Untargeted Profiling of Fungicide Resistance‐Related Extracellular Metabolites of Phytophthora infestans Using Two‐Dimensional Gas Chromatography Coupled With Time‐of‐Flight Mass Spectrometry

ABSTRACT Late blight, caused by the oomycete Phytophthora infestans , is the most devastating and economically significant disease of potatoes and tomatoes. Its management relies heavily on preventive fungicide applications, yet reports of fungicide resistance in P. infestans have been increasing worldwide. This study investigated extracellular metabolic changes following treatment with the fungicides metalaxyl‐M and fluazinam in US8 (fungicide‐resistant) and US23 (fungicide‐sensitive) isolates of P. infestans . A total of 143 extracellular metabolites were identified and quantified using untargeted metabolite profiling. Significant variations in metabolite composition were observed 14 days post‐inoculation and at higher fungicide concentrations. Metabolites associated with fungicide resistance included fatty acids (stearic, palmitic and oleic), sugars (sucrose and trehalose), amino acids (proline, serine, glutamic acid, tryptophan and isoleucine), organic acids (shikimic, citric, succinic and malic) and other compounds (ethanolamine, niacin, niacinamide and putrescine). Differentially expressed metabolites were upregulated or downregulated depending on fungicide dose, time of treatment and isolate sensitivity with distinct groupings most evident in the resistant isolate. The resistant isolate showed a stronger (time‐ and dose‐dependent) metabolic response under fungicide exposure, and resistance was associated with a distinct extracellular metabolic signature. Overall, this study reveals key metabolic responses of resistant versus sensitive P. infestans isolates to metalaxyl‐M and fluazinam, offering new insights into pathways by which the pathogen adapts to fungicide stressors. Such knowledge is critical for understanding the biochemical basis of fungicide resistance and informing the development of more sustainable management strategies for late blight.