with Congo red or sodium dodecyl sulfate, the observed mycelial growth inhibitions were lower or higher than expected, respectively. This suggests that difenoconazole may reduce the content of chitin and damage the plasma membrane integrity. Further analyses revealed that difenoconazole induced reactive oxygen species accumulation, decreased cell membrane permeability, reduced oxalic acid content, pectinases activity, and decreased ergosterol content (P < 0.05). No correlation was found between the sensitivity of S. sclerotiorum isolates to difenoconazole and three DMI fungicides. In both detached rapeseed leaves and potted rapeseed plants, the protective and curative efficacies of difenoconazole and carbendazim are slightly different. The relative expression levels of CYP51 and Ssabc genes significantly rose after treatment with difenoconazole at 2.2 μg/mL. These findings are crucial for developing effective resistance management strategies for sclerotinia stem rot.