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Article

Metabolic Response Induced by Methyl Jasmonate and Benzothiadiazole in Vitis vinifera cv. Monastrell Seedlings

by
Diego Paladines-Quezada
1,* and
Rocío Gil-Muñoz
2
1
Instituto de Ciencias de la Vid y del Vino (CSIC, Gobierno de La Rioja, Universidad de La Rioja), Ctra. de Burgos, km. 6, 26007 Logroño, Spain
2
Instituto Murciano de Investigación y Desarrollo Agrario y Medioambiental, Ctra. La Alberca s/n, 30150 Murcia, Spain
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(3), 277; https://doi.org/10.3390/horticulturae11030277
Submission received: 1 January 2025 / Revised: 16 February 2025 / Accepted: 27 February 2025 / Published: 4 March 2025
(This article belongs to the Special Issue Climate Change and Adaptive Modern Strategies in Viticulture)
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Abstract

:
This study evaluates the effects of the elicitors methyl jasmonate (MeJ) and benzothiadiazole (BTH) on the synthesis of phenolic compounds in Vitis vinifera cv. Monastrell seedlings over 72 h. Results show that both elicitors induce the accumulation of stilbenes and phenolic acids, essential compounds in plant defence against pathogens. Specifically, MeJ significantly increased the levels of astringin, T-resveratrol, and miyabenol C, known for their antifungal properties, indicating a rapid and effective activation of plant defences. Discriminant analysis revealed that MeJ and BTH markedly altered the phenolic profile, highlighting their role in modulating defence responses. However, their combined application exhibited antagonistic effects on some compounds, suggesting an adaptive metabolic response. The defence response was transient, with peak concentrations observed within the first 24 h, followed by down-regulation, optimising the plant’s energy resources. These findings suggest that MeJ and BTH can enhance the resistance of the Monastrell variety, potentially reducing pesticide use in sustainable viticulture. Further studies are needed to assess their long-term effects under field conditions, considering environmental variables and optimal application rates.

1. Introduction

The issues arising from pesticide use in viticulture are a global concern, affecting farmers’ health [1] and leaving residues in wine [2] that may disrupt yeast essential for production [3], thereby impacting wine quality. Moreover, their environmental impact extends to soil [4] and surface and groundwater [5]. However, fungal diseases in grape cultivation remain a significant issue, potentially reducing both the quality and yield of the berries through direct infection or by diminishing plant vigour [6]. Downy and powdery mildew (Plasmopara viticola and Erysiphe necator), black rot (Guignardia bidwellii), and grey mould (Botrytis cinerea) have recently attracted the attention of the European Union and have been identified as the vine diseases with the greatest impact across Europe [7]. Thus, the key challenge today lies in transitioning towards production systems that integrate sustainability, economic viability, and more environmentally friendly practices [8].
One alternative to achieving these objectives is the stimulation of the plant’s natural defence mechanisms through treatment with molecules known as elicitors [9]. These compounds, which include biotic elicitors (derived from plants or microbes) [10] and abiotic elicitoric factors, which encompass physical stimuli (such as osmotic stress and UV light) as well as chemical agents (such as methyl jasmonate and benzothiadiazole) [11], do not exhibit direct antimicrobial activity but enhance resistance by activating defence responses [12]. Upon elicitor perception, plant cells initiate signalling cascades that lead to hydrogen peroxide (H₂O₂) production, upregulation of defence-related genes, synthesis of pathogenesis-related (PR) proteins, accumulation of phytoalexins, and reinforcement of the cell wall [13,14,15]. The phenylpropanoid pathway plays a crucial role in this process, governing the biosynthesis of phenolic compounds, which exhibit antimicrobial and antifungal properties, contribute to cell wall strengthening, and function as signalling molecules in response to stress [16,17,18].
Among these, stilbenes are widely recognised as phytoalexins in the Vitaceae family and other plant groups due to their role in protecting plants against pathogenic fungi [19]. In grapevines, stilbenes are consistently present and can also be synthesised in response to various biotic and abiotic stress factors, particularly in leaves and berry skins [20]. In addition to stilbenes, certain phenolic acids play a crucial role in grapevine defence. Compounds such as coumaroyltartaric acid, 3-O-p-coumaroylquinic acid, and ellagic acid pentoside contribute to plant protection through antimicrobial activity [21], their participation in the phenylpropanoid pathway, and the accumulation of phytoalexins in response to pathogen attacks [22].
Several of the elicitors mentioned above have been trialled in vineyards, demonstrating positive effects on the synthesis of secondary metabolites in previous studies. For instance, Monastrell grapes exhibited increased levels of resveratrol and piceatannol following UV-C light irradiation [23], while post-harvest carbon dioxide treatments were found to enhance proanthocyanidin synthesis in wine grapes [24]. Additionally, osmotic stress induced by deficit irrigation has been shown to improve the phenolic compound content in wine grapes [25,26,27].
Building on this body of evidence, this study focuses specifically on two chemical elicitors, methyl jasmonate (MeJ) and benzothiadiazole (BTH), with the aim of further understanding their effects on grapevines. Previous trials have primarily demonstrated their potential to increase phenolic and aromatic compounds in grapes and wines [28,29,30,31,32,33,34].
MeJ, on the one hand, is involved in various plant responses to abiotic stress due to its crucial role in multiple communication and signalling processes within plant cells [35]. Studies on grapevines have shown that MeJ promotes callose deposition, the accumulation of pathogenesis-related (PR) proteins, and the production of salicylic acid in leaves and cultured suspension cells [36,37]. Furthermore, it is classified as a Generally Recognised as Safe (GRAS) substance by the U.S. Food and Drug Administration [38].
On the other hand, BTH, a functional analogue of salicylic acid, and its derivatives have been identified as compounds that stimulate plant defence mechanisms [39]. Moreover, BTH has a low toxicological risk [40], breaks down rapidly in plant tissues, and lacks antibiotic activity [41], all of which contribute to a significant reduction in the environmental impact of crop treatments [42].
However, there is limited information on how MeJ and BTH affect the phenolic composition of grapevine leaves, which play a crucial role in the plant’s defence functions [43]. Preliminary research has shown that these organs serve as reliable indicators of nutritional deficiencies [44], as well as resistance or susceptibility to pathogens [45], among other factors. Previous studies have demonstrated that MeJ and BTH activate defence mechanisms in Vitis vinifera. MeJ induces the expression of genes involved in the synthesis of defence-related enzymes, such as chitinase and glucanase, and promotes the accumulation of phytoalexins, enhancing resistance to powdery mildew (Erysiphe necator). Meanwhile, BTH stimulates genes involved in stilbene synthesis and PR protein production, reprogramming leaf metabolism to strengthen pathogen defence [6,46].
Based on these studies, we hypothesise that the application of these elicitors could also activate defence mechanisms in the Monastrell variety. To our knowledge, no trials have been conducted on seedlings of this variety, which is one of Spain’s major wine grapes and is known as ‘Mourvèdre’ in many other countries. Therefore, understanding how this variety responds to elicitation would be valuable, as it could ultimately contribute to reducing pesticide use in viticulture.
Thus, this study aimed to assess whether the external application of MeJ, BTH, or their combination to Vitis vinifera cv. Monastrell seedlings can trigger defence mechanisms in the leaves by stimulating the biosynthesis of phenolic compounds, the primary antimicrobial agents in grapevines.

