Induced Resistance in Tomato Plants against Verticillium Wilt by the

Mar 18, 2016 - A synergistic effect was observed between the ligand L and the transition metal Ni, with disease incidence reduced by 38% with L and 57...
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Induced Resistance in Tomato Plants against Verticillium Wilt by the Binuclear Nickel Coordination Complex of the Ligand 2,5-Bis(pyridin2-yl)-1,3,4-thiadiazole Hanane Zine,† Lalla Aicha Rifai,† Mohamed Faize,*,† Fouad Bentiss,‡ Salaheddine Guesmi,§ Abdelhakim Laachir,§ Amal Smaili,† Kacem Makroum,† Abdelaziz Sahibed-Dine,‡ and Tayeb Koussa† †

Laboratory of Plant Biotechnology, Ecology and Ecosystem Valorization, Faculty of Sciences, ‡Laboratory of Catalysis and Corrosion of Materials, Faculty of Sciences, and §Laboratory of Coordination and Analytical Chemistry, Faculty of Sciences, University Chouaib Doukkali, 24000 El Jadida, Morocco ABSTRACT: Verticillium wilt caused by Verticillium dahliae is a major limiting factor for tomato production. The objective of this study was to evaluate the effectiveness of ligand 2,5-bis(pyridin-2-yl)-1,3,4-thiadiazole (L) and its complex bis[μ-2,5bis(pyridin-2-yl)-1,3,4-thiadiazole-κ4N2,N3:N4,N5]bis[dihydrato-κO)nickel(II)] as activators of plant defenses in controlling Verticillium wilt. In the greenhouse, they protected tomato plants against V. dahliae when they were applied twice as foliar sprays at 100 μg mL−1. A synergistic effect was observed between the ligand L and the transition metal Ni, with disease incidence reduced by 38% with L and 57% with Ni2L2. Verticillium wilt foliar symptoms and vascular browning index were reduced by 82% for L and 95% for Ni2L2. This protection ability was associated with the induction of an oxidative burst and the activation of the total phenolic content as well as potentiation of the activity of peroxidase and polyphenol oxidase. These results demonstrated that L and Ni2L2 can be considered as new activators of plant defense responses. KEYWORDS: 2,5-bis(pyridin-2-yl)-1,3,4-thiadiazole, metal transition, plant defenses, protection, tomato, Verticillium

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INTRODUCTION Verticillium dahliae is a soil-borne fungus, which causes vascular wilts in a wide range of plant species worldwide. This disease is considered major economic problem because it prevents cultivation of susceptible crops in soils harboring propagules of the pathogen.1 The fungus invades host plants through the roots and spreads systemically, causing chlorosis of lower leaves and wilting of shoot tips.2 Severe defoliation, growth stunting, and plant death may occur.3 In tomato, this disease can be controlled by the use of cultivars carrying the Ve gene locus.4,5 Panoply of methods relying on crop rotation and chemical strategies has several limitations. For instance, the use of methyl bromide as a fumigant will be banned because of its adverse effects on human health and the environment.6 Crop rotation is ineffective as a control method in the long term because it has only a little effect on V. dahliae survival in soil. Active fungicides against Verticillium wilt, such as benomyl and carbendazim, became ineffective after continued and repeated use because of the development of resistant strains.7 Beside these conventional chemicals, there are some modern products that are able to protect crops against plant diseases by activating plant defense systems. For instance, the plant activator S-methyl benzo[1,2,3]thiadiazole-7-carbothioate (BTH), also named acibenzolar-S-methyl (ASM), has been reported to mediate resistance in various plant species against viral, bacterial, and fungal diseases, including V. dahliae in eggplant.8,9 The mechanism of action of BTH has been well-studied; it involves both direct activation of defense responses and priming. This second mechanism, also called potentiation, leads to a physiological state that enables plants to respond more rapidly and efficiently after exposure to © 2016 American Chemical Society

