Exogenous Methyl Jasmonate Enhances Chemical Defense in Blumea balsamifera Against Spodoptera litura by Boosting Phenylpropanoid and Flavonoid Metabolism

TL;DR

Applying methyl jasmonate boosts Blumea balsamifera's natural defenses against a pest by increasing specific chemical compounds.

Contribution

This study reveals how methyl jasmonate enhances plant resistance through metabolic pathway reprogramming.

Findings

Exogenous MeJA reduced pest feeding and growth in Blumea balsamifera.

MeJA increased enzymatic activity and antioxidant responses in the plant.

Metabolomic analysis showed elevated levels of defensive compounds like secoisolariciresinol diglucoside.

Abstract

Blumea balsamifera (L.) DC. is the primary source plant of natural borneol, an important ethnic medicine in China. But its quality and yield are severely threatened by the polyphagous pest Spodoptera litura Fabricius during cultivation. In order to elucidate the mechanism of the chemical defense response induced by methyl jasmonate (MeJA) in B. balsamifera plants against S. litura. This study investigated the MeJA-mediated chemical defense in B. balsamifera against S. litura by integrating insect bioassays, enzymatic analysis, and metabolomics. Results demonstrated that exogenous MeJA application significantly inhibited larval feeding preference and consumption, suppressed relative growth rates, and reduced pupal weights. Physiologically, MeJA treatment rapidly upregulated the activities of jasmonic acid (JA) biosynthetic and antioxidant enzymes. Crucially, metabolomic profiling revealed that MeJA triggered a metabolic reconfiguration, specifically promoting the accumulation of defensive secondary metabolites. Notably, key bioactive compounds, including secoisolariciresinol diglucoside and isochlorogenic acid C, were significantly enriched alongside elevated endogenous JA levels. These findings suggest that MeJA enhances the resistance of B. balsamifera not merely by activating enzymatic defense but by reprogramming the phenylpropanoid and phenolic metabolic pathways. The above results indicate that exogenous MeJA can induce the activation of the chemical defense system in B. balsamifera plants and enhance their resistance to S. litura.

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1. Introduction

Blumea balsamifera (L.) DC., a well-documented traditional medicinal plant, exhibits antimicrobial, antioxidant, and wound-healing properties with a long history of ethnopharmacological use across Southeast Asia and Southern China [1,2]. Spodoptera litura Fabricius, belonging to the family Noctuidae within the order Lepidoptera, is a polyphagous agricultural pest with strong migratory capabilities and an extensive dispersal range; moreover, it can feed on more than 400 plant species from over 110 families [3,4]. As a dominant herbaceous species in tropical understory habitats, B. balsamifera frequently encounters herbivory pressure from generalist pests, among which S. litura causes severe damage to leaf tissues of medicinal plants, directly threatening the yield and quality of bioactive compounds in B. balsamifera [5,6]. Currently, chemical pesticides are predominantly used to control S. litura in agriculture. However, these pesticides induce pesticide resistance in the pest and have detrimental impacts on the environment [7]. In particular, the use of chemical pesticides in the cultivation of medicinal plants, such as B. balsamifera, seriously compromises the quality of medicinal materials and the safety of their application. Therefore, there is an urgent need to develop green pest management strategies for the sustainable development of the B. balsamifera industry.

During the protracted adaptive coevolution of plants and herbivorous insects, sedentary plants have evolved a sophisticated, proactive set of physical and chemical defense mechanisms to combat herbivory [8]. Defensive proteins mainly comprise protease inhibitors, amylase inhibitors, lectins, proteases, amino acid enzymes, and oxidases [9,10]. Plants can use exogenous plant hormones, such as jasmonic acid (JA) and its derivatives, to induce and activate their own defense responses, thereby enhancing their resistance to insect feeding damage by forming physical barriers and inducing chemical responses [11,12,13]. Consequently, insect feeding, oviposition, digestion and utilization of food, and normal insect growth, development, and reproduction processes are disrupted, potentially leading to insect poisoning and death [10].