2. Materials and Methods

2.1. Plant Material

Wood cuttings of Vitis vinifera L. cv. Monastrell were collected in January 2020 from the experimental vineyard at the Murcia Institute of Agricultural and Environmental Research and Development (IMIDA), located in southeastern Spain. The cuttings were disinfected and stored in dark plastic bags in a refrigeration chamber at 4 °C. In early March 2020, the cuttings were planted in individual pots containing universal substrate (50–60% peat, 20–30% perlite, and 10–30% coconut fibre) and were placed in a climate-controlled chamber at IMIDA. The cultivation conditions were as follows: temperatures of 25 °C during the day and 18 °C at night (±2 °C); relative humidity of 75% (±10%); and a 16-h light photoperiod (550 ± 38 lux), measured with a T-10A illuminance meter (Konica Minolta, Tokyo Japan). The treatments were applied in May 2020, using seedlings with 10–12 leaves. For each treatment and replication, plastic trays with 30 seedlings/tray were prepared.

2.2. Preparation of Treatments

Four treatments were tested (3 repetitions/treatment), consisting of a single application of an aqueous suspension of (i) water (Control), (ii) MeJ (10 mM), (iii) BTH (0.3 mM), and (iv) a mixture of both MeJ + BTH (10 mM and 0.3 mM, respectively). All treatments, including the control, contained Tween 80 (0.1% v/v) as surfactant. MeJ (Methyl Jasmonate), BTH (S-methyl ester of benzo-(1,2,3)-thiadiazole-7-carbothioic acid), and Tween 80 were purchased from Sigma Aldrich (St. Louis, MO, USA). The water was of Milli-Q quality. Both MeJ and BTH were dissolved in water along with Tween 80 by sonication (room temperature; 30 min) using a Totech Sonicator LT-100-PRO (TierraTech, Cantabria-Spain). At 6 pm, trays with 30 plants were sprayed with 300 mL of the aqueous suspension (10 mL per plant).
For the analysis of phenolic compounds, leaves were collected at five different time points: before treatment (0 h) and after its application (12, 18, 24, and 72 h) to determine whether the elicitors could induce an immediate change in foliar biosynthesis and sustain it over time. Six seedlings were used per time point, and each replicate consisted of a mixture of six leaves (one per seedling) collected from alternating positions. Leaves from each sampling and treatment were frozen, ground in liquid nitrogen, and stored at −80 °C until further analysis.

