Plant material

We used 21-day old seedlings of tomato (Solanum lycopersicum) var. Tiny tim cherry, which were propagated from certified seed in a commercial greenhouse. The seedlings were taken to a growth chamber and kept at a temperature of 25 °C, under a 16 h light/8 h darkness (100 µmol m^-2 s^-1 of luminous intensity) photoperiod and 80% relative humidity. When the seedlings reached 28 days after germination, a 2% rooting stimulator (Magic root) was applied; 45 days after germination, the seedlings were inoculated with Forl and immediately transplanted to 3-kg bags containing prepared substrate (Nutrigarden). After the seedlings were transplanted, treatments were assigned and randomly applied to each group of plants.

Garlic extract (EA).

The extract was prepared using 10 g of fresh garlic cloves (var. Río Sonora) that were blended in 14 mL of distilled water for 1 min at room temperature. The mixture was centrifuged at 15000 x g at 4 °C for 20 min, then 6 mL of ethanol were added to the supernatant and the mixture was centrifuged again at 8000 x g at 4 °C for 20 min (^{Jansen et al., 1987}). The supernatant represented the garlic extract.

Obtaining Forl spores

The Fusarium oxysporum f. sp. radicis-lycopersici strain was molecularly identified using the ITS1-5.8S ADNr-ITS2 sequencing technique and/or D1/D2 domains of the 28S ADNr gene at the Genomic Biotechnology Center of the Instituto Politécnico Nacional (Access number: 90436915:DO452455.1 in the NCBI database). The Forl strain was donated by the Plant Biotechnology Laboratory of ITSON Centro’s CIBAA. It was grown in an agar-potato-dextrose (PDA) culture medium and incubated at 28 °C for 7 days. It was re-cultivated by taking a small piece of mycelium and inoculating it in fresh PDA at 28 °C for 7 days, or until the fungus produced enough spores to prepare a solution of 1x10⁷ spores/mL. The spore suspension was prepared by adding 5 mL of distilled water containing 0.01% of 0.02% Tween 80 on the surface of the dish containing the fungus; then the surface was scraped using a glass rod, and the liquid containing fungus spores was collected and filtered through gauze to remove the mycelium; the filtrate was centrifuged at 5000 x g. The collected spores were re-suspended in sterilized water until reaching a concentration of 1x10⁷ spores/mL, quantified with a Neubauer chamber.

Tomato seedlings inoculated with Forl.

Forty-five days after germination, the tomato seedlings were taken from the seedbed and their roots were gently washed to remove the substrate. Using a previously sterilized scalpel, small cuts were made on the adventitious roots, which were immediately submerged in a Forl inoculating solution (1X10⁷ spores/mL) for 30 min. The seedlings were then transplanted as described in the previous paragraph.

Treatments to induce resistance in tomato plants

Groups of 10 seedlings per treatment with three replications were formed. Six treatments were evaluated, as shown in Table 1. The first three treatments were used as controls. Seedlings were inoculated with Forl (1x10⁷ spores mL^-1) before transplanting. In treatments with EA, the extract was sprayed on the leaves 7 and 14 days after inoculation with Forl and transplanting.

Evaluation of disease severity

To evaluate the response of the tomato plants to the pathogen, we used a hedonic scale proposed by ^{Apodaca et al. (2004)} and ^{Clavijo-Castro (2014)}. Plants were classified after the fifth true leaf emerged, and each plant was evaluated using a scale of 1-5 (Table 2), according to disease intensity. To quantify disease severity, the parameters chlorophyll index (UC) and height (cm) were evaluated every 7 days after the treatment was applied for 49 days. Chlorophyll content was measured with a SPAD 502 meter (Minolta), a device that measures absorption in 650 and 940 nm wave lengths to estimate chlorophyll levels. Measurements were made in triplicate on the third leaf to obtain the chlorophyll index. Plant height was measured from the base of the stem to the tip using a flexometer.

The severity was calculated by applying the formula proposed by ^{Towsend and Heuberguer (1943)} (1).