pathogens or abiotic stress, and it results from improved perception and potentiation of signals involved in the induction of plant defense response.10−13 Heterocyclic compounds have gained great importance as a result of their potential biological activities. Among them, those containing a ring composed of three carbon atoms and two nitrogen atoms are known as diazoles. They include the isomer 1,3-diazole, known as imidazole. Its derivatives possess antibacterial, antifungal, antiviral, anthelmintic, and anticancer activities.14 The triazoles have in their ring two carbon atoms and three nitrogen atoms and are used as fungicides, inhibiting the pathway of ergosterol biosynthesis.15 Other heterocyclic compounds are characterized by the presence in their fivemembered heterocyclic system of one nitrogen atom and one sulfur atom, known as thiazoles, or two nitrogen atoms and one sulfur atom, known as thiadiazoles. Their derivatives and, particularly, those of 1,3,4-thiadiazoles are widely applied in pharmaceutical, agricultural, and materials chemistry.16 In agriculture, they have been reported to be active in vitro against various phytopathogens, including Rhizoctonia solani,17 Puccinia recondite,18 Xanthomonas campestris, Xanthomonas oryzae, Fusarium oxysporum,19 Fusarium graminearum, Botrytis cinerea, Sclerotinia sclerotiorum,20 and Alternaria solani.21 These thiadiazole derivatives were extensively used for the synthesis of numerous complexes with transition metals. Indeed, the donor sites of nitrogen and sulfur atoms of 1,3,4-thiadiazoles are able Received: Revised: Accepted: Published: 2661

January 11, 2016 March 17, 2016 March 18, 2016 March 18, 2016 DOI: 10.1021/acs.jafc.6b00151 J. Agric. Food Chem. 2016, 64, 2661−2667

Article

Journal of Agricultural and Food Chemistry

The fungi were cultured on potato dextrose agar (PDA) plates at 25 °C in the dark. NiCl2, L, and Ni2L2 were used for their antifungal activities at the concentrations of 0, 10, 20, 30, 50, and 100 μg mL−1 in agar medium. Thiophanate-methyl was used at 100 μg mL−1 as a standard fungicide. Stock suspensions of these compounds were prepared with sterile distilled water (DW), and specific aliquots of each suspension were added under sterile conditions to PDA medium after it was autoclaved and cooled. The amended media were poured into Petri dishes. Inoculums consisted of 5 mm plugs of agar and mycelium taken from actively growing cultures of fungus on PDA. Inverted plugs were placed onto the test medium in the center of the dishes. Petri plates were incubated in the dark at 25 °C. The mycelial growth was assessed by measuring two orthogonal diameters of each colony every day and during 5 days of incubation. Mycelial growth was compared to growth on non-amended PDA. The growth rate was determined, and the percentage of inhibition growth was expressed relative to the control grown in the same conditions. Plant Material, Chemical Treatments, Challenge Inoculation, and Disease Assessment. Tomato (Solanum lycopersicum) seeds of the variety ‘Pomodoro’ were subjected to superficial disinfection using 5% commercial bleach for 3 min, followed by extensive rinsing with sterile DW. Germination was carried out in black plastic poly pots (12.5 cm diameter and 17 cm length) containing a sterile mixture of peat and sand (3:1), and seedlings were grown in a greenhouse under a 12 h photoperiod at 26 °C and 70% relative humidity. L or Ni2L2 was applied at 100 μg mL−1 by spraying to runoff the aerial part of 2 week old tomato seedlings 2 times (7 and 3 days before pathogen inoculation). For controls, plants were sprayed with DW. The BTH was used at 100 μg mL−1 as a positive control. The inoculum of the strain SH of V. dahliae was obtained from a pure culture grown on PDA and incubated in the dark at 25 °C for 8 days. The colonies were scraped superficially with a sterile spatula after adding 3 mL of sterile DW. The obtained conidial suspension was filtered through two layers of cheesecloth to remove hyphal fragments. Conidial suspension was adjusted to 107 conidia mL−1 using a Malassez cytometer. At 7 days after the first treatment and 3 days after the second treatment, tomato seedlings growing in the greenhouse were inoculated by dipping their roots in spore suspension for 10 min as described by Zine et al.31 A total of 24 plants arranged in a randomized complete block were examined visually for disease evaluation. Disease incidence (DI) was calculated for 21 days as DI = (number of infected plants/total number of assessed plants) × 100. The expression of foliar symptoms on each plant was assessed through determination of Verticillium wilt foliar symptoms, which were measured periodically for 3 weeks as described by Daayf et al.32 The plant height was measured at the end of the assays to evaluate the stunting index (SI). It corresponds to the reduction in the size of the epicotyl in inoculated plants (Eplx) compared to control plants (Eplc) and is calculated using the formula SI = ((Eplc − Eplx)/Eplc) × 100. Vascular browning was assessed on cross-sections of the stem to quantify internal symptoms as described in Erwin et al.33 Biochemical Assays. For biochemical analyzes, plants were arranged, treated, and inoculated as described above for disease assessment. Their apical leaves were regularly sampled up to 15 days after treatment and used for extraction of enzymes, H2O2, and phenolic compounds according to Zine et al.31 Peroxidase (POX, EC 1.11.1.7) activity was determined spectrophtometrically at 470 nm according to Moerschbacher et al.34 and expressed as micromoles per minute per milligram of protein. Polyphenol oxidase (PPO, EC 1.10.3.1) activity was measured following the method described by Masia et al.35 and expressed as ΔDO per minute per milligram of protein. H2O2 was extracted and measured spectrophtometrically at 390 nm after reaction with KI according to the method of Alexieva et al.36 The H2O2 content was expressed as millimoles per gram of fresh weight. Total polyphenolic compounds were extracted with a solution of ethanol/water/chloroform (1:1:2, v/v/v). The water−ethanol phase