2.3. Extraction of Phenolic Compounds

A previous protocol was followed for the extraction of phenolic compounds [47]. Samples were prepared under light-protected conditions to prevent photochemical isomerization of trans-stilbenes into the less fluorescent cis forms [6]. Phenolic compounds were extracted from frozen ground leaves (100 mg). A mixture of diethyl ether:MeOH (70:30; v:v) pre-cooled (4 °C) was used as the extractant. Two re-extractions were performed with 10 mL of extractant each time by sonication (cold bath; 30 min) using a Totech Sonicator. The total supernatant was transferred to a Falcon tube (50 mL) and centrifuged (12,000 rpm; 4 °C; 10 min) using an Eppendorf 5018R Centrifuge (Eppendorf, Hamburg, Germany). Subsequently, the supernatant was filtered with Sep-Pack C18 cartridges (Waters Corporation, Milford, MA, USA) to remove chlorophyll. The extract was evaporated to dryness using a Techne™ DB3 concentrator (Techne, Staffordshire, UK) with nitrogen flow (Block temperature: 35 °C). The dry extract was diluted in 0.5 mL of MeOH with sonication (cold bath; 10 min). The sample was then filtered with nylon filters (0.22 µm) and transferred to amber vials with inserts.

2.4. Analysis of Phenolic Compounds

The determination of phenolic compounds was carried out as described in a previous protocol [48]. Briefly, 10 µL of the sample was injected into HPLC using a Waters 2695 liquid chromatograph (Waters, Lilford, PA, USA) equipped with a Photodiode Array Detector (PDA) Waters 2998 (Waters, Lilford, PA, USA) and a Li-Chrospher 100 RP-18 column (Merck, Darmstadt, Germany) (25 × 0.4 cm; 5 µm particle size). The mobile phases consisted of acidified water (1% formic acid) (Phase A) and acetonitrile (Phase B), with a flow rate of 0.7 mL min⁻1 and a column temperature of 25 °C. The working gradient was as follows: 0–4 min (100% A), 5–20 min (85% A and 15% B), 21–30 min (60% A and 40% B), 31–32 min (45% A and 55% B), and 33–42 min (100% A). The PDA working range was between 210–400 nm. The mass spectrometer was set in positive mode with a capillary voltage of 1.5 kV, while for negative mode, it was set at a capillary voltage of 0.3 kV. The cone voltage was set to 30 V, and the desolvation temperature was set to 350 °C in both modes. The spectra were acquired in the m/z range 100–1200, using an ACQUITY QDA Mass Detector (Waters Corporation, Milford, PA, USA).
The identification of the compounds present in the leaf samples was achieved through the analysis of several properties, including UV spectra (maximum absorbances), retention time, and spectral information, which included the identification of the molecular ion and several of its fragments. These properties were compared with those recorded by commercial standards, as well as with compounds reported in the literature and external databases. Given that some compounds exhibited overlapping retention times, additional spectral data from the PDA detector (UV absorption maxima) and mass spectrometry (molecular and fragment ion patterns) were used to ensure accurate differentiation.
All chemicals (solvents) used were of HPLC grade, analytical grade (>99%). The water was of Milli-Q quality. Standards piceatannol (3,3′,4,5′-tetrahydroxystilbene), trans-resveratrol (3,5,4′-trihydroxy-trans-stilbene), trans-piceid, and Ɛ-viniferin were purchased from Sigma Aldrich (St. Louis, MO, USA).
The quantification of phenolic compounds was expressed in Absorbance Units (AU) based on PDA detection rather than absolute concentrations. Consequently, the limit of detection (LOD) and the limit of quantification (LOQ) values were not determined. Instead, the focus of the study was on comparative analyses of metabolic responses rather than absolute quantification.
In addition, the different levels of identification were established in accordance with the methodology described by Summer et al. (2007) [49]. (1) Identified compounds confirmed using commercial standards. (2) Putatively annotated compounds, identified without chemical reference standards but based on physicochemical properties and/or spectral similarity to public or commercial spectral libraries. (3) Putatively characterised compound classes determined based on distinctive physicochemical properties of a chemical class or spectral similarity to known compounds within that class. (4) Unknown compounds, which, although not identified or classified, can still be distinguished and quantified using spectral data.

2.5. Statistical Analysis

Differences among treatments for each variable were evaluated using analysis of variance (ANOVA) with the Statgraphics 5.0 Plus software package (Statpoint Technologies, Inc., Warrenton, VA, USA). When ANOVA results were significant, Duncan’s test (p < 0.05) was performed to separate the means. Additionally, multivariate discriminant analysis was conducted to identify the variables with the highest discriminative power.

3. Results

3.1. Identification of Phenolic Compounds

From the 50 chromatographic peaks obtained, 19 compounds were identified as belonging to the families of flavanols, phenolic acids, and stilbenes (Table 1). Identification was established at level 1 for 4 compounds through comparison with standard patterns, level 3 for 15 tentatively identified compounds, and the remaining compounds were classified as unidentified (level 4) [49].
Subsequently, the relative abundance of the 50 chromatographic peaks was integrated (Table S1), from which 16 compounds were selected: 14, 15, 16, 20, 21, 24, 25, 26, 32, 33, 34, 40, 41, 45, 47, and 48, as they displayed statistically significant differences in at least one of the different sampling times. These 16 compounds were used to perform 5 discriminant analyses to visualise more intuitively the differences between the groups: General (Figure 1), 12 h (Figure 2A), 18 h (Figure 2B), 24 h (Figure 2C), and 72 h (Figure 2D).

