Abstract
Abstract The spread of fungicide-resistant pathogens has increased interest in plant-based antifungal agents for agricultural applications. We compared subcritical CO2 extraction (SbFE), ultrasound-assisted extraction (UAE), hot reflux extraction (HRE), and Soxhlet extraction (SE) to determine their effects on the metabolite composition of Tagetes erecta L. and antifungal efficacy against Botrytis cinerea. GC–MS and UHPLC–QTOF–MS analyses, combined with chemometric methods, revealed method-specific metabolite profiles. SbFE selectively enriched thiophene derivatives and sesquiterpenes, while UAE yielded the broadest range of phenolic acids, flavonoids, and terpenoids. HRE recovered thermostable terpenes, whereas SE concentrated phenolic compounds and sterols but lacked thiophenes due to thermal degradation. Despite these chemical differences, both SbFE and SE preparations produced complete inhibition of B. cinerea under the applied test conditions (30.0 mm inhibition zones), apparently through distinct mechanisms: phototoxic thiophene-terpenoid activity versus phenolic-sterol membrane disruption. Principal component analysis confirmed clear separation between extraction methods. These results demonstrate that extraction technique must be selected according to target compound classes and intended antifungal mechanisms: SbFE for phototoxic applications, SE for contact biocides, and UAE for broad-spectrum screening.