Thymbra spicata extracts against soilborne fungi: linking chemical composition, antifungal activity, and molecular...

In this study, the antifungal activities of essential oil, supercritical carbon dioxide (SC-CO₂) extract, methanol extract, and SC-CO₂+ethanol extract obtained from Thymbra spicata L. were evaluated in vitro against the soilborne fungal pathogens Sclerotinia sclerotiorum, Macrophomina sp., Rhizoctonia solani, and Fusarium oxysporum, which cause substantial economic losses in cultivated plants. The chemical profiles of the extracts were determined using GC-MS. Carvacrol and p-cymene were identified as the major constituents of the SC-CO₂ and SC-CO₂+ethanol extracts, accounting for 86.79% and 4.70%, respectively, of the SC-CO₂ extract and 72.07% and 4.6%, respectively, of the SC-CO₂+ethanol extract. Among the essential oils, carvacrol (89.61%) and trans-β-caryophyllene (1.86%) were identified as the predominant constituents, whereas the methanol extract was characterized by a greater proportion of non-volatile constituents, mainly carvacrol (33.93%) and linoleic acid (21.19%). Antifungal bioassays were conducted using the contact effect method at concentrations of 0.25, 0.50, 1.00, 2.00, and 4.00 µl/ml. T. spicata essential oil (1.00, 2.00, and 4.00 µl/ml) and the SC-CO₂ extract (2.00 and 4.00 µl/ml) completely inhibited (100%) the mycelial growth of S. sclerotiorum in a dose-dependent manner, indicating fungicidal activity. Similarly, complete inhibition of Macrophomina sp. was observed with essential oil at 2.00 and 4.00 µl/ml and with SC-CO₂ and SC-CO₂+ethanol extracts at 4.00 µl/ml. The essential oil and SC-CO₂ extracts (1.00, 2.00, and 4.00 µl/ml) completely inhibited the mycelial growth of R. solani and F. oxysporum, demonstrating fungicidal activity. These differences in inhibition rates were statistically significant (p ≤ 0.05). In contrast, the methanol extract had no inhibitory effect on any of the tested concentrations. ADMET analyses were performed to assess the acute toxicity profiles of carvacrol and p-cymene. Molecular docking against the 6CR2 target protein was conducted to assess ligand-protein interactions. Among the essential oil constituents evaluated, carvacrol and p-cymene exhibited the most favorable binding energies, supporting their potential as lead antifungal compounds.