Regarding induced defense, phytohormones such as JA, salicylic acid, and ethylene are central in regulating plant defense responses [8]. The JA signaling pathway is the core signal transduction route for plants to perceive and respond to insect feeding, and it plays a crucial role in modulating physiological processes through which plants defend against herbivorous insects [14]. Methyl jasmonate (MeJA), a JA derivative, is formed under the catalysis of JA carboxyl methyltransferase [15]. Exogenous MeJA and JA can be interconverted, and they can induce insect resistance by activating the endogenous JA signaling pathway in plants, thereby enhancing the plant’s insect resistance abilities. Previous studies have provided substantial evidence regarding the effectiveness of JA and MeJA in enhancing plant resistance. Pretreatment with JA could significantly increase the number of trichomes in insect-resistant genotype of Arachis hypogaea (ICGV 86699, ICGV 86031, ICG 2271 and ICG 1697), and reduce the oviposition of Helicoverpa armigera Hübner [16]. Furthermore, various genotypes of A. hypogaea demonstrated significant inhibitory effects on the feeding ability, larval weight, and survival rate of H. armigera after being treated with JA [17]. This phenomenon is closely related to the increased activities of peroxidase (POD), polyphenol oxidase (PPO), lipoxygenase (LOX), phenylalanine ammonia lyase (PAL), superoxide dismutase (SOD), ascorbate peroxidase, catalase (CAT), and trypsin proteinase inhibitor, as well as increased secondary metabolite content in peanut plants after JA treatment [16,17]. Similarly, MeJA treatment of tomato seeds increased PPO activity in tomato leaves and inhibited the growth rate of H. armigera [18]. In addition, spraying of 1 mM MeJA on cranberry plants in the field had a significant repellent effect on Sparganothis sulfureana adults [19].

Existing studies on MeJA-induced plant resistance to S. litura have yielded valuable insights [8]. Exogenous MeJA application and S. litura herbivory can boost PPO and TI activities in plants such as radish, pepper, and tomato [20]. Moreover, MeJA has obvious concentration- and distance-dependent effects on the induction of insect resistance in adjacent tomato plants. PPO, POD, and LOX activities can be effectively induced in tomato using MeJA at an appropriate concentration and distance range, thereby decreasing the weight growth rate of S. litura larvae [21]. In addition, MeJA can significantly induce the upregulation of a novel Kunitz trypsin inhibitor in tobacco plants in synergy with ethephon, thereby enhancing the resistance of tobacco plants against S. litura [22].

Currently, exogenous MeJA-induced plant insect resistance is mostly studied in a few crops, while research on medicinal plants remains extremely limited, and MeJA-induced specific defense mechanisms vary by plant species and insect type. This study took B. balsamifera as the experimental material to investigate the chemical defense mechanisms of MeJA-induced resistance against S. litura, focusing on B. balsamifera’s response to S. litura feeding and clarifying key physiological and molecular changes in its defense process. The findings are expected to fill gaps in medicinal plant defense research, deepen understanding of understudied medicinal plant defense strategies, provide new ideas for green pest control of B. balsamifera, and offer novel insights for sustainable pest management in its plantations.

2. Results

2.1. MeJA-Treated B. balsamifera Plants Inhibited the Feeding Preference and Growth and Development of S. litura

The results of the biological assays indicated that MeJA-treated B. balsamifera plants (Figure 1A) significantly inhibited the feeding selectivity, feeding amount, and growth and development of S. litura larvae (Figure 1). The feeding preference of third-instar S. litura larvae for MeJA-treated B. balsamifera leaves was reduced by 53.65% in contrast to the control group (t = 7.778, df = 10, p < 0.001) (Figure 1B). The feeding amounts of individual larvae on MeJA-induced leaves over a 12 h period were also significantly reduced by 31.24% (t = 4.003; df = 48; p < 0.001) (Figure 1C). When S. litura larvae were reared on B. balsamifera leaves treated with MeJA and deionized water separately, the pupal weight of those reared on MeJA-treated leaves was significantly reduced by 20.32% compared with that of larvae reared on deionized water (t = 3.656; df = 34; p < 0.001) (Figure 1D). Furthermore, the body weight and relative growth rate of S. litura larvae after feeding on MeJA-treated B. balsamifera leaves were significantly lower than those of the control group from day 2 to day 8 (p < 0.05) (Figure 1E,F).