3.2. General Discriminant Analysis

Sixteen discriminant functions were generated, with the first two explaining 68.4% of the observed variance (Function 1: 54.4%; Function 2: 14%) (Figure 1). The projections generated by this analysis allowed for a general anticipation of the differences among the established groups. However, given the inclusion of all treatments and their evolution across sampling points, a clear differentiation was challenging to discern. Consequently, individual sampling discriminant analyses are examined in more detail below.

3.3. Discriminant Analyses at Different Time Points

The projections generated by the two main functions are shown in Figure 2A–D. In general, a strong influence of the treatments on the biosynthesis of various analysed compounds was observed, as evidenced by the marked separation between the control group samples and those treated with elicitors. The time points of 18, 24, and 72 h were the most relevant in assessing the effects of the elicitors. At 18 and 24 h, the MeJ treatment showed the greatest differences compared to the control. However, at 72 h, all treatments showed significant differences compared to the control samples.
Below is a summary of the most significant parameters of the individual discriminant analyses (Table 2). In all samplings, only three functions were needed to explain nearly all the variance. Moreover, the percentages of variance explained by the first two functions exceeded 97.5%, indicating that the features enabling discrimination among the different groups are robust. The most significant standardised coefficients were Coumaroyltartaric acid, Ellagic acid pentoside, Astringin, compound no. 16 (unknown), Miyabenol, and 3-O-p-Coumaroylquinic acid.

3.4. Effect of Elicitors on the Evolution of Various Phenolic Compounds

Among the 16 compounds that showed significant differences between the different samples, only 9 compounds were identified, including compounds of the stilbene family and phenolic acids (Figure 3A–I).

3.4.1. Stilbenes

  • Astringin: Figure 3A shows a general decrease in astringin concentration across all analysed samples during the first 12 h of the study. At 18 h, astringin concentration continued to decrease in the Control, BTH, and MeJ + BTH groups, except for the MeJ-treated samples, which reached their maximum concentration at this time. From 18 to 24 h, astringin concentration increased in the control, BTH, and MeJ + BTH samples, while it began to decrease in the MeJ-treated samples. After this point, the concentration of astringin declined in all samples until the end of the study (72 h). It is worth noting that MeJ-treated plants were the only ones showing a significantly higher concentration of astringin at the 18 and 24-h samplings compared to the other treatments.
  • Miyabenol C: The general decrease in Miyabenol C concentration was recorded during the first 12 h of treatment (Figure 3B) and observed in all analysed samples. At the 18-h sampling, the Control, BTH, and MeJ + BTH samples continued to decrease, except for the MeJ-treated samples, which experienced a significant increase in Miyabenol C concentration. After 24 h, all samples showed higher levels of Miyabenol C, with MeJ and MeJ + BTH-treated plants showing statistically significant differences from Control plants. From this point, Miyabenol C concentration decreased in all leaves until the end of the study.
  • T-piceid: In Figure 3C, an overall increase in T-piceid concentration was observed in all samples during the first 12 h, with the exception of a slight decrease in the MeJ + BTH group. At the 18-h sampling, this upward trend persisted, particularly notable in the MeJ group, which reached its peak concentration at this point. At 24 h after treatment initiation, all samples continued to show an increase in T-piceid concentration, except for the MeJ group, which began to decrease. At the end of the study, the Control and BTH samples continued to increase T-piceid concentration, while the MeJ group decreased to levels similar to the other groups. In contrast, the MeJ + BTH samples showed a decline to the lowest levels compared to the other treatments.
  • Piceatannol: During the first 12 h, Piceatannol concentration increased in all treated plants (Figure 3D) except for the control group. At the 18-h sampling, the MeJ and MeJ + BTH groups continued to show an increase in concentration, contrasting with the control group, which continued to decline, while the MeJ + BTH group began to decrease. In the subsequent sampling (24 h), the MeJ, BTH, and MeJ + BTH samples continued to decrease in Piceatannol concentration, except for the control group, which showed a slight increase. At the end of the study, a general increase in Piceatannol concentration was observed in all samples, being more pronounced in the treated plants. Specifically, the MeJ and MeJ + BTH treatments were statistically distinguishable from the control samples.
  • T-Resveratrol: In the first 12 h, an increase in T-resveratrol concentration was observed in all treatments (Figure 3E), with this increase being especially notable in the MeJ and MeJ + BTH samples, which reached concentrations much higher than the other groups. At the 18-h sampling, this compound continued to increase in MeJ and BTH treatments, although to a lesser extent in the latter. Conversely, a marked decrease was recorded in the MeJ + BTH-treated plants, with a slight decrease in the control group. In the 24-h sampling, T-resveratrol concentration dramatically decreased in MeJ-treated samples, while in BTH-treated samples, it also declined but less markedly. Conversely, the control and MeJ + BTH groups showed a slight increase in T-resveratrol concentration. At the end of the study, T-resveratrol concentration levelled across all samples, except in MeJ + BTH samples, which showed the lowest concentration of this compound.

