Methodological insights for assessing the antifungal activity of eutectic systems as sustainable biopesticides

The environmental concerns and the emergence of fungal strains resistant to conventional fungicides underscore the urgent need for sustainable agriculture alternatives. Natural Deep Eutectic Solvents (NADES) have gained attention as promising, biodegradable systems with potential antifungal activity. However, the absence of standardized methodologies for assessing their biological efficacy has limited progress toward their practical implementation. This study assessed the antifungal activity of four NADES against Monilinia fructicola using a comprehensive in vitro approach combining solid and liquid methods, providing a more accurate interpretation of their biological effects. Disk diffusion method revealed limitations, the results being non-comparable with the other methods evaluated. For amended agar assay and liquid methods, all NADES completely inhibited mycelial growth and conidial germination, while individual components showed lower activity than their corresponding eutectic systems. LGH (lactic acid, glucose and water) was the most effective, achieving total inhibition at 2% in solid medium and 1% in liquid medium. ChClUH (choline chloride, urea and water) exhibited moderate activity (3-5% for full inhibition), whereas CGlyH (citric acid, glycerol and water) and ChClGly (choline chloride and glycerol) were markedly less effective, requiring 10-15%. Physicochemical characterization revealed that dilution significantly affected viscosity, polarity, and pH, highlighting the influence of water addition and the importance of evaluating NADES at biologically relevant dilutions. Microscopic analyses confirmed that LGH reduced conidial viability and caused pronounced morphological alterations in conidia and hyphae. Furthermore, in vivo assays on plums demonstrated LGH efficacy of 38-63% depending on concentration. Overall, these findings contribute to establishing methodological criteria for assessing NADES as biopesticides and strengthen the scientific basis for their sustainable use in crop protection.