Persistence of antifungal activity in stored tobacco (Nicotiana tabacum L.) pyrolysis oil despite the minor contribution of nicotine: insights into metabolite interactions against Fusarium proliferatum

The chemical characteristics and antifungal activity of tobacco ( Nicotiana tabacum ) pyrolysis crude oil were evaluated following extended storage and multi-stage fractionation. The crude oil was separated into 100% n-hexane (H100), n-hexane:ethyl acetate 99:1 (H99E1), n-hexane:ethyl acetate 95:5 (H95E5), 100% ethyl acetate (E100), and 100% methanol (M100) fractions. Characterization was performed using FTIR, spectrophotometry, LC-MS, HPTLC densitometry, particle size analysis (PSA), and GC–MS. Antifungal activity against Fusarium proliferatum was assessed by measuring mycelial growth inhibition, mycelial dry weight, spore density, and fluorescence. The crude oil exhibited the strongest and most consistent antifungal activity, with inhibition reaching 47.83% at 10,000 ppm but remaining below 50%. Fractionation resulted in change to metabolite distribution, functional groups, particle size, and nicotine content among the fractions. Notably, the E100 fraction maintained antifungal activity even when nicotine was undetectable. Pearson correlation analysis demonstrated that no single chemical or physicochemical parameter consistently accounted for antifungal activity, and smaller particle size did not necessarily enhance inhibition. These findings indicate that antifungal activity was associated with differences in the comprehensive chemical composition of the crude oil and its fractions, rather than with nicotine, particle size, or any single measured parameter.