Severity= (Σnv/ 5N) x 100 (1)

Where:

S= Severity

n= Plants per category

v= Value of the category

N= Plants per experimental unit

Quantification of salicylic acid and jasmonic acid

The content of SA and JA was determined on leaves before and after transplanting, both on treated and non-treated plants, every 7 days for 49 days. The leaves were cut from the plants, wrapped in aluminum foil, immediately frozen in liquid nitrogen and stored at -80 °C until they were analyzed. To quantify metabolites, we used the methodology proposed by ^{Guzmán-Téllez et al. (2014)} and ^{Arbona and Gómez-Cárdenas (2008)}. We started by cutting 1 g of leaves and placing them in tubes (30 mL) to which 10 mL of deionized water were added. This was homogenized with an Ultra-Turrax dispersing machine. The tubes were centrifuged (Galaxy 7D Centrifuge, VWR, USA) at 4000 g for 45 min at 4 °C. The supernatant was collected, and its pH adjusted to 3.0 with 15% acetic acid. Two 3-ml samples of the pH 3 supernatant were prepared, and 2 mL of ethyl ether were added to each tube. The tubes were shaken to recover the organic phase, which was then dried under a stream of gaseous nitrogen. The dry residue obtained was re-suspended in 500 µL of the A/B (50/50) mobile phase that was prepared for determination in high performance liquid chromatography (HPLC). It was then filtered using a 0.45 µm PTFE filter (Pall Gelman Acrodisc filters). The filtrate was used to determine SA and JA content with a liquid chromatography system (Agilent Technologies Model 1260 Infinity) controlled by the OpenLab ChemStation software (Agilent Technologies). The equipment consists of a quaternary pump, a diode-array detector and a 7725i Rehodyne manual injector; we used a 250 x 4.6 mm C18 column (Phenomenex Luna) of 5 µm particle. The mobile phase consisted of an A phase containing 94.9% H₂O, 0.1% HCOOH (formic acid) and 5% CH₃CN (acetonitrile) and a B phase containing 5% H₂O, 94.9% CH₃CN and 0.1% HCOOH, at a 60 (A):40 (B) ratio, for which we used an isocratic method at 1 mL per min during 20 min at room temperature. The detection wave length used for both compounds was 303 nm. Pure salicylic acid and jasmonic acid compounds were used to determine the calibration curves (Sigma Aldrich), the retention time of each compound and calculate the concentration. The calibration curves ranged as follows: 0.02-0.1 µg g^-1 and 0.1-1.0 µg g^-1 for AS, and 0.05-0.1 µg g^-1 and 0.1-1.0 µg g^-1 for AJ.

Experimental design and data analysis

We used a completely randomized design with 6 treatments and three replications. The experiment was conducted twice for validation. Data were analyzed using ANOVA (one-way) and NCSS statistical software version 2007 (Number Cruncher Statistical System, Kaysville, Utah). Seven-week data on the chlorophyll index, height, severity, and salicylic and jasmonic acid concentration were variable responses. The means were compared using the Tukey-Kramer method at a p≤0.05 probability level. The chlorophyll index and height were analyzed using descriptive statistics.

Chlorophyll content in tomato plants

The chlorophyll content in all tomato plants a week after transplanting was between 41 and 44 UC (Figure 1). These values are within the range (41.3-56.11 UC) reported for tomato plants that were measured under conditions similar to ours (^{Mendoza et al., 1998}; ^{Peñuelas-Rubio et al., 2017}). The chlorophyll content in plants used as absolute controls and controls with roots with cuts on them decreased starting on the third week after transplanting, a fact that is associated with the plants’ demand for nitrogen during their rapid vegetative growth (^{Nurzyeki, 2013}). However, the control plants that had roots with cuts and that had been inoculated with Forl showed a significant decrease in UC beginning the second week after transplanting, with significantly lower chlorophyll values (p≤0.05) than the other two control groups (Figure 1). The decrease in UC could be due to the presence of Forl, as reported by ^{Mendoza et al. (1998)} and ^{Hiderman et al. (1992)}.

Figure 1 Chlorophyll content in tomato plants non-inoculated and inoculated with Forl and treated with different concentrations of garlic extract. The values are the means of three repetitions of 10 plants each. The vertical bars represent the standard deviation (n=3). Different letters indicate a significant difference between treatments, according to the Tukey-Kramer method at a p≤0.05 probability level. The experiment was conducted twice. 

Plants treated with EA were able to increase the UC. Specifically, plants treated with 1 and 2% of EA had significantly increased values of UC (p≤0.05), so during the last two weeks of evaluation they were able to reach the values of the absolute control, whose chlorophyll value was of 41.3 UC, and plants treated with 1 and 2% of EA with values of 41.5 and 40.5 UC, respectively. ^{Massacci et al. (2008)} reported that in plants infected with F. oxysporum, photosynthetic processes do not take place, and this decreases the photosynthetic assimilation rate. These results are in agreement with data obtained in this evaluation, where the lowest chlorophyll values (24.9 UC) were observed in the group of control plants that had roots with cuts and that had been inoculated but not treated with EA so that they were not able to recover the chlorophyll values as the plants treated with EA did. UC is an indirect measure of the amount of N in a plant (^{Vázquez et al., 2012}), so it is assumed that plants treated with EA developed well even when they were infected with Forl.

