The development of natural protective agents as alternatives to chemical fungicides is currently in the spotlight. In the present investigation, chemical composition and antifungal activities of thyme, cinnamon, rosemary and marjoram essential oils (EO), as well as synergism of their possible double and triple combinations were investigated. The compositions of the oils were determined by GC/MS. For determination of antifungal activity against Penicillium expansum and Botrytis cinerea, a broth microdilution method was used. The possible interactions of some essential oil combinations were performed by the two and three-dimensional checkerboard assay and isobologram construction. An in vivo antifungal assay was performed by artificial wounding of pear fruits. The maximum antifungal activity was demonstrated by thyme and cinnamon oils which displayed lower MIC values whereas rosemary and marjoram oils with MIC range between 2500 and 10,000 μg/mL exhibited weak antifungal activities against tested fungi. In synergy testing, some double combinations (thyme/cinnamon, thyme/rosemary, cinnamon/rosemary) were found to be synergistic (FICi ≤0.5). The triple combination of thyme, cinnamon and rosemary was synergistic for B. cinerea and P. expansum (FICi values of 0.5 and 0.375, respectively); while combination of cinnamon, marjoram and thyme exhibited additive and synergistic effect against P. expansum (FIC =0.625) and B. cinerea (FIC =0.375) respectively. The usage of a mathematical Gompertz model in relation to fungal kinetics, showed that the model could be used to predict growth curves (R2 =0.993 ± 0.05). For B. cinerea, Gompertz parameters for double and triple combination treatments showed significant increase in lag phase (1.92 and 2.92 days, respectively) compared to single treatments. Increase lag time up to 2.82 days (P < 0.05) also observed in P. expansum treated by triple combination of EOs. Base on the results, the lowest maximum growth rate (0.37 mm/day) was observed in B. cinerea treated by triple combination of thyme, cinnamon and rosemary. The in vivo test also demonstrated considerable inhibitory effects of EO combination treatments. Average lesion diameter of pears treated with triple combination of cinnamon/rosemary/thyme (78, 1250, 39 μg/mL) was 6 mm and 8 mm against B. cinerea and P. expansum respectively, in 10 days at 25 °C. Results also showed that double combination of thyme/cinnamon (78, 156 μg/mL) has more inhibitory effect than single EO treatments.

Keywords