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Article

Sustainable Management of Bacterial Leaf Spot in Bell Pepper by Biological and Chemical Resistance Inducers

by
Pisut Keawmanee
1,
Ratiya Pongpisutta
2,
Sujin Patarapuwadol
1,
Jutatape Watcharachaiyakup
3,
Sotaro Chiba
4,
Santiti Bincader
5 and
Chainarong Rattanakreetakul
1,*
1
Department of Plant Pathology, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Nakhon Pathom 73140, Thailand
2
Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand
3
Center for Agricultural Biotechnology, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand
4
Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan
5
Program in Plant Science, Faculty of Agricultural Technology and Agro-Industry, Rajamangala University of Technology Suvarnabhumi, Phra Nakhon Si Ayutthaya 13000, Thailand
*
Author to whom correspondence should be addressed.
Agriculture 2025, 15(17), 1859; https://doi.org/10.3390/agriculture15171859
Submission received: 27 July 2025 / Revised: 26 August 2025 / Accepted: 30 August 2025 / Published: 31 August 2025
(This article belongs to the Section Crop Protection, Diseases, Pests and Weeds)
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Abstract

Bacterial leaf spot, particularly in chili peppers, is major concern worldwide, particularly in chili peppers. Enhancing pepper resistance to bacterial leaf spot addresses a key agricultural challenge while minimizing chemical usage. In this study, the efficacy of plant resistance inducers (PRIs) in controlling bacterial leaf spot in peppers was evaluated through molecular and secondary metabolite analyses. Pepper plant seedlings were treated with salicylic acid (SA), acibenzolar-S-methyl, β-aminobutyric acid, chitosan, Bacillus subtilis B01, and B. velezensis CH6 and inoculated with Xanthomonas euvesicatoria pv. euvesicatoria. Disease severity was assessed, and the expression level of genes (PR-1, PR-2, PR-4, and CAT) and the abundance of secondary metabolites were analyzed via quantitative PCR (qPCR) and gas chromatography-mass spectrometry (GC-MS), respectively. Soil drenching with B. subtilis B01 produced the best effects, reducing the disease severity by 80% and significantly inducing PR-1 expression 24–48 h post-treatment. SA was similarly effective in inducing systemic acquired resistance (SAR), while β-aminobutyric acid primed antioxidative defenses through sustained catalase (CAT) expression, and chitosan induced PR-4. GC-MS analysis revealed secondary metabolites associated with systemic resistance pathways including SAR and induced systemic resistance (ISR). Herein, B. subtilis B01 and SA were identified as potent resistance inducers that reduce the disease severity of bacterial leaf spot and activate key defense pathways in pepper plants. These findings contribute to the development of sustainable, integrated disease management strategies.