3.4.2. Phenolic Acids

  • Coumaroyltartaric Acid and 3-O-p-Coumaroylquinic Acid: The evolution of these two compounds (Figure 3F,G) followed a similar pattern throughout the study, although the signal detected for 3-O-p-Coumaroylquinic acid was considerably higher than that for Coumaroyltartaric acid. In general, during the first 12 h, all samples except for the control group experienced a decrease in the concentration of these two compounds. At the 18-h sampling, this decreasing trend continued, except for the MeJ-treated plants, which reached their maximum concentration in both cases. At 24 h, the control, BTH, and MeJ + BTH groups increased their concentration of Coumaroyltartaric acid and 3-O-p-Coumaroylquinic acid, whereas MeJ-treated samples began to experience a decrease in the concentration of these compounds, similar to the majority of the treatments, until the end of the study (72 h).
  • Ellagic Acid Pentoside: At the 12-h sampling, a general decrease in Ellagic acid pentoside concentration was observed across all samples (Figure 3H). After this point, this compound’s concentration continued to decline in most samples, except for MeJ-treated plants, where an increase was observed. Between the 18-h and the 24-h samplings, significant increases were noted in MeJ, BTH, and MeJ + BTH-treated samples. However, concentrations subsequently began to decrease, except in the control samples, which continued to increase. By the end of the study, the Ellagic acid pentoside concentration in the control samples was notably lower than in the other treatments.
  • Dihydroxybenzoic acidhexosyl pentoside: At 12 h after the start of the trial, a general increase in the concentration of this compound was observed across all treatments (Figure 3I), except for those treated with MeJ. By the 18-h sampling, control group samples continued to show a significant increase in dihydroxybenzoic acid hexosyl pentoside concentration, with a slight rise also seen in MeJ-treated samples. However, samples from the BTH and MeJ + BTH groups exhibited a slight decrease in concentration. At the 24-h mark, control group samples displayed a notable increase in this compound’s concentration, whereas BTH and MeJ + BTH group samples began to show a slight rise, in contrast to the slight decrease observed in MeJ-treated samples. By the end of the trial, the concentration of dihydroxybenzoic acid hexosyl pentoside in control samples had nearly tripled its initial value. It is worth noting that although samples from the treated groups also experienced an increase in this compound’s concentration, the increase was lower compared to the control group. Specifically, samples from the MeJ + BTH group showed the least variation in concentration from the start of the trial.