to coordinate ligands for transition metal ions. The transition metal nickel is found in several enzymes, such as superoxide dismutase, urease, hydrogenase, glyoxylase, acireductase, dehydrogenase, and acetyl coenzyme A (CoA) synthase. It is considered as a strong Lewis acid and can activate coordinated ligands for reactivity.22 The nickel sites in these enzymes show extreme plasticity in coordination and redox chemistry. They are able to cycle through three redox states (1+, 2+, and 3+), allowing catalyzes from simple hydrolytic to multistep redox reactions over a potential range of 1.5 V.23 Resulting complexes of ligand−metal can be monomeric24 or binuclear.25 Although these complexes are widely tested against human and animal pathogens,26−28 to the best of our of knowledge, there is no report on their activities against plant pathogens. Moreover, neither thiadiazole nor their transition metal complexes were tested for their ability to induce plant defense response against plant pathogens. In this work, we examined the ability of the ligand 2,5bis(pyridin-2-yl)-1,3,4-thiadiazole (L) and its binuclear transition metal nickel complex bis[μ-2,5-bis(pyridin-2-yl)-1,3,4thiadiazole-κ4N2,N3:N4,N5]bis[dihydrato-κO)nickel(II)] tetrachloro trihydrate [Ni2L2(H2O)4]Cl4·3H2O (Ni2L2) to protect tomato seedlings against Verticillium wilt in the greenhouse and to activate plant defense responses.

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MATERIALS AND METHODS

Chemical Synthesis. The heterocyclic ligand 2,5-bis(pyridin-2-yl)1,3,4-thiadiazole (L) (Figure 1A) was synthesized according to Lebrini

Figure 1. Chemical structures of the (A) ligand 2,5-bis(pyridin-2-yl)1,3,4-thiadiazole (L) and (B) its metal complex bis[μ-2,5-bis(pyridin2-yl)-1,3,4-thiadiazole-κ4N2,N3:N4,N5]bis[dihydrato-κO)nickel(II)] tetrachloro trihydrate (Ni2L2). et al.29 2-Pyridine carboxaldehyde (0.02 mol) was mixed with sulfur (0.03 g) and hydrazine hydrate (0.08 mol) in ethanol (20 mL) and irradiated for 1 h (300 W) at 150 °C under pressure. Ethanol was then evaporated under reduced pressure, and the residue was dissolved in chloroform. The chloroform solution was washed with water, dried, filtered, and then evaporated by rotary evaporation. The resulting residue was crystallized from ethanol. Synthesis of the complex Ni2L2 (Figure 1B) was carried out as described by Bentiss et al.24 Briefly, nickel(II) chloride hexahydrate (1.5 mmol, 0.36 g) dissolved in 8 mL of hot water was added to ligand (0.42 mmol, 0.1 g) dissolved in 8 mL of hot ethanol. The solution was filtered and left to stand at ambient temperature. After 24 h, a green crystallized compound was obtained, washed with water, and vacuum-dried. All of the crystal data and structures were described by Bentiss et al.25 Fungal Isolates and Mycelial Growth. The strain SH of V. dahliae isolated previously from olive trees in Morocco was used.30 2662