2.2. MeJA and S. litura Feeding Induce the Activation of Biosynthetic Enzymes of the JA Signaling Pathway in B. balsamifera

The activities of LOX, AOS, and AOC, three key enzymes in the JA signaling pathway, were increased in B. balsamifera leaves from the MeJA, control + SL, and MeJA + SL treatment groups, with the most significant upregulation observed in the MeJA + SL treatment group (Figure 2). The LOX activities in B. balsamifera leaves from the MeJA, control + SL, and MeJA + SL groups were 3.19, 4.44, and 5.49 times that of the control group, respectively (F3, 16 = 16.268; p < 0.001) (Figure 2A), while the AOC activities were 1.83, 1.56, and 2.21 times that of the control group, respectively (F3, 16 = 451.187; p < 0.001) (Figure 2B), and the AOS activities were upregulated by 1.98, 1.84, and 2.35 times compared to the control group, respectively (F3, 16 = 46.573; p < 0.001) (Figure 2C).

2.3. Impact of MeJA and S. litura Feeding on the Defense Enzyme Activity of B. balsamifera

In B. balsamifera leaves, the CAT, POD, PPO, and PAL activities were enhanced in the MeJA, control + SL, and MeJA + SL treatment groups. However, the SOD activity was significantly higher only in the MeJA + SL treatment group compared to the other groups (Figure 3C). Compared with the control group, the CAT activities in B. balsamifera leaves from the MeJA, control + SL, and MeJA + SL groups were increased by 1.5, 1.8, and 2.34 times, respectively (F3, 16 = 33.382; p < 0.001) (Figure 3A). The POD activities were upregulated by 1.62, 1.52, and 1.97 times compared to the control group, respectively (F3, 16 = 56.230; p < 0.001) (Figure 3B), while the PPO activities were 1.36, 1.39, and 1.59 times that of the control group, respectively (F3, 16 = 12.410; p < 0.001) (Figure 3D), and the PAL activities were upregulated by 1.63, 2.28, and 2.95 times compared to the control group, respectively (F3, 16 = 92.200; p < 0.01) (Figure 3E).

2.4. Effects of MeJA and S. litura Feeding on the Secondary Metabolites of B. balsamifera

Compared with the B. balsamifera leaves in the control group, those in the MeJA, control + SL, and MeJA + SL treatment groups showed a significant increase in the content of flavonoids, reaching 1.62, 1.25, and 1.47 times that of the content in the control group, respectively (F3, 8 = 132.533; p < 0.001) (Figure 4A). Moreover, B. balsamifera leaves in the MeJA + SL treatment group showed a significant increase in the content of lignin, reaching 1.28 times that in the control group (F3, 8 = 8.703; p < 0.01) (Figure 4B). Furthermore, B. balsamifera leaves in the MeJA and MeJA + SL treatment groups showed a significant increase in the total phenolic content, reaching 1.73 and 1.42 times that in the control group, respectively (F3, 8 = 428.323; p < 0.001) (Figure 4C). However, alkaloid and tannin contents showed no significant differences between each treatment group and the control group, indicating that the experimental treatments did not affect the accumulation of these two secondary metabolites (Figure 4D,E).

2.5. Effects of MeJA and S. litura Feeding on the Metabolic Level of B. balsamifera Plants

2.5.1. Analysis of Differences in Metabolite Profiles Among Different Treatments

Orthogonal partial least-squares discriminant analysis (OPLS-DA) indicated that results revealed no overfitting in the original model, and the model robustness was good (when the R^2^ value is smaller than the Q^2^ value and the Y-intercept of the Q^2^ regression line is positive) (Figure 5). This result indicates that significant differences in metabolites were detected in the four comparison groups: MeJA vs. control, Control + SL vs. Control, MeJA + SL vs. MeJA, and MeJA + SL vs. Control + SL.

In this study, 1335 metabolites were identified from 16 experimental samples in the MeJA, MeJA + SL, Control + SL, and Control groups (Table S1). Between the MeJA and Control groups, 83 differential metabolites, including 50 upregulated and 33 downregulated, were detected; between the MeJA + SL and Control + SL groups, 216 differential metabolites, including 98 upregulated and 118 downregulated, were detected; between the Control + SL and Control groups, 68 differential metabolites, including 27 upregulated and 41 downregulated, were detected; and between the MeJA + SL and MeJA groups, 116 differential metabolites, including 46 upregulated and 70 downregulated, were detected (Differentially expressed variables were identified with the thresholds of VIP > 1.0, FC > 1.2 or FC < 0.833, and a significance level of p < 0.05) (Table 1 and Figure S1).