4. Discussion

This study examined the effects of methyl jasmonate (MeJ) and benzothiadiazole (BTH), as well as their combination, on the biosynthesis of phenolic compounds in Vitis vinifera cv. Monastrell seedlings over a 72-h period. The results confirmed that MeJ and BTH induce the accumulation of specific phenolic metabolites, suggesting an effective activation of defence mechanisms. This aligns with studies on Cabernet Sauvignon, where similar effects of these elicitors were reported [6,46]. However, as this is the first report on Monastrell seedlings, it highlights varietal specificity. While responses in Monastrell resemble those in Cabernet Sauvignon, differences in phenolic accumulation patterns, particularly in stilbenes and phenolic acids, were evident [6]. These findings underscore the importance of studying individual cultivars, as genetic and physiological traits influence metabolic responses. Understanding these differences could help optimise elicitor applications in Monastrell and other grapevine varieties.
Discriminant analysis (Figure 2) clearly distinguished elicitor-treated plants from controls, demonstrating the ability of MeJ and BTH to modify the phenolic profile of Monastrell leaves. The metabolic response was time-dependent, with MeJ inducing the most pronounced divergence at 18 and 24 h, suggesting rapid activation of phenolic metabolism. By 72 h, all treatments differed significantly from the control, indicating a sustained effect. While MeJ triggered a faster response, BTH appeared to contribute to long-term metabolic shifts. The convergence of all treatments at 72 h suggests the activation of complementary pathways over time, consistent with the idea that plants require a rapid supply of defensive compounds and cross-flow of biosynthetic intermediates as a survival strategy [60]. These temporal changes highlight the need to analyse multiple time points to fully understand elicitor-induced responses.
MeJ treatment significantly increased phenolic acids such as coumaroyltartaric acid, 3-O-p-coumaroylquinic acid, and ellagic acid pentoside (Figure 3), along with stilbenes including astringin, miyabenol C, T-piceid, piceatannol, and T-resveratrol. These compounds play key roles in plant defence, reinforcing the effectiveness of MeJ in enhancing secondary metabolism. However, certain stilbenes, such as T-piceid and piceatannol, showed different accumulation patterns compared to resveratrol, suggesting alternative biosynthetic pathways or regulatory mechanisms requiring further investigation.
A notable observation was the initial decrease in astringin and miyabenol C during the first 12 h, followed by a significant increase at 24 h (Figure 3A,B). This may be due to early enzymatic degradation or conjugation processes, which regulate phenolic metabolism. Stilbenes, particularly dimeric forms like miyabenol C, undergo rapid metabolic transformations, including glycosylation or oxidation, in response to elicitor-induced stress [61]. The subsequent increase at 24 h likely reflects the activation of stilbene synthase (STS), a key enzyme in stilbene biosynthesis, which exhibits delayed expression peaks after elicitor application [62]. Similarly, the decrease in T-resveratrol at 24 h in MeJ- and BTH-treated samples (Figure 3E) may be associated with its conversion into bioactive derivatives such as piceatannol or resveratrol oligomers, which play essential roles in plant defence. MeJ and BTH have been reported to promote the enzymatic transformation of resveratrol into viniferins and other oligomeric stilbenes mediated by peroxidases and laccases [61]. This would explain the decline in T-resveratrol without implying degradation but rather its integration into interconnected biosynthetic pathways.
MeJ treatment appears to enhance Monastrell’s defence capacity by increasing the production of antifungal compounds. The higher concentration of stilbenes detected within the first 24 h supports this observation. Previous studies under pathogen infection conditions have identified defence biomarkers resulting from pathogen–grapevine interactions [7]. Future research should explore whether this early accumulation translates into enhanced disease resistance under field conditions.
The interaction between MeJ and BTH exhibited antagonistic effects on certain phenolic compounds, though not all metabolites responded similarly. For instance, combined treatment reduced T-resveratrol levels at 72 h and further decreased dihydroxybenzoic acid hexosyl pentoside compared to individual treatments. This suggests a compensatory regulation within phenolic biosynthesis, where multiple defence signals trigger an adaptive response to balance secondary metabolite production [63].
Additionally, unidentified compounds (no. 16, 32, 45, and 48; Table 1) exhibited similar increasing trends to the identified phenolic compounds, with concentrations rising primarily between 18 and 24 h. Their continued accumulation beyond 72 h remains to be investigated.
The temporal accumulation patterns provide insights into the kinetics of elicitor-induced responses. The concentration peaks of astringin, miyabenol C, T-piceid, and piceatannol at 18 and 24 h in MeJ-treated samples suggest rapid activation of the stilbene pathway, likely mediated by defence-related genes in the phenylpropanoid pathway [64]. This rapid response could be advantageous against pathogen attacks, allowing swift mobilisation of defences. The subsequent decline in these compounds, regardless of treatment, suggests a negative regulatory mechanism after initial accumulation, consistent with other studies on induced defence responses in grapevines [6,46]. Once an adequate level of defensive metabolites is reached, biosynthesis is likely downregulated to optimise energy expenditure, a crucial balance for plant fitness under stress conditions [65]. The biosynthesis of major phenolic groups, such as stilbenes, lignins, and flavonoids, is tightly regulated at the transcriptional level, with transcription factors playing a pivotal role in this process [66].
The use of MeJ and BTH as elicitors in Vitis vinifera cv. Monastrell has shown promise in enhancing the production of antimicrobial phenolic compounds such as astringin, miyabenol C, T-piceid, piceatannol, and T-resveratrol. These compounds exhibit antifungal activity against Plasmopara viticola, Erysiphe necator, and Botrytis cinerea [66], major pathogens in viticulture. This natural activation of defence mechanisms aligns with integrated pest management (IPM) strategies, reducing reliance on synthetic pesticides and mitigating environmental and health risks [67].
By strengthening plant resilience, elicitors like MeJ and BTH contribute to vineyard sustainability. Additionally, they enhance grape quality, as phenolic compounds, particularly stilbenes, are associated with improved antioxidant properties and higher commercial value [68]. This underscores the potential of elicitors as a sustainable alternative for pest management. However, challenges remain under field conditions, where environmental factors and pathogen dynamics may influence treatment efficacy [69]. Further long-term studies are needed to optimise these applications and explore interactions with other agricultural practices to maximise their benefits. The observed increase in polyphenol levels supports the hypothesis of a positive modulation of metabolites that contribute to stress resistance.

5. Conclusions

This study provides the first evidence that methyl jasmonate (MeJ) and benzothiadiazole (BTH) regulate the biosynthesis of phenolic compounds in the leaves of Vitis vinifera cv. Monastrell seedlings. The results confirm that both elicitors significantly enhance the accumulation of phenolic compounds, particularly stilbenes such as astringin, miyabenol C, T-piceid, piceatannol, and T-resveratrol, which are recognised for their antimicrobial properties and protective role against fungal pathogens.
A rapid and specific metabolic response was observed, with peak concentrations occurring between 18 and 24 h, particularly in MeJ-treated plants. This pattern suggests a swift activation and efficient regulation of phenolic defences, likely mediated by the phenylpropanoid pathway and controlled at the transcriptional level. These findings underscore the potential of MeJ and BTH as sustainable alternatives for enhancing the natural defence mechanisms of Monastrell, potentially reducing dependence on synthetic pesticides. By stimulating the production of natural antimicrobial compounds, these elicitors contribute to improving vineyard resilience within a sustainable viticulture framework.
While these results are promising, they are limited to the first 72 h post-treatment. Further long-term studies are necessary to fully elucidate the prolonged effects of these elicitors and their potential role in managing chronic or recurrent infections in vineyards.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/horticulturae11030277/s1, Table S1: Relative areas of the different compounds analysed in the leaves of Vitis vinifera cv. Monastrell treated with methyl jasmonate and benzothiadiazole.

