Citral-based acylthiourea derivative enhances the efficacy of thiophanate-methyl against Rhizoctonia solani through membrane destabilization.

Rhizoctonia solani is a highly destructive fungal pathogen of rice, and effective management currently depends heavily on chemical fungicides such as thiophanate-methyl (TM). To establish a sustainable approach for enhancing fungicide efficacy, we screened 22 citral-based acylthiourea derivatives and identified compound e3 as a highly effective synergist of thiophanate-methyl (TM), achieving a synergistic ratio of 3.88. When the molar ratio of e3 is 20 %, the mixture of e3 and TM (SETM) exhibits a pronounced antifungal activity against R. solani, both in vivo and in vitro. Integrated transcriptomic and metabolomic analyses demonstrated that steroid biosynthesis and membrane lipid metabolism were the primary pathways affected following the addition of e3. Notably, genes including Cytochromes P450 and sterol reductase ERG24 were significantly upregulated and exhibited strong correlations with multiple membrane lipid metabolites. Physiological and biochemical analyses revealed that e3 could induce an increase in ergosterol content, promote the accumulation of reactive oxygen species (ROS), and enhance the activity of antioxidant enzymes. Furthermore, SETM exhibited additional effects by increasing membrane permeability, causing intracellular substance leakage, and inhibiting energy metabolism. Importantly, the incorporation of e3 did not enhance the cytotoxicity of TM toward mammalian cells. These findings show that e3 is a highly effective synergist that disrupts fungal membrane lipid homeostasis and impairs key physiological functions, significantly enhancing TM's antifungal activity. The study advances understanding of fungicide-synergist interactions and offers a sustainable strategy to improve efficacy without sacrificing safety.