Antifungal Activity of Organic Acid-Based Natural Deep Eutectic Solvents against Necrotrophic Phytopathogen Agroathelia rolfsii.

Natural deep eutectic solvents (NADES) have become a popular green solvent due to their environmentally friendly properties. The present study investigated the biocompatibility of organic acid-based NADES systems, specifically glucose: citric acid mixtures, and their methanolic blends (20% v/v) against the phytopathogen Agroathelia rolfsii. A. rolfsii is a necrotrophic phytopathogen affecting more than 500 plant species. Physicochemical analysis, including conductivity, viscosity, polarity, and density, revealed that methanol addition increased viscosity, decreased conductivity, and caused minimal changes in polarity and density. Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopy confirmed the enhanced hydrogen bonding in methanol-mixed NADES, while differential scanning calorimetry (DSC) demonstrated the characteristic amorphous thermal behavior of the glucose-citric acid NADES, supporting their successful formation. Among the three NADES formulations (glucose:citric acid in molar ratios of 1:1, 1:2, and 2:1), the 1:2 ratio demonstrated the highest antifungal activity, even at low concentrations (25-50 μL/mL). Conductivity and nucleic acid leakage assays showed increased release of intracellular ions and nucleic acids from treated fungal cells, confirming membrane damage and loss of cellular integrity. Scanning electron microscopy revealed disrupted, uneven hyphae in A. rolfsii treated with NADES, while methanol-enriched NADES completely inhibited fungal growth. These findings suggest that hydrogen bonding and electrostatic interactions in the solvents compromised fungal cell walls and membranes, disrupting cellular integrity. This study demonstrates that economical sugar:organic acid-based NADES exhibited notable antifungal activity against phytopathogens under in vitro conditions, supporting their further exploration as sustainable antifungal formulations.