2.5.2. Hierarchical Clustering Analysis of Differential Metabolites

Among the top 20 upregulated metabolites with the greatest fold changes in the MeJA vs. Control group, five lipids and lipid-like molecules, four organoheterocyclic compounds, two lignans, neolignans, and related compounds, one alkaloid and its derivative, one nucleoside, nucleotide, and analog, and seven undefined metabolites were identified (Figure 6A and Table S2). Among the top 20 upregulated metabolites with the greatest fold changes in the MeJA + SL vs. Control + SL group, three lipids and lipid-like molecules, two organoheterocyclic compounds, three phenylpropanoids and polyketides, one nucleoside, nucleotide, and analog, one benzenoid, and ten undefined metabolites were identified (Figure 6B, Table S3). Among the top 20 upregulated metabolites with the greatest fold changes in the Control + SL vs. Control group, six lipids and lipid-like molecules, two phenylpropanoids and polyketides, one benzenoid, two organoheterocyclic compounds, two organic oxygen compounds, and seven undefined metabolites were identified (Figure 6C and Table S4). Among the top 20 upregulated metabolites with the greatest fold changes in the MeJA + SL vs. MeJA group, four nucleosides, nucleotides, and analogs, three phenylpropanoids and polyketides, one organic acid and its derivative, and twelve undefined metabolites (Figure 6D and Table S5).

We retrieved and analyzed the physicochemical properties of differential metabolites using PubChem, while integrating findings from previously reported literature on defensive metabolites. The results indicated that the metabolites JA, (+)-dihydrojasmonic acid, indole-3-acetic acid, secoisolariciresinol diglucoside, isochlorogenic acid C, isatin, zerumbon and phloretic acid, which were substantially upregulated by exogenous MeJA, possess insecticidal properties. The metabolites artemisinin, β-caryophyllene, gallic acid, and nitenpyram, which were substantially upregulated after MeJA pretreatment and subsequent feeding with S. litura compared with the Control + SL group, also exhibited insecticidal effects. The metabolites isovitexin and leonurine, which were substantially upregulated in the Control + SL group compared with the Control group, also exhibited insecticidal properties. The metabolite sakuranetin, which was substantially upregulated in the MeJA + SL group compared with the MeJA group, also exhibited insecticidal properties.

2.5.3. Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Enrichment Analysis

KEGG analysis revealed that the differentially accumulated metabolites (DAMs) in the four comparison groups were primarily enriched in eight metabolic pathways (Figure 7). The DAMs between the MeJA and Control group were markedly enriched in histidine metabolism, indole alkaloid biosynthesis, aminoacyl-tRNA biosynthesis, glycine metabolism, serine metabolism, and threonine metabolism, cysteine and methionine metabolism, and plant hormone signal transduction pathways (p < 0.05) (Figure 7A). The DAMs between the MeJA + SL and Control + SL group were significantly enriched in c5-branched dibasic acid metabolism (p < 0.05) (Figure 7B). Between the Control + SL and Control groups, DAMs were mainly enriched in the flavonoid biosynthesis and isoquinoline alkaloid biosynthesis pathways (Figure 7C). Between the MeJA + SL and MeJA groups, DAMs were primarily enriched in starch and sucrose metabolism (p < 0.05) (Figure 7D).

3. Discussion

The JA signaling pathway is a core defense mechanism of plants against herbivorous insect damage. The activation of this pathway can regulate the synthesis of various defense-related proteins and secondary metabolites, thereby inhibiting the feeding, digestion, growth, and development of insects and generating defensive responses [23]. The results of this study indicate that MeJA treatment significantly inhibits the feeding selectivity, feeding amount, growth, and development of S. litura larvae on B. balsamifera leaves. Previously, the exogenous application of MeJA and MeSA significantly inhibited the development and reproduction of the two-spotted spider mite Tetranychus urticae on Phaseolus vulgaris [24]. Similarly, after S. litura larvae fed on MeJA-treated tomato (Lycopersicon esculentum) leaves, their relative growth rate was significantly reduced, indicating that JA induction can effectively inhibit the growth and development of phytophagous insects [21]. These results further confirm the broad applicability of MeJA-mediated defense responses in various crops and medicinal plants.

