**3. Discussion**

In this study, these in vitro assays for the antifungal activities of these seven essential oils on mycelial growth of two fungi showed that the lemongrass (*C.cit*) essential oil was the most effective. The mycelial growth of *A. alternata* was totally inhibited by application of *C.cit* at a moderate concentration, while *S. cucurbitacearum* was completely inhibited at the highest *C.cit* concentration, with fungicidal activity seen in both cases. Only a few studies have investigated lemongrass essential oils and these fungi, with most studies focused on the essential oil activity rather than its composition. Shafique et al. [29] reported total inhibition of *A. alternata* by a *C. citratus* essential oil, with an IC50 of 279.13 μL/L, as also reported by Jie et al. [31]. In the same year, Guimarães et al. [32] confirmed in vitro fungitoxic activity on *A. alternata*, with the essential oil rich in citral (69.3%) and myrcene (23.8%). For the second fungus here, *S. cucurbitacearum*, Fiori et al. [30] reported 100% inhibition of mycelial growth and spore germination at a rate of 20 μL *C. citratus* essential oil. Seixas et al. [28] reported the same result at 0.25, 0.5, 0.75, 1, and 1.25 mg/mL *C. citratus* essential oil. A number of studies have reported these high proportions of the two isomers α-citral and β-citral in *C. citratus* essential oils, even when collected from different countries [33–37], as also confirmed by the present study. Brügger et al. [38] reported that in addition to the high proportion of citral, their commercial *C. citratus* essential oil showed relevant amounts of nonan-4-ol (6.5%) and camphene (5.2%). These two compounds were completely absent in the *C.cit* essential oil used in the present study. A Brazilian commercial *C. citratus* essential oil also indicated a different composition, which was rich in nonterpenes, as especially 4,8-dimethyl-3,7-nonadien-2-one (25.0%), 1-heptadec-1-ynyl-cyclopentanol (9.6%), and 7,7-dimethyl-bicycloheptan-2-ol (8.0%); here, the proportion of citral was less than 37% [39]. The antifungal activity of *C. citratus* essential oil has also been reported against other fungi, including *Aspergillus flavus*. This activity can be ascribed to the presence of various components such as citral, geraniol, and βmyrcene [37,40]. According to some studies, citral and geranol can indeed inhibit the mycelial growth of *Fusarium oxysporum*, *Colletotrichum gloeosporioides*, *Bipolaris* sp. and *A. alternata* [41,42]. These major compounds in the *C. citratus* essential oil also have antioxidant and antimicrobial activities [43]. Furthermore, Kurita et al. [44] defined the fungicidal action of citral as due to its ability to receive electrons from the fungus cell, through charge transfer with an electron donor in the cell, which results in death of the fungus. In previous studies, β-myrcene and geraniol were found in *C.cit* essential oil. These compounds with citral can contributed to inhibit the mycelial growth of *A. alternata* and *S. cucurbitacearum*.

The cultivated lavender *L. dentata* essential oil showed eucalyptol (63.5%) as the major compound in the present study, which was higher than that previously reported for both inflorescences (46.3%) and the aerial parts (40.4%) [45]. Iranian lavandin (*L. hybrida*) has also been characterized by high proportions of oxygenated monoterpenes, with eucalyptol (41.1%) as the main component, followed by borneol (20.7%) and camphor (10.8%) [46]. All of these components were present in *L.hyb* in the present study, although in lower amounts (6.5%, 4.4%, 9.3%, respectively), with the main component here being linalool (33.7%) and linalyl acetate (27.7%). The antifungal activity of linalool on *Candida* species was recently studied by Dias et al. [47], who indicated the potential use of this unsaturated monoterpene as a strong candidate with antifungal potency. According to Pitarokili et al. [48], linalyl acetate was inactive against all of the fungi they studied, although it showed weak activity against only *Sclerotinia sclerotiorum*. On the contrary, they confirmed the antifungal effects of linalool.