DOI: 10.1021/acs.jafc.6b00151 J. Agric. Food Chem. 2016, 64, 2661−2667

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Journal of Agricultural and Food Chemistry containing phenolic compounds was separated from the organic phase containing lipids, chlorophylls, and other pigments. Ethanol was removed using a Rotavapor at low pressures at 35 °C. Phenolic compounds were dissolved in water, and their concentrations were determined using the Folin−Ciocalteu reagent by reading the absorbance at 765 nm according to the method of Ainsworth and Gillespie.37 Gallic acid was used as the standard, and the results were expressed as milligrams of gallic acid equivalent per gram of fresh weight. Statistical Analysis. Data were subjected to one-way analysis of variance (ANOVA), and means were compared by Tukey’s honest significant difference (HSD) test (p < 0.05).

Table 1. Inhibition Rates of Mycelial Growth V. dahliae by the Ligand 2,5-Bis(pyridin-2-yl)-1,3,4-thiadiazole (L) and Its Complex Bis[μ-2,5-bis(pyridin-2-yl)-1,3,4-thiadiazoleκ4N2,N3:N4,N5]bis[dihydrato-κO)nickel(II)] Tetrachloro Trihydrate (Ni2L2) growth rate (cm day−1) negative 0.86 ± 0.01 da control NiCl2 (μg mL−1) 0.82 ± 0.06 d 10 0.81 ± 0.02 d 50 0.74 ± 0.03 cd 100 L 0.7 ± 0.05 c 10 0.61 ± 0.01 b 20 0.51 ± 0.04 b 30 0.54 ± 0.1 b 50 0.54 ± 0.1 b 100 Ni2L2 (μg mL−1) 0.74 ± 0.14 cd 10 0.69 ± 0.03 c 20 0.56 ± 0.05 b 30 0.51 ± 0.03 b 50 0.46 ± 0.06 b 100 thiophanate-methyl (μg mL−1) 0.02 ± 0.005 a 100

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RESULTS In Vitro Activity of L and Ni2L2 on Mycelial Growth of V. dahliae. Growth rates, calculated from the growth curves, and mycelial growth inhibition were determined after culture of the strain SH of V. dahliae at 25 °C in PDA medium amended with different concentrations of the ligand L and its complex Ni2L2 (Table 1). The antifungal activities of NiCl2·6H2O and thiophanate-methyl were also studied. When the transition metal Ni was added, as NiCl2·6H2O, to the PDA medium, only very weak inhibition of growth (12%) but significant was recorded at 100 μg mL−1. The percentage of inhibitions varied from 14 to 33% for L and from 10 to 39% for Ni2L2 and were significantly different from the control at concentrations starting from 20 μg mL−1. Nevertheless, these inhibitions are by far significantly much lower than that obtained with thiophanate-methyl at 100 μg mL−1. L and Ni2L2 Stimulate Plant Resistance to V. dahliae Infection. Disease development was evaluated in the greenhouse on plants treated with L and Ni2L2 and compared to DW (as a negative control) and BTH (as a positive control). Results showed that two applications of L, Ni2L2, or BTH at 100 μg mL−1 at 7 and 4 days before inoculation with V. dahliae induced a reduction in disease incidence compared to DW treatment. This reduction was significant from the eighth day of inoculation (Figure 2A). At the end of the experiment, disease incidence reached 62 and 67% in plants pretreated with the ligand L and BTH, respectively, whereas it decreased to 43% in plants pretreated with Ni2L2. Similarly, disease severity was reduced in plants pretreated by the three chemicals. Progress of Verticillium wilt foliar symptoms was affected over time (Figure 2B). Plants pretreated by BTH, ligand L, and its complex Ni2L2 showed a significantly much reduced severity of foliar symptoms when compared to the control from the eighth day until the end of the experiment. However, protection afforded by L and Ni2L2 was significantly higher than that induced by BTH. At 21 days after inoculation, the percentage of protection was about 82% with the ligand L, 91% with the complex Ni2L2, and only 70% with BTH. Stunting index and vascular browning index of vessels were also recorded at 21 days after inoculation with V. dahliae to monitor disease severity. The fungus reduced the growth of plants pretreated with DW by 22%, whereas this was significantly less affected in plants pretreated with BTH, L, or Ni2L2 (Figure 2C). Likewise, the vascular browning index of vessels was inhibited by 95% in plants pretreated with Ni2L2 and 79 and 82% in those sprayed with L and BTH, respectively (Figure 2D). An illustration of protection afforded by Ni2L2 is shown in Figure 2E.