LOX, AOS, and AOC are key enzymes in the JA biosynthetic pathway. LOX catalyzes the conversion of linolenic acid to hydroperoxy fatty acid, AOS catalyzes the formation of the epoxide intermediate, and AOC catalyzes the conversion of the epoxide intermediate to the JA precursor [14]. To elucidate the induction effects of MeJA and S. litura feeding on the JA defense system of B. balsamifera, we measured the LOX, AOS, and AOC activities in B. balsamifera leaves. The results revealed that exogenous MeJA treatment and S. litura feeding significantly enhanced LOX, AOS, and AOC activities in B. balsamifera leaves. Previous studies have demonstrated that the upregulation of JA pathway marker genes and changes in the activity of JA biosynthetic enzymes in plants can induce the production of defensive proteins, secondary metabolites, or volatiles, which in turn can exert direct repellent or toxic effects on pests [17,25]. The exogenous application of MeJA enhances LOX activity in Chrysanthemum morifolium and modulates the plant’s JA signaling pathway, thereby promoting endogenous JA accumulation, activating the transcription of defense-related genes, and ultimately conferring resistance against aphids [26]. In this study, by measuring the changes in key enzyme activities, we confirmed that exogenous MeJA treatment and S. litura feeding can significantly activate the JA biosynthetic pathway in B. balsamifera.

CAT, POD, SOD, PPO, and PAL are important defense enzymes involved in the regulation of plant defense responses. POD can remove the H_2_O_2_ generated by the catalysis of SOD, reducing the accumulation of reactive oxygen species [17]. This study revealed that MeJA treatment, S. litura feeding, and combined MeJA and S. litura significantly increase CAT, POD, PPO, and PAL activities in B. balsamifera leaves. MeJA and S. litura feeding also significantly increased the SOD activity in leaves. This process subsequently promotes the lignification of plant cells, wound healing, and the production of secondary metabolites, enhancing the plant’s resistance to herbivorous insects. Studies have found that JA and feeding by the fall armyworm (Spodoptera frugiperda) significantly upregulated POD and SOD activities in maize, with the most significant upregulation occurring under the combined stress of JA treatment and S. frugiperda feeding [27]. PPO could lead to a decline in the nutritional quality of plant tissues, inhibit the digestion of plant proteins by insects, and reduce insect digestibility, thus playing a central role in plant defense against insects [28]. Applying exogenous MeJA to tomato plants (Lycopersicon esculentum) significantly increases PPO activity in their leaves [29]. PAL is a key enzyme in the phenylpropanoid metabolic pathway in plants. It promotes the synthesis of defensive secondary metabolites, including flavonoids and lignin, which are toxic to herbivorous insects [30]. Research has found that treating Rosa rugosa with different MeJA concentrations increases POD, PPO, and PAL activities in its leaves [31]. These treatments enhance the activity of defense enzymes, thus exerting toxic effects on S. litura.

Exogenous MeJA can activate the JA defense network in plants, and the secondary metabolites within the JA defense network are directly toxic to insects. Flavonoids exert antifeedant and toxic effects on insects by disrupting their feeding behavior, digestive system, or physiological metabolism, thereby inhibiting their growth, development, and reproduction [32]. After MeJA induction, plants activate the JA signaling pathway, which promotes lignin accumulation and enhances cell wall synthesis [33]. This process increases the hardness of plant tissues, making it more challenging for insects to feed. Phenolic compounds are important secondary metabolites in plants. They can bind with insect digestive enzymes and reduce their activity, thereby disrupting the insects’ digestion and food absorption. Phenolic compounds are crucial in plant resistance to insect herbivory [34]. Research has revealed that after the application of exogenous MeJA to roses (Rosa chinensis), leaf tannin and total phenol contents significantly increased, whereas the feeding amount of adult two-spotted leaf beetles (Monolepta hieroglyphica) on the roses was reduced [35]. In this study, MeJA and S. litura feeding significantly increased flavonoid, lignin, and total phenol contents in B. balsamifera leaves. MeJA induction alone increased flavonoid and total phenol contents. These results indicate that MeJA treatment and S. litura feeding induce an increase in the content of certain defensive secondary metabolites in B. balsamifera leaves, disrupting the feeding and digestion of S. litura and consequently enhancing the plant’s resistance to S. litura.