Height of the tomato plants

The results of measuring the height of the tomato plants are shown in Figure 2. During the first three weeks after inoculation and transplanting, all the plants grew and there were no significant differences between the plants non-treated and treated with EA (p≤0.05). After the fourth week, there were marked differences in growth within the groups of plants; for example, plants treated with 1 and 2% EA grew the most, and plants treated with 2% EA reached 67.8 cm, while plants treated with 1% EA reached 62.2 cm; these values were similar (p≤0.05) to the value of the absolute control: 61.9 cm. Plants treated with 0.5% EA reached an average height of 53.5 cm, which is higher than the 34.9 cm reached by the control with cut + inoculated roots. Both groups showed values that are statistically lower (p≤0.05) than the values of treatments with 1 and 2% of EA (Figure 2). Growth decrease is a characteristic symptom of plants infected by Forl (^{Apodaca et al., 2004}; ^{Peñuelas-Rubio et al., 2017}), due to the proliferation of the pathogen in the plant’s vascular bundles, which means the spores are disseminated towards the apex in the xylem flow (^{Hadian et al., 2011}). This is in agreement with the results observed in control plants with cuts in the roots + Forl inoculation that were not treated with EA; the disease established itself and was propagated intercellularly. The treatment with 0.5% EA induced the lowest level of resistance in tomato plants because the group of plants sprayed with EA were the lowest in height of the three EA treatments. The effect of garlic as a promoter of vegetative and reproductive growth in pathogen-infected plants was reported by ^{Fatema and Ahmad (2005)} in peanut plants infected by nematodes, and by ^{Chohan and Perveen (2015)} in tomato plants infected with Fol. In both cases, aqueous garlic solutions improved the plant’s growth response and were able to control the pathogen. In this study, 1 and 2% concentrations of EA improved plant development and induced resistance to Forl.

Figure 2 Evaluation of the height of tomato plants non-inoculated and inoculated with Forl and treated with different concentrations of garlic extract. The values are the means of three repetitions with 10 plants each. The vertical bars represent the standard deviation (n=3). Different letters indicate a significant difference between treatments, according to the Tukey-Kramer method at a p≤0.05 probability level. The experiment was conducted twice. 

Disease severity of tomato plants infected with Forl

Results of the evaluation of disease severity caused by Forl in tomato plants are shown in Figure 3. All plants inoculated with Forl showed characteristic symptoms of the disease at different levels, including leaf wilt and defoliation, slow growth and stem damage in the form of spots and necrotic lesions on vascular bundles. As expected, the absolute control and the plants with cut roots did not develop disease symptoms because they were not inoculated with the fungus (Figure 3). The minimum value of disease severity was 26.4% in the group of plants treated with 1 and 2% EA, followed by 33.4% in plants treated with 0.5% EA. The most affected plants were those in the control group with cut roots + inoculation, which showed visible symptoms starting the second week after inoculation and 100% severity in the seventh week. These results show the positive effect of applying EA, because disease severity was reduced by 66 to 73% compared to the severity level in the control plants (cut + inoculated), which means that the plants treated developed fewer symptoms. The effect on disease severity caused by Forl in tomato plants treated with EA could be due to the accumulation of metabolites such as SA and JA, which induce defense mechanisms, as demonstrated by Shah and ^{Zeier (2013)} and ^{El Oirdi et al. (2011)}, where these metabolites induced the expression of genes involved in plant defense under stress conditions. Based on this result, we can conclude that the exogenous application of EA acted as an activator of systemic acquired resistance in tomato plants.

Figure 3 Percentage of disease severity caused Forl in non-inoculated and inoculated tomato plants that were treated with different concentrations of garlic extract. The values are the means of three replications of 10 plants each. Different letters indicate a significant difference between treatments, according to the Tukey-Kramer method at a p≤0.05 probability level. The experiment was conducted twice. 

Quantification of salicylic acid (SA)