percentage of inhibition

diameter of growth (cm)

0 ± 0.01 d

4.46 ± 0.04 d

3.3 ± 0.11 d

4.33 ± 0.37 d

5.2 ± 1.03 d

4.23 ± 0.03 d

12.4 ± 2.3 c

3.91 ± 0.01 cd

14 ± 3.15 c

3.82 ± 0.03 c

25 ± 2.85 b

3.35 ± 0.02 b

33 ± 1.73 b

2.98 ± 0.14 b

33 ± 1.31 b

2.98 ± 0.34 b

32 ± 1.96 b

3.02 ± 0.05 b

10 ± 4.5 cd 16 ± 1.9 c 28 ± 4.8 b 33 ± 2 b 39 ± 8.3 b 85 ± 1.31 a

4 ± 0.57 cd 3.73 ± 0.1 c 3.2 ± 0.23 b 3 ± 0.11 b 2.79 ± 0.26 b 0.7 ± 0 a

Values are means ± confidence intervals (α = 5%) from three replicates. Means with different letters are significantly different according to Tukey’s HSD test (p < 0.05). The growth rate (in centimeters per day) was calculated from growth curves. The diameter of inhibition growth (in centimeters) was determined at 5 days after incubation. The negative control is from unamended PDA. Thiophanate-methyl is used as a standard fungicide. a

L and Ni2L2 Potentiate the Activity of Defense Enzymes. We examined if the ligand used in this study and its derivative were able to induce plant defenses by analyzing kinetics of activation of two enzymes, POX and PPO. Time course analysis of POX activity was determined in plants pretreated twice with 100 μg mL−1 ligand L and its metallic complex Ni2L2 (Figure 3). In non-inoculated plants pretreated with L or Ni2L2, POX activities started to increase from 6 h, began to stabilize from 1 day until 7 days after treatment, and then increased again to reach their maximum at 11 days. These increases were significantly higher in plants treated with L and Ni2L2 when compared to the control at all analyzed time points, except at 7 days after treatment for L. At 11 and 15 days, POX activities induced by L and Ni2L2 were 4.6 and 7.6 times higher than the control, respectively. In inoculated plants, when compared to the control, L and Ni2L2 increased POX activity by 3 and 12 times, respectively, from the second until the seventh day post-inoculation. 2663

DOI: 10.1021/acs.jafc.6b00151 J. Agric. Food Chem. 2016, 64, 2661−2667

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Journal of Agricultural and Food Chemistry

Figure 2. Effect of the ligand L and its metal complex Ni2L2 on Verticillium wilt of tomato. (A) Progress of disease incidence. (B) Progress of Verticillium wilt foliar symptoms. (C) Stunting index at 21 days post-infection (dpi). (D) Vascular browning at 21 dpi. (E) Verticillium wilt symptoms in plants treated with Ni2L2 (right side) or DW (left side). Tomato seedlings were treated with DW, BTH (used as a positive control), ligand (L), or its complex Ni2L2 at 7 and 3 days before root-dip inoculation with 107 conidia mL−1 of V. dahliae. Values are means ± confidence intervals (α = 5%) from 24 replicates. Means with different letters are significantly different according to Tukey’s HSD test (p < 0.05).