Primary metabolites are synthetic precursors of secondary metabolites and are one of the defense tools of plants against pests [12]. Previous studies have shown that exogenous MeJA can induce the upregulation or downregulation of plant metabolites, enhancing plant defense against herbivorous insects [36]. To further elucidate the chemical defense of B. balsamifera against S. litura, we discovered through metabolomics analysis that exogenous MeJA treatment and S. litura feeding induce significant differences in metabolites in B. balsamifera plants. Comparing the MeJA and control groups, 50 metabolites were significantly upregulated, and the DAMs were significantly enriched in the pathways of indole alkaloid biosynthesis, plant hormone signal transduction, and multiple amino acid metabolic pathways. Between MeJA + SL and control + SL, 98 metabolites were significantly upregulated, and the DAMs were significantly enriched in the alpha-Linolenic acid metabolism, nicotinate and nicotinamide metabolism, flavone and flavonol biosynthesis pathways. These results indicate that exogenous MeJA and S. litura feeding can induce the activation of JA signaling defense system in B. balsamifera plants. Because plant hormone signal transduction, linoleic acid metabolism and biosynthesis of various amino acids are closely related to JA biosynthesis [37]. Moreover, the accumulation of alkaloids, nicotinamide, flavonoids and other metabolites has a certain degree of biological toxicity and antioxidant effect on pests [12,37].

Our experiments revealed that upregulated metabolites, namely nitenpyram and sakuranetin, exerted insect-repellent effects [37,38]. Meanwhile, the levels of key defense-related substances (JA and (+)-dihydrojasmonic acid) were also significantly increased; this observation aligns with the reported role of JA in plant insect resistance [39]. The results of this study indicate that exogenous MeJA and S. litura feeding can significantly upregulate the content of defense-related metabolites, enhancing the plant’s resistance to S. litura.

4. Materials and Methods

4.1. Insect and Plant Cultivation

Healthy B. balsamifera seeds were selected for seedling cultivation. The seeds were grown on sterilized nutrient soil and transplanted at the 3–4 leaf stage after budding into vegetative pots (8.5 cm diameter, 9.5 cm height) using sterilized nutrient soil. Subsequent plant culture was completed in a greenhouse (27 °C ± 1 °C, 60% ± 5% relative humidity [RH], and 16/8 h light/dark photoperiod). When the plants reached the 8–12 leaf stage (approximately three months), healthy B. balsamifera plants were selected as experimental materials.

The lab-reared strain of S. litura has been reared in the laboratory for more than 20 generations on an artificial diet. The breeding conditions for S. litura were 27 °C ± 1 °C, 60% ± 5% RH, and 16/8 h light/dark photoperiod. The artificial diet provided for the insects was replaced every 2 days to ensure their freshness and nutritional adequacy for the healthy growth of the larvae.

4.2. Plant Treatment

Based on previous studies, 1.0 mmol/L MeJA ((±)-MeJA, Sigma-Aldrich (Shanghai) Trading Co., Ltd. (Shanghai, China)) was selected as the treatment concentration [40]. The preparation of MeJA solution was according to the method of Zhang et al. [41]. Healthy and pest-free B. balsamifera plants with uniform growth were selected. The entire B. balsamifera plant was sprayed with approximately 5 mL of MeJA solution (1.0 mmol/L). B. balsamifera plants sprayed with deionized water containing the same amount of ethanol were used as controls. The Control and MeJA treatment groups were placed in two greenhouses with the same planting conditions, and the S. litura infection test was conducted after 3 days. The experimental design included four treatment groups. (1) MeJA treatment group: 1.0 g leaves were cut as test samples 72 h after MeJA treatment. (2) MeJA + SL treatment group: plants were pretreated with MeJA for 66 h, after which second-instar S. litura larvae fed on the leaves for 6 h (two larvae per plant); 1.0 g leaves were cut as test samples. (3) Control + SL treatment group: plants were pretreated with deionized water for 66 h, after which second-instar S. litura larvae were fed on the leaves for 6 h (two larvae per plant); 1.0 g leaves were cut as test samples. (4) Control group: 1.0 g leaves were cut as test samples 72 h after B. balsamifera plants were sprayed with deionized water.