Good antifungal effects on mycelial growth of *A. alternata* were also seen here using the *Origanum* essential oils. Even though the *O. majorana* essential oils tested here had the same classes of compounds shown in a Brazilian species studied by Chaves et al. [49], they did not show pulegone as the main compound. An Italian species investigated by Della Pepa et al. [50] was in partial agreemen<sup>t</sup> with the present study for the high amount of terpinen-4-ol (29.6%), while a Tunisian oregano species were characterized by similar

terpinen-4-ol content [51,52]. Moreover, Busatta et al. [53] showed that an Egyptian essential oil that was extracted by hydrodistillation of dried leaves of *O. majorana* showed the same dominance of terpinen-4-ol (31.8%) and γ-terpinene (13.0%). Although most studies on the composition of *Origanum* essential oils have agreed on the main compounds from *O. majorana* [54–57], these have indeed varied. The effectiveness of *Origanum* might be due to its high content of terpinen-4-ol, a monoterpene alcohol that is known to have good antifungal activity, as previously reported against *Fusarium avenaceum*, *Fusarium moniliforme*, *Fusarium semitectum*, *F. solani*, *F. oxysporum*, and *F. graminearum* [58,59]. Its fungicidal activity was also reported by Morcia et al. [60], who analyzed its potency on mycotoxigenic plant pathogens. However, Ebani et al. [61] showed weak activity of a tea tree essential oil on *Aspergillus fumigatus* even though terpinen-4-ol was present at relatively high levels. This might be explained by the synergistic effects among all of the different components in each of the essential oils.

The *M. alternifolia* essential oil in the present study was characterized by high levels of terpinen-4-ol (41.1%) and γ-terpinene (16.0%). These are comparable with the data reported by Elmi et al. [62] and Silva et al. [63]. In their investigations of Italian and Brazilian, commercial essential oils, they confirmed the predominance of terpinen-4-ol (41.5%, 43.1%, respectively) and γ-terpinene (20.6%, 22.8%, respectively). They also reported relatively high levels of both α-terpinene (9.6%, 9.3%, respectively) and α-terpineol (4.4%, 5.2%, respectively), while in the present study these two compounds were present in lower amounts (6.1%, 3.7%, respectively). It is also interesting to note the high proportion of p-cymene (9.3%) in the present study. In a more recent study, α-terpineol (4.4%) and 1,8-cineol (4.0%) were found in relatively high amounts, together with terpinen-4-ol (30.2%) and γ-terpinene (16.9%) [61]. In the present study, 1,8-cineol was also present but at a lower amount (2.8%).

The *L.nob* profile in the present study was in good agreemen<sup>t</sup> with that reported by Dhifi et al. [64], where they also showed high proportions of oxygenated monoterpenes (64.3%), with eucalyptol as the main constituent (46.8%) in their Tunisian species.

#### **4. Materials and Methods**

#### *4.1. Origin of the Essential Oils*

The lemongrass (*Cymbopogon citratus* (DC.) Stapf), lavender (*Lavandula dentata* L.), sweet marjoram (*Origanum majorana* L.), and bay laurel (*Laurus nobilis* L.) essential oils were provided by different laboratories (see Table 1), where the dried aerial parts of the plants were hydrodistilled using a Clevenger apparatus, as recommended by the European Pharmacopeia. The lavandin (*Lavandula hybrida* E.Rev. ex Briq) and tea tree (*Melaleuca alternifolia* (Maiden & Betche) Cheel) essential oils were from Flora Srl (Lorenzana, Pisa, Italy). The selection of these essential oils was based initially on their availability in our laboratory, and then on the studies in the literature that have reported in vitro activities of some of these against pathogen growth [65–67].

#### *4.2. Fungal Strains*

The *A. alternata* (GenBank accession: MK497774) and *S. cucurbitacearum* (GenBank accession: MF401569) strains used in the present study were isolated from infected squash seeds [2]. Pure cultures were transferred into Petri dishes (diameter, 90 mm) with potato dextrose agar (PDA; 42 g/L; Liofilchem Srl, Roseto degli Abruzzi, Italy) and incubated at 22 ± 2 ◦C with a photoperiod of 12/12 h dark/ ultraviolet light (TL-D 36W BLB 1SL; Philips, Dublin, Ireland).