L and Ni2L2 Induce a Strong H2O2 Accumulation. In this experiment, we wanted to see if the ligand and its metallic complex were able to induce an oxidative burst. To do so, time course analysis of the H2O2 content was determined in plants pretreated twice with 100 μg mL−1 ligand L and its metallic complex Ni2L2 (Figure 5). In non-inoculated plants, H2O2 increased over time either after pretreatment with DW, ligand L, and Ni2L2 but significantly higher contents were recorded from plants pretreated with these two chemicals. No differences were observed between L and Ni2L2 in H2O2 activation. In inoculated plants, higher concentrations of H2O2 were obtained in plants pretreated with L and Ni2L2. Such an increase seemed to be slightly superior with Ni2L2 than L. In all cases, these increases did not exceed 1.6-fold in both inoculated and non-inoculated plants.

As a second marker of plant defense, we monitored the time course analysis of PPO activity in plants pretreated twice with 100 μg mL−1 ligand L and its metallic complex Ni2L2 (Figure 4). In non-inoculated plants, an early activation of PPO activity was observed at 6 h after treatment. It reached a maximum at 2 days, where these activities were 3.3 and 4.6 times higher in plants pretreated with L and Ni2L2 than in the control, respectively. This was followed by a sharp decrease at 4 and 7 days after treatment before reaching a second maximum with L at 11 days after treatment. With Ni2L2, PPO activity continued to increase at 11 and 15 days. At this moment, activities were 5.7 and 7.6 times higher than those recorded from the control. These increases intensified after inoculation. At the end of inoculation, values were 7.8 and 11 times higher than the control, indicating a potentiation of plant defense after V. dahliae inoculation. 2664

DOI: 10.1021/acs.jafc.6b00151 J. Agric. Food Chem. 2016, 64, 2661−2667

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Journal of Agricultural and Food Chemistry

L and Ni2L2 Induce Phenolic Accumulation. Total phenolic compounds were determined in plants pretreated twice with 100 μg mL−1 ligand L and its metallic complex Ni2L2 (Figure 6). In non-inoculated plants, an increase was observed

Figure 3. Effect of the ligand L and its metal complex Ni2L2 on POX activity in tomato leaves. Tomato seedlings were treated with DW, ligand (L), or its complex Ni2L2 at 7 and 3 days before root-dip inoculation with 107 conidia mL−1 of V. dahliae. Values are means from three replicates ± confidence intervals (α = 5%). Means with different letters are significantly different according to Tukey’s HSD test (p < 0.05).

Figure 6. Effect of the ligand L and its metal complex Ni2L2 on the accumulation of total phenolic compounds in tomato leaves. Tomato seedlings were treated with DW, ligand (L), or its complex Ni2L2 at 7 and 3 days before root-dip inoculation with 107 conidia mL−1 of V. dahliae. Values are expressed as milligrams of gallic acid equivalent per gram of fresh weight and are means from three replicates ± confidence intervals (α = 5%). Means with different letters are significantly different according to Tukey’s HSD test (p < 0.05).

during the 15th day of the experiment in the control and in plants pretreated with L and Ni2L2. However, these increases were significantly higher in plants treated with these two products. The phenolic compound content in plants treated with Ni2L2 was significantly higher than in plants pretreated with L alone at 15 days after treatment, being 1.8 and 2.5 times higher than the control at the 15th day, respectively. In inoculated plants, L and Ni2L2 induced a significantly elevated phenolic content when compared to plants pretreated with DW. This content was higher in inoculated plants pretreated with Ni2L2 than with L.

Figure 4. Effect of the ligand L and its metal complex Ni2L2 on PPO activity in tomato leaves. Tomato seedlings were treated with DW, ligand (L), or its complex Ni2L2 at 7 and 3 days before root-dip inoculation with 107 conidia mL−1 of V. dahliae. Values are means from three replicates ± confidence intervals (α = 5%). Means with different letters are significantly different according to Tukey’s HSD test (p < 0.05).