4.3. Effect of MeJA Induction on B. balsamifera Against S. litura

Feeding preference experiment: B. balsamifera leaves treated with deionized water (control) or 1.00 mmol/L MeJA solution for 3 days were used for bioassay experiments according to the method of Han and Lei [42]. The leaf disks were made from modified transparent plastic boxes (diameter: 25 cm, with holes punched in the lid to ensure ventilation). Leaf disks were divided into two equal parts; one part was placed with MeJA-treated plant leaves, and the other part was placed with deionized water-treated plant leaves. Third-instar S. litura larvae, which had been starved for 6 h, were placed in the center of each leaf disk. At one replicate per leaf disk (20 larvae). After 24 h, the number of larvae distributed on the two types of leaves was observed and recorded. The experiment was set up with 6 biological replicates.

Measurement of S. litura feeding amount: B. balsamifera leaves treated with MeJA or deionized water for 72 h were weighed and placed into specially designed plastic Petri dishes (diameter 15 cm, with holes in the lid covered with 200-mesh gauze to ensure ventilation). Third-instar S. litura larvae, which had been starved for 6 h, were released to feed on the leaves. After 12 h of feeding, the weight of the consumed leaves was measured. The feeding amount was calculated by subtracting the final leaf weight from its initial weight. Each treatment included 25 biological repeats (1 larva per replicate).

Larval weight determination: B. balsamifera leaves treated with MeJA or deionized water for 72 h were weighed and placed into specially designed plastic Petri dishes. Individual third-instar S. litura larvae, previously weighed, were placed into each Petri dish and marked. Every 24 h, fresh B. balsamifera leaves treated as described above were replaced, and the weight of each larva was recorded every 2 days for 8 days [29]. The change in larval weight and the relative growth rate were calculated. The change in larval weight was calculated as the weight of the larva on day N minus the weight on day 0. The relative growth rate was calculated as the change in larval weight divided by the weight on day 0. Each treatment included 25 biological repeats (1 larva per replicate).

Pupal weight determination: Third-instar S. litura larvae were selected and fed with B. balsamifera leaves treated with MeJA or deionized water until pupation. The pupae were weighed 48 h after pupation [43]. Each treatment included 18 biological repeats (1 larva per replicate).

4.4. Enzyme Activity Assay

Leaves from the same position of B. balsamifera plants treated according to the procedures described in Section 4.2 were collected. For each plant, 0.1 g of leaf tissue was sampled to determine the activities of CAT, POD [44], SOD [45], PPO [46], PAL [47], and the JA biosynthetic enzyme LOX [48] in B. balsamifera leaves. The activities of allene oxide synthase (AOS) and allene oxide cyclase (AOC) were determined using an enzyme-linked immunosorbent assay kit obtained from Ruixin Biotechnology Co., Ltd. (Quanzhou, China). Each experimental treatment included five biological replicates, with one plant serving as one replicate.

4.5. Determination of Secondary Metabolite Content

B. balsamifera leaves from the four treatment groups described in Section 4.2 were used to determine secondary metabolite contents. Leaves (0.1 g) were collected from the same position on each B. balsamifera plant. Tannin, total phenol, and flavonoid contents were determined via the sodium tungstate–phosphomolybdic acid colorimetric, folin-phenol, and sodium nitrite–aluminum nitrate colorimetric methods, respectively [41]. Lignin content was determined using the acetylation method, whereas alkaloid content was determined using the bromocresol green indicator method [41]. Each experimental treatment was conducted with three biological replicates, where each B. balsamifera plant served as one replicate.

4.6. Metabolite Analysis of B. balsamifera Plants Treated with MeJA and S. litura Feeding

According to the treatment method described in Section 4.2, 1.0 g of B. balsamifera leaf, each from the four treatment groups, was cut as a test sample for metabolomics analysis. Each experimental treatment was performed with four replicates, with each B. balsamifera plant serving as one replicate. First, 100 mg of B. balsamifera leaf tissue was weighed and ground to a powder in liquid nitrogen, then transferred to an EP tube and mixed with 500 μL of 80% methanol. The mixture was vortexed vigorously, chilled on ice for 5 min, and centrifuged at 15,000× g and 4 °C for 20 min. The supernatant was diluted with water to 53% methanol, recentrifuged under identical conditions, and analyzed via UHPLC–MS/MS (Thermo Fisher, Langenselbold, Germany).