Author Contributions

All the authors have approved the final version of the manuscript. Conceptualization, D.P.-Q. and R.G.-M.; data curation, D.P.-Q.; formal analysis, D.P.-Q.; funding acquisition, R.G.-M.; investigation, D.P.-Q.; methodology, D.P.-Q. and R.G.-M.; project administration, R.G.-M.; resources, R.G.-M.; writing—original draft, D.P.-Q.; writing—review and editing, D.P.-Q. and R.G.-M. All authors have read and agreed to the published version of the manuscript.

Funding

Part of this work has been funded through grants from the State Plan for Scientific, Technical, and Innovation Research for the period 2021–2023 within the framework of the Recovery, Transformation, and Resilience Plan. Diego Paladines holds a Juan de la Cierva Training Postdoctoral contract (Reference: FJC2021-046437-I).

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors thank the staff of the “Oenological Station of Jumilla” (Murcia, Spain) for their partial support of this study.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of this study, the collection, analyses, or interpretation of data, the writing of the manuscript, or the decision to publish the results.

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Figure 1. General discriminant analysis of the phenolic compounds analysed in Monastrell seedling leaves after treatment with methyl jasmonate (MeJ), benzothiadiazole (BTH), and a combination of both (MeJ + BTH).
Figure 1. General discriminant analysis of the phenolic compounds analysed in Monastrell seedling leaves after treatment with methyl jasmonate (MeJ), benzothiadiazole (BTH), and a combination of both (MeJ + BTH).
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Horticulturae 11 00277 g002aHorticulturae 11 00277 g002b
Figure 2. Individual discriminant analyses (different time points) of phenolic compounds analysed in Monastrell seedling leaves after treatment with methyl jasmonate (MeJ), benzothiadiazole (BTH), and a combination of both (MeJ + BTH). Analysis at (A) 12 h; (B) at 18 h; (C) at 24 h; and (D) at 72 hours after treatment.
Figure 2. Individual discriminant analyses (different time points) of phenolic compounds analysed in Monastrell seedling leaves after treatment with methyl jasmonate (MeJ), benzothiadiazole (BTH), and a combination of both (MeJ + BTH). Analysis at (A) 12 h; (B) at 18 h; (C) at 24 h; and (D) at 72 hours after treatment.
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Figure 3. Changing patterns of phenolic compound contents in the leaves of Vitis vinifera cv. Monastrell treated with methyl jasmonate (MeJ), benzothiadiazole (BTH), and their combination (MeJ + BTH). (A) Astringin; (B) Miyabenol C; (C) T-piceid; (D) Piceatannol; (E) T-resveratrol; (F) Coumaroyltartaric acid; (G) 3-O-p-Coumaroylquinic acid; (H) Ellagic acid pentoside; (I) Dihydroxybenzoic acid hexosyl pentoside.
Figure 3. Changing patterns of phenolic compound contents in the leaves of Vitis vinifera cv. Monastrell treated with methyl jasmonate (MeJ), benzothiadiazole (BTH), and their combination (MeJ + BTH). (A) Astringin; (B) Miyabenol C; (C) T-piceid; (D) Piceatannol; (E) T-resveratrol; (F) Coumaroyltartaric acid; (G) 3-O-p-Coumaroylquinic acid; (H) Ellagic acid pentoside; (I) Dihydroxybenzoic acid hexosyl pentoside.
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Table 1. Identified compounds in Monastrell seedling leaves after treatment with methyl jasmonate (MeJ), benzothiadiazole (BTH), and a combination of both (MeJ + BTH).
Table 1. Identified compounds in Monastrell seedling leaves after treatment with methyl jasmonate (MeJ), benzothiadiazole (BTH), and a combination of both (MeJ + BTH).
NotR, minCompound NameChemical GroupMolecular FormulaLevel of IdentificationFragmentsWavelength (max. Absorbance) (nm)Ref.
16.923Myricetin 3-O-hexocideFlavanolsC21H19O13-3479-317264-353-389[50,51]
27.153Gallic acid hexosidePhenolic AcidsC13H15O10-3331276-309[50]
37.292unknown 4 282-268
47.86unknown 4 274
58.114unknown 4 259
68.307unknown 4 278
