**2. Results**

#### *2.1. In Vitro Inhibition of Fungal Growth by the Seven Essential Oils*

The effects of increasing concentrations of seven essential oils on mycelial growth of the fungi *A. alternata* and *S. cucurbitacearum* were investigated. These essential oils were from various sources, and are defined as (see Table 1): *C.cit*, *Cymbopogon citratus* (lemon grass); *L.dent*, *Lavandula dentata* (lavender); *L.hyb*, *Lavandula hybrida* (lavandin); *M.alt*, *Melaleuca alternifoglia* (tea tree); *L.nob*, *Laurus nobilis* (bay laurel); *O.maj1/2*, *Origanum majorana* 1/2 (majoram).

As can be seen in Figures 1 and 2, and as summarized in Tables 2 and 3, all of these essential oils inhibited the growth of these two fungi in a dose-dependent manner. The greatest inhibitory activity was shown by the *C.cit* essential oil, with 100% inhibition of mycelial growth of both *A. alternata* and *S. cucurbitacearum* reached at 0.6 mg/mL and 0.9 mg/mL, respectively (Table 2). *A. alternata* was generally more sensitive to these essential oils than *S. cucurbitacearum*, and at 1 mg/mL essential oils, its mycelia growth was inhibited by 55.0%, 71.5%, 68.2%, 36.1%, 74.2%, and 90.5% by *L.dent*, *L.hyb*, *M.alt*, *L.nob*,

*O.maj1*, and *O.maj2*, respectively (Table 2). At the same essential oil concentration, *S. cucurbitacearum* radial growth was inhibited by 73.5%, 74.0%, 73.7%, 65.3%, 60.0%, and 67.3%, respectively (Table 3). The positive control of the fungicide combination of 25 g/L difenoconazole plus 25 g/L fludioxonil completely inhibited the mycelial growth of *A. alternata* at all concentrations tested. Against *S. cucurbitacearum*, this fungicide combination at 0.1, 0.5, and 1 mg/mL inhibited the mycelial growth by 75.7%, 84.9%, and 86.7%, respectively.

In addition, the *C.cit* essential oil had a fungicidal effect against *S. cucurbitacearum* from 900 μg/mL. Indeed, for *A. alternata*, *C.cit* had fungistatic effects at 0.6 mg/mL and 0.7 mg/mL, and it was fungicidal from 0.8 mg/mL (Table 4). These data show that the *C.cit* had potent antifungal activity against *A. alternata* and *S. cucurbitacearum* with IC50 values of 0.315 mg/mL and 0.102 mg/mL, respectively (Figure 3). The essential oils of *L.dent*, *L.hyb*, *M.alt*, *O.maj1*, and *O.maj2* showed moderate antifungal activities against *A. alternata*, with IC50 values from 0.473 mg/mL to 0.893 mg/mL, as similarly against *S. cucurbitacearum*, with IC50 values from 0.322 mg/mL to 0.884 mg/mL. However, *L.nob* showed only weak antifungal activities against both *A. alternata* and *S. cucurbitacearum*, as seen by its relatively high IC50 values of 1.310 mg/mL and 1.248 mg/mL, respectively (Figure 3).

**Table 1.** Details of the essential oils included in this study.


**Figure 1.** Representative experiment showing inhibition of *Alternaria alternata* mycelial growth by *Cymbopogon citratus* essential oil at 0.1 to 1 mg/mL and by the fungicide combination of 25 g/L difenoconazole plus 25 g/L fludioxonil at 0.1, 0.5 and 1 mg/mL, as seen after 8 days of incubation at 22 ± 2 ◦C.

at 0.1, 0.5 and 1 mg/mL, after 7 days of incubation at 22 ± 2 ◦C.

**Figure 2.** Representative experiment showing inhibition of *Stagonosporopsis cucurbitacearum* mycelial growth by the sevenessential oils: *C.cit*, *Cymbopogon citratus*; *L.dent*, *Lavandula dentata*; *L.hyb*, *Lavandula hybrida*; *M.alt*, *Melaleuca alternifolia*; *L.nob*,*Laurus nobilis*; *O.maj1/2*, *Origanum majorana* 1/2, at increasing concentrations (right to left; as indicated) from 0 mg/mL(control) to 1 mg /mL, and by the fungicide combination of 25 g/L difenoconazole plus 25 g/L fludioxonil (positive control)


**Figure 3.** Inhibitory concentration for 50% reduction (IC50) of mycelial growth of *Alternaria alternata* (**A**) and *Stagonosporopsis cucurbitacearum* (**B**) by the seven essential oils: *C.cit, Cymbopogon citratus; L.dent*, *Lavandula dentata; L.hyb*, *Lanvandula hybrida; M.alt*, *Melaleuca alternifolia*; *L.nob*, *Laurus nobilis; and O.maj1/2, Origanum majorana* 1/2. Data with different letters (**<sup>a</sup>**–**f**) are significantly different between treatments (*p* ≤ 0.05; Fisher's LSD).

#### *2.2. Chemical Profiles of the Essential Oils*

The 41 components given in Table 5 were identified as comprising from 97.7% (*O.maj1*) to 100% (*L.hyb*) of these essential oils. The oxygenated monoterpenes dominated in all of the essential oils, even though these belonged to different plant families and species. They represented more than two-thirds of the fraction in three of the four *Lamiaceae*: *L.dent* (81.1%), *L.hyb* (90.8%), and *O.maj2* (66.8%). The oxygenated monoterpenes were also the highest proportionally in *C.cit* (88.5%) and *L.nob* (70.3%). On the other hand, the compositions of *M.alt* and *O.maj1* were divided mainly between oxygenated monoterpenes as the main class (48.1%, 49.7%, respectively) and monoterpene hydrocarbons in similar proportions (40.4%, 44.3%, respectively). In more detail, *C.cit* showed α-citral (geranial; 51.6%) and β-citral (neral; 26.0%), whereby these two major oxygenated monoterpenes represented together over three-quarters of the total composition. In the *Lamiaceae*, almost two-thirds of *L.dent* was eucalyptol (63.5%) and β-selinene (4.1%). Instead, the total composition of *O.maj1* and *O.mag2* included around half and over two-thirds as terpenen-4-ol (44.8%) and *p*-cymene (68.2%), respectively, followed by γ-terpinene (12.6%) for *O.maj1* and α-terpineol (5.4%) for *O.maj2*. For the two commercial essential oils, the main compounds of *L.hyb* were linalool (33.7%) and linalyl acetate (27.7%), followed by camphor (9.3%), while *M.alt* showed terpinen-4-ol (41.1%) as 86% of its oxygenated monoterpene, with γ-terpinene (16.0%), p-cymene (9.3%), and α-terpinene (6.1%), together representing 78% of the monoterpene hydrocarbons. Finally, more than half of the identified fractions of the *Lauraceae L.nob* were characterized by the combination of eucalyptol (47.9%) and α-terpinyl acetate (10.2%).