The results of SA quantification in tomato plants are shown in Figure 4. There were variations in the SA content beginning in the first week of inoculation and basal levels of 0.10 µg g^-1 in the absolute control plants, a value within the range (0.10-10 µg g^-1) reported for Solanaceae (^{Rivas-San Vicente and Plasencia, 2011}). In cut and inoculated plants, an initial concentration of 0.24 µg g^-1 was quantified, which was twice the concentration in the absolute control. In plants inoculated with Forl and treated with 1 and 2% of EA, there was a significant increase (p≤ 0.05) in the SA content in the third week after inoculation. In plants treated with 2% EA, we found values that were 11 and 6 times higher than the values of the control treatments within the same week; for the treatment with 1% EA, we found values 8 and 4.5 times higher than the values of their respective controls. After that increase, SA levels decreased in the following weeks but increased again in the sixth week (Figure 4). As of the fifth week, SA accumulation in the control plants (absolute control and cut + inoculated plants) increased and reached a maximum concentration of 0.78 y 0.82 µg g^-1, respectively. These increases in the control treatments occurred two weeks after the increase in the group of plants treated with 1 and 2% EA. Increases in SA content were between 2 and 5 times higher than the basal values reported in plants at the onset of flowering, and in plants infected with necrotic pathogens such as Fusarium oxysporum (^{Swarupa et al., 2014}). This fact may explain the increase in SA content in the control plants from week five to seven. However, increases five times higher than SA concentration that occurred in the third week in plants treated with 1 and 2% EA may be associated with the treatment applied rather than with the presence of the pathogen, given that the maximum SA values in treatments with 1 and 2% of EA were consistent with the reduced symptoms observed when disease severity was evaluated (Figure 3). In support of this deduction, ^{Ojha and Chatterjee (2012)} applied SA to tomato plants infected with F. oxysporum and observed a significant increase in plant resistance to pathogen attack when the activity of enzymes related to the defense system increased. The greatest increases in enzyme activity took place 21 days after SA application. This result is in agreement with the results of the present study, which show that the highest SA values were quantified the third week after treatment application. The protective effect against Forl in plants treated with EA, which improved their growth and development, was also reported by ^{Hayat et al., (2012)}, who by increasing endogenous SA concentrations were able to significantly improve plant growth characteristics, including height, leaf number and color, shoot diameter, dry and fresh weight, and photosynthetic rate.

Figure 4 Concentration of salicylic acid in tomato plants inoculated and non-inoculated with Forl and treated with different concentrations of garlic extract. The values are the means of three repetitions with 10 plants each. The vertical bars represent the standard deviation (n=3). Different letters indicate a significant difference among treatments, according to the Tukey-Kramer method at a p≤0.05 probability level. The experiment was conducted twice. 

Based on the results of our study, increased SA concentration coincides with an improvement in plant development, as well as resistance to the pathogen, which agrees with the important protective role of SA against pathogens, probably because of its ability to induce the transcription of defense proteins (PR) which codify enzymes such as chitinases, glucanases, endohydrolases and β-1, 3-glucanase that contribute to systemic acquired resistance (SAR) against different pathogens because of its antifungal activity (^{Heil and Bostock, 2002}; ^{Hayat et al., 2010}).

Quantification of jasmonic acid (JA)

The results of JA quantification in tomato plants are shown in Figure 5. Plants with cut roots + plants inoculated with Forl, as well as those to which EA was applied increased their JA content, with initial values of 0.89 µg g^-1 in plants cut and/or inoculated with Forl, which was significantly higher (p≤0.05) than the value of 0.74 µg g^-1 of the absolute control plants. As of the second week, application of 1 and 2% EA produced an increase 2.5 times the value of the absolute control, and 1.8 times the value of the cut + Forl control. These JA values remained significantly higher (p≤0.05) in the third week after inoculation and after transplanting plants treated with 1 and 2% EA. In all treatments, except for the absolute control, JA content was gradually reduced as of the fourth week until the end of the evaluation but maintained high levels, which indicates that the plant was responding to a stress condition (^{Ryan and Moura, 2002}). From this result we can infer that 1 and 2% EA concentrations are enough to increase endogenous JA concentrations in tomato plants, which in turn can induce plant defense responses (^{Kravchuk et al., 2011}). JA is known to be a key regulator that stimulates defense responses in synergy with SA and reduces symptoms of diseases caused by pathogens (^{Pieterse et al., 2012}) by activating the expression of genes involved in plant defense (^{Ryan, 2000}). It has been reported that JA induces protease and polyphenol oxidase inhibitors, and shikimate pathway compounds specifically in tomato plants, which reduces pathogens (^{El Oirdi et al., 2011}). It is well accepted that plants need to activate induced systemic resistance that depends on JA to fight necrotrophic pathogens (^{Glazebrook, 2005}). Therefore, protecting tomato plants from Forl as was done in this study could be related to rapid JA induction and quantified endogenous concentrations (^{Rahman et al., 2012}). However, simultaneous activation of the defense systems that depend on SA and JA results in increased resistance to pathogens (^{Mur et al., 2006}). This is in agreement with the observed increases in endogenous SA and JA and the reduction of disease symptoms caused by Forl in tomato plants.

Figure 5 Concentration of jasmonic acid in tomato plants non-inoculated and inoculated with Forl and treated with different concentrations of garlic extract. The values are the means of three repetitions with 10 plants each. The vertical bars represent the standard deviation (n=3). Different letters indicate a significant difference between treatments, according to the Tukey-Kramer method at a p≤0.05 probability level. The experiment was conducted twice.