78.465unknown 4 277-259
88.564unknown 4 262
98.715Dihydroxybenzoic acid hexosidePhenolic AcidsC13H15O9-3315-153-135319-241[50,52]
108.879unknown 4 263
119.04unknown 4 268
129.265unknown 4 277
139.395unknown 4 287
149.543AstringinStilbenesC20H22O9-3405-243-159324-295[53]
159.942Miyabenol CStilbenesC42H32O9-3451-359-345329-293-246
1610.23unknown 4 323-289
1710.402unknown 4 287
1810.706unknown 4 290-312
1911.011unknown 4 264
2011.3Coumaroyltartaric acidPhenolic AcidsC13H11O8-3295-163-140-119311[50]
2111.4863-O-p-Coumaroylquinic acidPhenolic AcidsC16H17O8-3337-163-119314-289[50]
2211.866unknown 4 277
2312.03Feruloyltartaric (fertaric) acidPhenolic AcidsC14H13O9-3325-193-149327-296-249[50]
2412.408Ellagic acid pentosidePhenolic AcidsC19H13O12-3433-301-257-213284-326[50]
2512.676Dihydroxybenzoic acidhexosyl pentosidePhenolic AcidsC18H23O13-3447-315-285275[50]
2612.876Caffeoylshikimic acidPhenolic AcidsC16H15O8-3335-179-135272-260[50]
2713.097unknown 4 262-354
2813.346unknown 4 272
2913.683unknown 4 258
3013.786Chlorogenic acid hexosidePhenolic AcidsC22H27O14-3299-137-353-341258[50,54]
3114.097Kaempferol 3-O-hexuronidemethyl etherFlavanolsC22H19O12-3475-327-285259-354[50]
3214.392unknown 4 264
3314.601T-piceidStilbenesC20H22O8-1227-210-225329-302[55]
3414.894QuercetinFlavanolsC15H9O7-1301-271-179256-355[50,56,57]
3515.458unknown 4 265-352
3615.624unknown 4 268-294
3715.916unknown 4 265-297-354
3816.233unknown 4 252-321-358
3916.355unknown 4 264-349
4016.464Kaempferol 7-O-hexuronideFlavanolsC21H17O12-3461-285-267-239-241265-347[50]
4116.641PiceatannolStilbenesC14H12O4-1243-159-227-213-202323 [58]
4216.923unknown 4 257-355
4317.681unknown 4 278
4417.796unknown 4 251
4518.265unknown 4 245-329
4618.956unknown 4 288
4719.836T-resveratrolStilbenesC14H11O3-1227-185-159-143311-317[50,58]
4820.499unknown 4 291
4922.349Ellagic acid 3301298-368[57,59]
5023.472unknown 4 333
Table 2. Summary of the parameters of the individual discriminant analyses.
Table 2. Summary of the parameters of the individual discriminant analyses.
SamplingGenerated Discriminant FunctionsPercentage Variance Explained
Function 1Function 2Function 1
+
Function 2
12 h377%
Most significant standardised coefficients:
- Coumaroyltartaric acid
- Ellagic acid pentoside		20.5%
Most significant standardised coefficients:
- Dihydroxybenzoic acidhexosyl pentoside
- Compound No. 16 (unknown)		97.5%
18 h397.1%
Most significant standardised coefficients:
- Astringin
- Compuesto nº16 (desconocido)		2.9%
Most significant standardised coefficients:
- 3-O-p-Coumaroylquinic acid
- Astringin		100%
24 h393.5%
Most significant standardised coefficients:
- Miyabenol
- 3-O-p-Coumaroylquinic acid		6.2%
Most significant standardised coefficients:
- Astringin
- Miyabenol		99.7%
72 h388.3%
Most significant standardised coefficients:
- Astringin
- 3-O-p-Coumaroylquinic acid		11.2%
Most significant standardised coefficients:
- Astringin
- Miyabenol		99.5%
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Paladines-Quezada, D.; Gil-Muñoz, R. Metabolic Response Induced by Methyl Jasmonate and Benzothiadiazole in Vitis vinifera cv. Monastrell Seedlings. Horticulturae 2025, 11, 277. https://doi.org/10.3390/horticulturae11030277

AMA Style

Paladines-Quezada D, Gil-Muñoz R. Metabolic Response Induced by Methyl Jasmonate and Benzothiadiazole in Vitis vinifera cv. Monastrell Seedlings. Horticulturae. 2025; 11(3):277. https://doi.org/10.3390/horticulturae11030277

Chicago/Turabian Style

Paladines-Quezada, Diego, and Rocío Gil-Muñoz. 2025. "Metabolic Response Induced by Methyl Jasmonate and Benzothiadiazole in Vitis vinifera cv. Monastrell Seedlings" Horticulturae 11, no. 3: 277. https://doi.org/10.3390/horticulturae11030277

APA Style

Paladines-Quezada, D., & Gil-Muñoz, R. (2025). Metabolic Response Induced by Methyl Jasmonate and Benzothiadiazole in Vitis vinifera cv. Monastrell Seedlings. Horticulturae, 11(3), 277. https://doi.org/10.3390/horticulturae11030277

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