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DISCUSSION In this report, we have showed that the ligand L and its binuclear metallic complex Ni2L2 displayed low inhibitory effects against V. dahliae. Although growth rates obtained with these two chemicals were significantly different from that observed with the control, they were extremely higher when compared to the benchmark fungicide thiophanate-methyl. In addition, no synergistic effect was observed between the ligand and its transition metal complex for the in vitro growth inhibition of the pathogen because differences were not significant mainly at concentrations higher than 20 μg mL−1. These results are somehow contradictory to previous reports from other thiadiazole derivatives against animal and human pathogens.26,27,38 However, they suggest that the ligand alone plays a major role in this weak inhibitory effect. The weak antifungal activity for the ligand L and the complex Ni2L2 made them suitable candidates as plant resistance activators. Greenhouse experiments revealed that they reduced disease incidence and severity caused by V. dahliae in tomato. These parameters were similarly reduced by the ligand and commercialized plant activator BTH.31 However, they seem to be more reduced upon coordination with Ni. The ligand that we used and its derivative share the thiadiazole ring with BTH. It has been suggested that the carboxylate group on the phenyl

Figure 5. Effect of the ligand L and its metal complex Ni2L2 on the accumulation of reactive oxygen species (ROS) in tomato leaves. Tomato seedlings were treated with DW, ligand (L), or its complex Ni2L2 at 7 and 3 days before root-dip inoculation with 107 conidia mL−1 of V. dahliae. Values are means from three replicates ± confidence intervals (α = 5%). Means with different letters are significantly different according to Tukey’s HSD test (p < 0.05).

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DOI: 10.1021/acs.jafc.6b00151 J. Agric. Food Chem. 2016, 64, 2661−2667

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Journal of Agricultural and Food Chemistry ring is critical to the biological activity of BTH,39 and substitution of methyl by fluorine atoms in the esterified carboxylate group resulted in compounds with efficacy higher than BTH against Erysiphe cichoracearum and Colletotrichum lagenarium in cucumber.40 In our study, it seems that substitution of the carboxyphenyl group with two pyridyl rings is also of great importance. Recently, a pyrimidin-type plant activator, 5-(cyclopropylmethyl)-6-methyl-2-(2-pyridyl)pyrimidin-4-ol, sharing the 2-pyridyl group with our ligand, has been reported to reduce bacterial growth of Pseudomonas syringae pv. maculicola in Arabidopsis thaliana and an earlier and stronger oxidative burst compared to plants pretreated with BTH.41 L and Ni2L2 were all able to promote H2O2 production in tomato plants. It has been shown that BTH was able to inhibit the activity of H2O2-scavenging enzymes, such as catalase and ascorbate peroxidase, and to increase concentrations of H2O2.42 In plants, it participates in various resistance mechanisms. For instance, H2O2 functions as a substrate involved in strengthening of cell walls by cross-linking glycoproteins to resist pathogen invasion and also has a direct effect on pathogens.43 It can act as a diffusible signal for the induction or potentiation of defensive genes in adjacent cells to limit pathogen invasion. L and Ni2L2 applications induced a significant increase of PPO and POX activities in tomato plants, two enzymes involved in cell wall reinforcement and pathogen inhibition.44,45 Activation of PPO with phenolic compounds by the ligand and its complex may lead to quinones, known for their toxicity to the plant pathogens.46,47 Moreover, POX and PPO activities were dramatically increased in inoculated plants pretreated with these two chemicals. This clearly demonstrates that L and Ni2L2 were able to act by priming, as reported for BTH.10−13 Interestingly, Ni2L2 was more efficient than L. These data suggest that coordination enhances plant defense responses maybe by altering the redox status of the cells. Taken together, our results clearly showed that coordination of the ligand with transition metal Ni enhanced its ability to protect tomato against Verticillium wilt and to activate plant defenses. Reduction of symptom development in plants pretreated with these products as foliar spray implies that they are translocated and lasted long enough in the xylem to activate plant defenses. Altogether, this study offers new structural insights into designing novel plant activators in the field of plant protection.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +212-671-276-617. Fax: +212-523-342-187. Email: [email protected]. Funding

This work was supported by the University of Chouaib Doukkali, El Jadida, Morocco. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank S. Qsaib for his technical assistance. REFERENCES

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