Antifungal Activity and Chemical Composition of Seven Essential Oils to Control the Main Seedborne Fungi of Cucurbits

Essential oils represent novel alternatives to application of synthetic fungicides to control against seedborne pathogens. This study investigated seven essential oils for in vitro growth inhibition of the main seedborne pathogens of cucurbits. Cymbopogon citratus essential oil completely inhibited mycelial growth of Stagonosporopsis cucurbitacearum and Alternaria alternata at 0.6 and 0.9 mg/mL, respectively. At 1 mg/mL, Lavandula dentata, Lavandula hybrida, Melaleuca alternifolia, Laurus nobilis, and two Origanum majorana essential oils inhibited mycelia growth of A. alternata by 54%, 71%, 68%, 36%, 90%, and 74%, respectively. S. cucurbitacearum mycelia growth was more sensitive to Lavandula essential oils, with inhibition of ~74% at 1 mg/mL. To determine the main compounds in these essential oils that might be responsible for this antifungal activity, they were analyzed by gas chromatography–mass spectrometry (GC-MS). C. citratus essential oil showed cirtal as its main constituent, while L. dentata and L. nobilis essential oils showed eucalyptol. The M. alternifolia and two O. majorana essential oils had terpinen-4-ol as the major constituent, while for L. hybrida essential oil, this was linalool. Thus, in vitro, these essential oils can inhibit the main seedborne fungi of cucurbits, with future in vivo studies now needed to confirm these activities.


Introduction
Cucurbits are an important source of income for countries in the Mediterranean basin, with a total production of nearly 3,356,669 tonnes in 2018 [1]. Squash (Cucurbita maxima Duchesne; Cucurbita moschata Duchesne) is one of the major cucurbits grown in tropical and temperate regions. Cucurbita spp. can be affected by a number of fungal pathogens, which can cause major economic losses [2]. The majority of these fungi are seedborne, such as gummy stem blight (with foliar symptoms) and black rot (with fruit symptoms), which are caused by Stagonosporopsis cucurbitacearum (Fr.) Aveskamp, Gruyter & Verkley (anamorph: Phoma cucurbitacearum (Fr.) Sacc.), synonym Didymella bryoniae (Fuckel) Rehm, and which represent serious diseases that are a major constraint to cucurbit production worldwide [2][3][4]. Nuangmek et al. [5] reported that losses in cantaloupe can also reach 100% under conditions conducive to S. cucurbitacearum. Alternaria alternata (Fr.) Keissl. is the 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 IC 50 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 IC 50 values from 0.473 mg/mL to 0.893 mg/mL, as similarly against S. cucurbitacearum, with IC 50 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 IC 50 values of 1.310 mg/mL and 1.248 mg/mL, respectively ( Figure 3).  (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).

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 IC 50 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][34][35][36][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 agreement 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][55][56][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 L.nob profile in the present study was in good agreement 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.

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][66][67].

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): (1) where dc and dt represent the mean diameter of the mycelial growth of the control and treated fungal strains, respectively. Moreover, the IC 50 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.

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][70][71][72][73][74].

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).

Conclusions
The management of plant diseases using natural compounds is a great and important need nowadays. The present study has demonstrated the in vitro activities of seven essential oils and their efficacies against the fungi A. alternata and S. cucurbitacearum. These data show that the chemical compositions of essential oils can affect their antimicrobial activities. These essential oils were characterized by high proportions of oxygenated monoterpenes followed by monoterpene hydrocarbons. Essential oil with citral, β-myrcene, and geraniol as major components (i.e., lemongrass [C.cit],) controlled these fungi most effectively, followed by essential oils containing terpinen-4-ol or linalool (i.e., marjoram [O.maj1/2], tea tree [M.alt], lavandin [L.hyb]). Antifungal activity of essential oils can be ascribed to individual effect of major components, and/or due to a synergistic effect of its minor components. Further studies are required to determine the effects of these oils as seed treatments, to evaluate their potential as preventive and curative treatments.