Alternaria tenuissima (Nees & T. Nees: Fr.) Wilshire is a fungus that causes losses in the production of crops such as apple (Malus x domestica Borkh.) and pear trees (Pyrus communis L.), broccoli (Brassica oleracea var. italica), and others. The problem becomes more important due to the production of toxins (^{Jones and Aldwinckle, 2002}; ^{Fraire-Cordero et al., 2010}; ^{Agamy et al., 2013}). Alternaria is a widely distributed genus in agricultural areas and it has saprophytic, endophytic, and phytopathogenic species. Likewise, as a product of its activity, about 70 secondary metabolites have been identified, all toxic to plants, and some may affect human health, relating to cases of allergies and esophagus cancer (^{Pavón-Moreno et al., 2012}; ^{Woudenberg et al., 2015}).

Traditionally, to prevent and control of the invasion of phytopathogenic fungi in agriculture, synthetic fungicides have been used (^{Campbell and López-Ortíz, 2014}). The most widely used against Alternaria are azoxystrobin, difenoconazole, mancozeb, tebuconazole, and others (^{Malandrakis et al., 2015}; ^{Savitha and Ajithkumar, 2016}). However, these fungicides have adverse effects on the environment and human health. It is therefore necessary to search for environmentally friendly alternatives for the control of phytopathogenic fungi (^{Blair et al., 2015}).

Some compounds from botanical sources have the potential to be used for the control of plant pathogens in crops of agricultural interest. Considering that about 374,000 plant species have been described, there is a wide window of opportunity for the development of antifungal substances from the extracts of these plants (^{Cowan, 1999}; ^{Christenhusz and Byng, 2016}). Among the compounds of plants with antifungal capabilities, a few that stand out are essential oils (^{Isman et al., 2011}), some of which have proven antifungal capabilities, such as those based on lemon (Cymbopogon citratus (D.S.) Stapf.), eucaliptus (Eucalyptus spp.), cinnamon (Cinnamomum verum L.), clove teas (Syzygium aromaticum L.), and others (^{Calo et al., 2015}). Garlic (Allium sativum L.) contains an essential oil composed of aromatics such as diallyl disulfide, diallyl trisulfate and other sulfurous compounds with antimicrobial activity with a potential to be used in fungal control (^{Casella et al., 2013}; ^{Kocić-Tanackov et al., 2012}). The aim of this study was to evaluate the antifungal activity in vitro of garlic essential oil against A. tenuissima.

The study was conducted in the laboratories of the Centro de Investigación en Alimentación y Desarrollo, AC (Food and Development Research Center), Culiacán unit. The A. tenuissima strain was provided by the Department of Food Research and Postgraduate Studies of the University of Sonora, previously identified by ^{Quintana-Obregón et al. (2013)}. It was reactivated and grown in a potato-dextrose-agar (PDA) medium at 25 °C with light periods of 12 h light-darkness for seven days. Later, spore suspensions were prepared with a Tween^® 20 at 0.1% (v/v), and finally, the concentration was determined using a hematometer (Neubauer camera, BRAND ^® Germany).

The garlic essential oil used was of an analytical grade (Sigma-Aldrich^® lot MKBB8390V). Oil volatiles were identified using a GC-7890B (Agilent Technology^®, USA) gas chromatographer linked to an Agilent 240 (CG-MS) selective mass detector with an electric ionization system of 70 eV. The capillary column used was DB-5 (50 m*0.25 mm) (J&C Scientific, Agilent Technologies^®, Pennsylvania, USA) and it was conditioned at 60 °C for 10 min. The temperature was raised (20 °C/ min) up to 180 °C for 2 min and finally (4 °C/min) up to 250 °C for 4.5 min. Helium carrier gas was used at a flow of 2 mL min^-1. The temperatures of the ionization chamber and of the line of transfer were 220 and 280 °C, respectively. The constituents were identified by comparing linear retention indices based on a mixture of n-alkanes and times of retention of the spectra obtained using database NIST 98 (National Institute of Standard and Technology, Maryland, EUA).

Radial Growth. The PDA with Tween^® 80 (1% v/v) was sterilized for 15 min at 15 psi, then cooled at 45 °C and mixed with garlic essential oil at different concentrations (0, 10, 100, 500, 1,000 and 10,000 ppm). In addition, PDA was prepared without Tween^® 80 under the same conditions, and without mixing it with garlic essential oil, each mixture was placed, 15 mL at a time, in Petri dishes with diameter of 50 mm. Later, using a sterilized glass Pasteur pipette, a 6 mm perforation was made in the center of the solidified culture medium and it was inoculated with 25 µL of the A. tenuissima spore solution (105 spores). Finally, the cultures were incubated at 25 °C with photoperiods of 12 h light-darkness and the radius of the fungal colony was measured every 24 h until it covered 95% of the control (PDA without Tween^® 80) taken as a reference. The minimum inhibiting (CMI) and average inhibiting (CI₅₀) conditions were determined at 120 h post-inoculation of the fungus with a Probit analysis with the statistical program NCSS 2000 (Number Cruncher Statistical Systems, Utah, USA).

Production of Biomass. The Petri dishes with oil concentrations described above were inoculated with 25 µL of the A. tenuissima spore suspension (105 spores) in the central section, spreading them by surface diffusion and kept in the same conditions as described above. After 120 h, the dry weight was taken of the cultures that grew in the culture medium, and for this, the mycelium was separated from the Petri dish and transferred to a precipitate beaker with 50 mL of distilled water; it was sterilized and the mycelium was separated with Whatman^® # 2 filter paper. The filter paper, along with the mycelium, was dried in an air convection over at 105 °C for 2 h and collected in a desiccator. The dry weight was expressed in mg dish^-1 (^{Larralde-Corona et al., 1997}; ^{López-Insunza et al., 1997}).

