4.3.3. Antifungal Activity

Incorporation assay. The possible fungicidal activity of the studied EO was evaluated at three different doses (0.1, 1 and 5 mg/mL) following the incorporation method [43–45] as explained below. The EO was incorporated into Potato Dextrose Agar (PDA) medium at 45 ± 2 ◦C. Fungal disks (0.5 cm) from each of the phytopathogenic fungi (96 h fresh culture) were deposited in the centre of each Petri dish. All plates were incubated at 22 ± 2 ◦C for 96 h in darkness. As negative control, PDA plates without any treatments were inoculated only with each fungus. The diameter of fungal mycelium growth was measured in mm ± Standard Deviations (SDs) between the three replicates [46] and the percentage of growth inhibition (PGI%) was calculated using Equation (1) [47] compared to synthetic fungicides Azoxystrobin (0.8 μL/mL), a large spectrum fungicide, as control according to the international limit of microbicide standards.

$$\text{PGI} \left( \% \right) = \frac{\text{GC} - \text{GT}}{\text{GC}} \times 100 \tag{1}$$

where PGI is the percentage of growth inhibition, GC is the average diameter of fungal mycelium in PDA negative control and GT is the average diameter of fungal mycelium on the oil-treated PDA dish.

### *4.4. Cell Membrane Permeability*

The CMP effect of the mint EO and its two main principals was determined by measuring the potential of electrical current transport through water as molar conductivity (MC) or electrolytic conductivity (EC) as reported by Elshafie et al. [21]. This assay was performed by transferring five mycelial discs (0.5 cm diameter) from fresh culture of each tested fungus into Potato Dextrose Broth (PDB) medium and incubated under shaking condition (180 rpm/min), at 28 ◦C for 96 h. A gram and half of dried mycelia from each fungal species was re-suspended into 20 mL of each tested EO concentration at 1, 3, 5 and 7 mg/mL or single components at 0.1, 0.2, 0.4, 0.8 and 1.6 mg/mL and incubated at 22 ± 2 ◦C. EC values have been measured after 72 h of incubation. The IP% of EC value was calculated following Equation (2) [21].

$$\text{IP (\%)} = \frac{\text{EC t}}{\text{EC ctrl}} \times 100\tag{2}$$

where EC t. is the electrical conductivity of the treated sample and EC ctrl. is the electrical conductivity of the PDB broth culture.

#### *4.5. Fungicidal Microdilution Broth Assay*

The MFC was carried out against the four tested pathogenic fungi using a 96-well microplate (Nunc MaxiSorp®, Vedbaek, Denmark) by a micro-dilution method [22,48]. Four millilitres of liquid suspension from fresh fungal cultures (96 h) was prepared at 10<sup>8</sup> spore/mL. The tested EO was dissolved in PDB at 1.0, 3.0, 5.0 and 7.0 mg/mL, whereas the tested concentrations for menthol and menthone were 0.1, 0.2, 0.4, 0.8 and 1.6 mg/mL. The proposed concentrations of this assay were selected according to the obtained results from the preliminary in vitro antifungal assay. One hundred microlitres/well from each prepared concentration of EO and 100 μL/well of PDB media were added into the 96-well microplates then 30 μL/well of fungal suspension from each tested fungus was uploaded per all wells. All plates were incubated at 24 ± 2 ◦C. The absorbance was measured at λ = 450 nm using microplate reader instrument (DAS s.r.l., Rome, Italy) after 24 h. The MGP percentage was calculated using Equation (3). The whole experiment was repeated in triplicate ± SDs.

$$\text{MGI} \left( \% \right) = \frac{\text{Abs. t}}{\text{Abs. c}} \times 100 \tag{3}$$

where Abs. t: is the value of the absorbance at 450 nm for each treatment; Abs. c: is the value of the absorbance at 450 nm for the PDA + fungi as control.

## *4.6. Statistical Analysis*

The obtained results of the biological assays were subjected to one-way ANOVA for the statistical analysis. Then, the significance level of the findings was checked by applying *Tukey* B Post Hoc multiple comparison test with a probability of *p* < 0.05 using statistical Package for the Social Sciences (SPSS) version 13.0 (Prentice Hall: Chicago, IL, USA, 2004).