#### *4.3. In Vitro Antifungal Activities on Mycelial Growth*

The antifungal activities of these *C.cit*, *L.dent*, *L.hyb*, *O.maj1, O.maj2*, *M.alt*, and *L.nob* essential oils were determined according to their contact phase effects on mycelial growth of *A. alternata* and *S. cucurbitacearum*. For these tests, the essential oils were dissolved in sterilized distilled water with 0.1% (*v*/*v*) Tween 20 (Sigma Aldrich, Steinheim, Germany), to obtain homogeneous emulsions. The autoclaved PDA medium (cooled to 40 ◦C) had the essential oil emulsions added to obtain the final concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1 mg/mL. The negative control was PDA containing 0.1% (*v*/*v*) Tween 20. The positive control was provided by three concentrations (0.1, 0.5, 1 mg/mL) of fungicides as 25 g/L difenoconazole plus 25 g/L fludioxonil (Celest Extra 50 FS; Cambridge, UK). The PDA was mixed and poured immediately into Petri dishes (diameter, 90 mm; 20 mL/plate), and after medium solidification, each plate was inoculated under aseptic conditions with 6 mm plugs of *A. alternata* or *S. cucurbitacearum*, taken from the edges of actively growing cultures. The experiments were carried out as three replicates per concentration and treatment. The inoculated plates were sealed with Parafilm and incubated for 7 days at 22 ± 2 ◦C with a photoperiod of 12/12 h dark/ ultraviolet light (TL-D 36W BLB 1SL; Philips, Dublin, Ireland). The orthogonal diameters of the colonies were measured daily until the control plates were completely covered by the mycelia. Mycelial growth inhibition was calculated based on Equation (1):

$$\text{[Mycelial growth in inhibition (\%)} = \text{[(dc -- dt)/dc]} \times 100\tag{1}$$

where dc and dt represent the mean diameter of the mycelial growth of the control and treated fungal strains, respectively. Moreover, the IC50 for mycelial growth inhibition of the fungi was determined from the linear regression between the essential oil concentrations and the mycelial growth inhibition.

Experiments were performed to differentiate between the fungicidal and fungistatic activities of the elevated essential oil concentrations against fungi. Here, each of the completely inhibited fungal plugs were transferred to fresh PDA plates to note their viability after 7 days of incubation under the same conditions.

#### *4.4. Gas Chromatography-Mass Spectrometry Analysis*

The volatile constituents of each essential oil were analyzed by GC-MS as previously reported [68]. They were processed using a gas chromatograph (Agilent 7890B; Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a capillary column (Agilent HP-5MS; 30 m × 0.25 mm; coating thickness, 0.25 μm; Agilent Technologies Inc., Santa Clara, CA, USA) and a single quadrupole mass detector (Agilent 5977B; Agilent Technologies Inc., Santa Clara, CA, USA). The analytical conditions were as follows: injector temperature, 220 ◦C; transfer line temperature, 240 ◦C; oven temperature programmed, from 60 ◦C to 240 ◦C at 3 ◦C/min; carrier gas, helium at 1 mL/min; injection volume, 1 μL (in 0.5% HPLC grade n-hexane solution); split ratio, 1:25. The full scan acquisition parameters were as follows: scan range, 30 *m/z* to 300 *m/z*; scan time, 1.0 s (See supplementary materials).

Identification of the constituents was based on comparisons of retention times with those of the authentic standards, with comparisons of their linear retention indices relative to the series of n-hydrocarbons. Computer matching was also used against commercial (NIST 14, Adams) and laboratory developed mass spectra libraries built for pure substances and components of known oils, and against the mass spectrometry literature data [69–74].

#### *4.5. Statistical Analysis*

Analysis of variance was calculated using SPSS (version 20, IBM, Armonk, NY, USA). The data were analyzed by analysis of variance (ANOVA). Means were compared using Fisher's test protected least significant difference at *p* ≤ 0.05. All of the trials were repeated at least twice, and data are means ± standard error (SE).