Spore Germination. An evaluation was carried out on the effect of the CI₅₀ and CMI of the essential oil obtained with a Probit analysis against the A. tenuissima spores. For this, 25 μL of the spore suspension (105 spores) were distributed on the surface of the culture medium in the Petri dish in controls and treatments with essential oils. The Petri dishes were incubated at 25 ºC with a photoperiod of 12 h light-darkness for up to 24 h. Random samples of each were taken every 4 h and the number of germinated and not germinated spores were counted, out of a total 200 spores, under a light microscope. The spore was considered germinated when the length of the germinative tube was at least 50% as long as the not germinated spore (^{Dantigny et al., 2006}).

Experimental Design and Statistical Analysis. A randomized design was used in the experiment, in which the treatments were: PDA, PDA-Tween^® 80 (controls) and concentrations of garlic essential oils in PDA-Tween^® 80 (10, 100, 250, 500 and 1,000 ppm). The seven treatments were evaluated against A. tenuissima , where the experimental unit was a Petri dish. The response variables were radial growth and production of biomass. CI₅₀ and CMI were evaluated in the spore germination stage. All experiments were carried out three times. Data were analyzed using the statistical program JMP version 5.0 (^{SAS, 2002}) for the analysis of variance (ANOVA) and the averages of the treatments were separated using a Tukey test (p≤0.05).

Table 1 shows the tentative main volatile components of the garlic extracts obtained by CG-MS with relative proportions ≥1%. There is a predominance of compounds with sulfur in their structures, some of the most important of which are diallyl sulfate and diallyl disulfate, which derive from the activity of the enzyme allinase when released by rupture from the tissue during the oil extraction (^{Kocić-Tanackov et al., 2012}).

The radial growth of A. tenuissima in the treatment with PDA medium only covered 95% of the Petri dish after 120 h, reaching a radius of 22 mm. There were no significant differences in the radial growth in treatments PDA and PDA-Tween^® 80 after 120 h. The essential oil achieved a significant reduction of the radial growth of A. tenuissima with concentrations equal to or greater than 250 ppm, achieving, at 1,000 ppm, an inhibition of 100% in regard to the control PDA-Tween^® 80 (Table 2).

In the production of biomass, medium PDA-Tween^® 80 with 10 ppm of essential oil showed a significant increase in regard to the PDA alone. However, when increasing the concentration of essential oil to 500 and 1,000 ppm, there were significant differences with the controls, presenting inhibition on the growth of A. tenuissima at these concentrations, with the inhibition of biomass production being of 86.20% at 1,000 ppm of oil in regard to the control PDA-Tween^® 80 (Table 2). The increase in biomass in a culture medium with Tween^® 80 at 10 ppm could be due to the interaction between Tween^® 80 and the essential oil. Reportedly, Tween^® 80 can increase cell permeability, favoring nutrient absorption; in Aspergillus fumigatus it has been suggested that the compound is used as a source of carbon (^{Inouye et al., 2001}; ^{Taoka et al., 2011}). Since the concentration of Tween^® 80 is greater than the essential oil (10 ppm) the surface of mycelial contact is competed for by Tween^® 80, reducing the antifungal activity of the oil on its own (Inouye et al., 2001). When increasing the concentration of essential oil (500 and 1,000 ppm) the oil-mycelium interaction increases, favoring its bioactivity, shown by the significant reduction of biomass at 500 and 1,000 ppm in regard to the PDA-Tween^® 80 control (Table 2).

The CI₅₀ and CMI of 229 ppm and 1,023 ppm, respectively, were obtained from the radial growth. The kinetics of the germination of A. tenuissima spores exposed to the CI₅₀ and CMI of garlic essential oil (Figure 1) showed significant differences with those of the control averages (PDA and PDA-Tween^® 80) with spore germination inhibitors of 88.89 and 94.17% for CI₅₀ y CMI, respectively.

Figure 1 Kinetics of the germination of A. tenuissima spores at 25 °C and photoperiods of 12 h in PDA, PDA-Tween^® 80, CI₅₀ and CMI of essential garlic oil. 

According to ^{Inouye et al. (2001)}, the inhibiting capacity of the volatile compounds of essential oils stands out for having three stages of fungal development: spore germination, vegetative mycelia, and reproductive mycelia. This effect of garlic essential oil has been reported in growth stages of Aspergillus versicolor and Penicillium funiculosum (^{Kocić-Tanackov et al. 2012}; ^{Li et al., 2014}). When evaluating the CMI of the essential oil versus A. tenuissima, a fungistatic effect was observed. It is possible that garlic essential oil interacts with the cell membrane, and when its concentration is reduced in time by the volatility of its components, the interaction diminishes and the fungus normalizes its metabolism. ^{Tian et al. (2012)} found alterations in the cell membrane of Aspergillus flavus, particularly ergosterol, when treated with dill (Anethum graveolens). The antimicrobial action mechanism of sulfured compounds has been related the interaction with hydrogen sulfide groups of the cell and the disulfide bonds that may form (^{El-Sayed et al., 2017}); it is possible that the inhibition in A. tenuissima is due to this type of chemical interactions. However, additional studies are necessary to elucidate mechanisms of interaction of the garlic essential oil on the in vitro growth of A. tenuissima.