UHPLC-MS/MS analyses were conducted on a Vanquish UHPLC system (Thermo Fisher, Langenselbold, Germany) coupled to an Orbitrap Q Exactive™ HF, Exploris 480, or Q Exactive™ HF-X mass spectrometer (all Thermo Fisher, Langenselbold, Germany) at Novogene Co., Ltd. (Beijing, China). Samples were injected onto a Hypersil Gold column (100 × 2.1 mm, 1.9 μm) and separated via a 12 min linear gradient (0.2 mL/min flow rate). The mobile phase for positive/negative ionization modes comprised eluent A (0.1% formic acid in water) and eluent B (methanol), with the gradient program: 2% B (0–1.5 min); 2–85% B (1.5–4.5 min); 85–100% B (4.5–10 min); 100–2% B (10.0–10.1 min); 2% B (10.1–12 min). Mass spectrometry was operated in positive/negative modes with parameters: spray voltage, 3.5 kV; capillary temperature, 320 °C; sheath gas flow, 35 psi; auxiliary gas flow, 10 L/min; S-lens RF level, 60; auxiliary gas heater temperature, 350 °C.

4.7. Data Analysis

One-way analysis of variance (ANOVA) was conducted via SPSS 21.0 software (IBM Corporation, Armonk, NY, USA) to compare defense enzyme activities, JA signaling pathway biosynthetic enzyme activities, and secondary metabolite contents across treatments, with Tukey’s test applied to identify significant differences (p < 0.05). An independent samples t-test was used to assess differences in larval feeding selectivity, feeding amount, larval weight, relative growth rate, and pupal weight. Data visualization was conducted using Origin 2024 software (Origin Lab Corporation, Northampton, MA, USA).

For untargeted metabolomics studies, UHPLC–MS/MS (Thermo Fisher, Langenselbold, Germany) was applied. The raw files (.raw) obtained from MS detection were imported into Compound Discoverer 3.3 software (Thermo Fisher, Langenselbold, Germany) for spectral processing and database matching, enabling qualitative and quantitative analysis of metabolites. Partial least squares discriminant analysis (PLS-DA) was used to establish the relationship model between metabolite expression levels and sample categories. PLS-DA models were constructed for each comparison group and validated through seven cycles of cross-validation and model sorting; the closer R^2^ and Q^2^ are to 1, the more stable and reliable the model is. A t-test was performed to calculate the p-value and evaluate the significance of observed differences. Metabolites with a variable importance in the projection (VIP) > 1.0, Fold change (FC) > 1.2 or FC < 0.833, and p < 0.05 were considered significantly different. PubChem (https://pubchem.ncbi.nlm.nih.gov/, accessed on 4 June 2025) was used to retrieve and analyze the physicochemical properties of the differential metabolites. Finally, the biological significance of the metabolites was elucidated through metabolic pathway analysis.

5. Conclusions

This study establishes that exogenous MeJA acts as a potent immune elicitor in B. balsamifera, significantly enhancing its resistance against S. litura by orchestrating a systemic defense response. The observed resistance is driven by a multi-layered mechanism: the rapid activation of JA biosynthetic (LOX, AOS, AOC) and antioxidant enzymes (POD, PPO) serves as the initial defense, while the subsequent metabolic reprogramming constitutes the core durable resistance. Crucially, our metabolomic profiling revealed that MeJA treatment does not merely increase general defense metabolites (flavonoids and phenols) but specifically upregulates distinct bioactive compounds, including isatin and zerumbone. The accumulation of these metabolites—known for both their anti-herbivore properties and pharmacological (e.g., antitumor) activities—underscores a synergistic effect: MeJA elicits a defense state that concurrently enhances the medicinal quality of the herb. Therefore, this study validates MeJA application as a promising “dual-function” strategy for green pest management and quality improvement in the good agricultural practice for Chinese crude drugs cultivation of B. balsamifera. However, this study also has some limitations. For example, the optimal MeJA concentration for activating the defense response of B. balsamifera plants needs to be further optimized; the function of defensive metabolites with potential insect resistance needs to be further verified. Future research should focus on deciphering the molecular transcriptional networks regulating these specific metabolic pathways to further optimize this bio-elicitation technique.