*2.4. Fungal Cell Membrane Permeability Assay*

This assay was carried out to explain the possible mechanism of the antifungal activity of the tested EO. In general, the fungicidal effect of EO depends on the destruction of the fungal cell membrane that increases the cell permeability. For that reason, the current assay was performed to investigate the effect of mint EO and its main single constituents on the CMP of the tested phytopathogenic fungi by measuring their electric conductivity (EC) [21,22].

Figure 2 showed the effect of peppermint EO at different doses on the mycelium electrical conductivity (MEC) as indication of the cell membrane permeability (CMP) of the four tested fungi. Generally, the effect of the studied EO on the CMP of all tested fungi was dose-dependent. In particular, the highest tested concentration (7.0 mg/mL) showed the EC values 87.2, 85.3, 92.3 and 85.1 S/cm corresponding to the CMP of *M. fructicola*, *B. cinerea*, *A. niger* and *P. expansum*, respectively. On the other hand, the concentration 7.0 mg/mL showed a significant increase in the CMP in the case of *M. fructicola* and *B. cinerea*, whereas

there was no significant difference between the two doses 5.0 and 7.0 mg/mL regarding *A. niger* and *P. expansum.* In addition, there was a dramatical increase in the CMP in the case of *P. expansum* after treatment with 5.0 mg/mL.

Tested concentrations of Mentha EO (mg/mL)

**Figure 2.** The effect of peppermint EO on mycelium electrical conductivity of the tested fungi. Where, (**A**): *Monilinia fructicola*; (**B**): *Botrytis cinerea*; (**C**): *Aspergillus niger*; (**D**): *Penicillium expansum*. C-ve: negative control (potato dextrose broth). Differences between the tested concentrations for each tested fungus indicate mean values significantly different at *p* < 0.05 according to one-way ANOVA for each fungus combined with Duncan post hoc multiple comparison test.

Figure 3 showed the effects of two single constituents of peppermint EO (menthol and menthone) at different doses on the MEC of the four tested fungi. In the case of menthol, it showed a dose-dependent effect on CMP of *M. fructicola*, *B. cinerea* and *A. niger,* whereas the CMP was highly decreased after treatment with 0.8 mg/mL in the case of *P. expansum*. The EC values were 35.9, 37.2, 39.0 and 45.5 S/cm for *M. fructicola*, *B. cinerea*, *A. niger* and *P. expansum*, respectively. Regarding the menthone, it showed a dose-dependent effect on the CMP against *M. fructicola*, *B. cinerea* and *P. expansum*. The EC values were 38.9, 44.5, 37.8 and 34.0 S/cm for *M. fructicola*, *B. cinerea*, *A. niger* and *P. expansum*, respectively.

Tested concentrations of Mentha EO (mg/mL)

**Figure 3.** The effect of single constituents of peppermint EO on mycelium electrical conductivity of the tested fungi. Where (**A**): *Monilinia fructicola*; (**B**): *Botrytis cinerea*; (**C**): *Aspergillus niger*; (**D**): *Penicillium expansum*. C-ve: negative control (potato dextrose broth). Differences between the tested concentrations for each tested fungus indicate mean values significantly different at *p* < 0.05 according to one-way ANOVA for each fungus combined with Duncan post hoc multiple comparison test.