1. Introduction

Bacterial leaf spot, caused by Xanthomonas spp., is a major concern worldwide, particularly in chili peppers and bell peppers. Various species of Xanthomonas, including X. euvesicatoria pv. euvesicatoria (Xee), X. euvesicatoria pv. perforans (Xep), X. hortorum pv. gardneri, and X. vesicatoria, are causative agents of bacterial leaf spot and contribute to its widespread occurrence in chili peppers and tomatoes of the Solanaceae family. These short, motile, Gram-negative, rod-shaped bacteria can infect the leaves, fruits, stems, and seeds, leading to necrotic lesions, chlorosis, and defoliation. The symptoms of this disease on plant leaves include necrotic spots, with occasional yellowing of the lesion edges and signs of water soaking. Crop yield losses due to bacterial leaf spot can be severe and have been reported to reach 66% [1,2,3]. Bacterial leaf spot remains among the most destructive diseases affecting pepper production worldwide. Disease management has traditionally relied heavily on the prophylactic application of copper-based bactericides, and in some regions, antibiotics [1,4,5,6]. However, these chemical controls have become increasingly ineffective because of the emergence of copper- and antibiotic-resistant Xanthomonas strains as well as regulatory restrictions and environmental concerns associated with chemical use [4,7].
Identifying compounds that can stimulate plant resistance to plant diseases by inducing plants to exploit their inherent resistance will reduce disease severity, and these compounds could be substitutes for pesticides or minimize their use. This, in turn, will contribute to more cost-effective agricultural management [8]. Many research and development efforts have focused on the use of induced resistance agents to prevent plant diseases including investigations to assess resistance at the molecular level. These findings have provided insights into the internal cellular mechanisms of plant response.
Some agents that induce plant resistance can transmit signals via salicylic acid (SA), leading to the expression of disease resistance genes and pathogenesis-related (PR) proteins within plant cells. This, in turn, enables plants to inhibit the destructive activities of pathogens. The use of induced resistance agents represents a viable approach for disease prevention [9,10]. One example of the potential efficacy of this approach is the registration of probenazole, the first commercially approved inducer, which is marketed under the trade name Oryzemate in Japan. The application of probenazole for controlling rice blast disease represents an important milestone in leveraging inducers to increase plant defense mechanisms [11]. Recent advances in plant immunity research have paved the way for the development and commercial use of numerous plant resistance inducers (PRIs) to manage devastating pathogens in agriculture [12].
Biological agents, especially Bacillus, are among the most effective biological inducers of plant defense, triggering induced systemic resistance (ISR) that enhances host resilience against a broad spectrum of pathogens including bacteria, fungi, and viruses [13]. ISR elicited by Bacillus spp. predominantly engages the jasmonic acid (JA) and ethylene (ET) signaling pathways, leading to the rapid activation of defensive gene networks and the accumulation of antimicrobial metabolites [14]. The multifaceted mode of action makes Bacillus spp. an invaluable component of sustainable disease management programs, offering durable protection with minimal environmental impact.
Recently, plant resistance inducers or plant biostimulants have been widely used among farmers, and certain compounds, including SA, acibenzolar-S-methyl, β-aminobutyric acid, chitosan, and Bacillus species, have been reported to effectively induce disease resistance in chili peppers and tomatoes. Although plant resistance inducers have shown efficacy against a wide spectrum of pathogens, their comparative performance, optimal integration, and impact at the molecular level under tropical or high-disease-pressure environments have not been fully clarified, particularly for the bacterial leaf spot of pepper. Furthermore, evaluating the efficacy of resistance-inducing agents against bacterial leaf spot disease provides valuable data and insights into the associated mechanisms. This information is crucial for explaining the rationale behind disease prevention strategies, allowing for informed decisions for disease control to be made that are beneficial for both the producers and environment within a country and contributes to increasing the safety standards for consumers.

2. Materials and Methods

2.1. Plant Pathogens and Pathogenicity Test

Two isolates of the X. euvesicatoria pv. euvesicatoria (Xee XCVKK-C-1, gene bank accession number JBHJCF000000000 and Xee 20HP1591, gene bank accession number JBHJBX000000000) and two isolates of X. euvesicatoria pv. perforans (Xep 62XPKK2, gene bank accession number JBHJAQ000000000 and Xep XCVKK-T-31, gene bank accession number JBHJAR000000000) were received from the Center for Agricultural Biotechnology, Kasetsart University Kamphaeng Saen Campus, Nakhon Pathom, Thailand [15]. The bacterial strains were cultured on nutrient agar (NA) at 30 °C for 48 h. The bacterial suspensions were then removed from the agar surface by washing with sterile, deionized water, and the cell density was adjusted to OD600nm = 0.2 or approximately 107 CFU/mL. Each bacterial suspension was subsequently inoculated onto approximately 4–5 true leaves of bell pepper (California) and chili pepper (Tavee 60) seedlings by spraying, and the plants were placed in plastic bags for 48 h. Disease assessment was conducted 14 and 21 days after inoculation. Symptoms were evaluated according to the visual symptom severity scale described by Kousik and Ritchie [16] as follows: 0 = no lesions observed; 1 = less than 1% of the leaf area was diseased; 2 = 1 to 10% of the leaf area was diseased; 3 = 11 to 20% of the leaf area was diseased; 4 = 21 to 35% of the leaf area was diseased; 5 = 36 to 50% of the leaf area was diseased; 6 = 51 to 65% of the leaf area was diseased; 7 = 66 to 80% of the leaf area was diseased; 8 = 81 to 99% of the leaf area was diseased; and 9 = complete defoliation. The disease severity index (DSI) was calculated as follows: DSI = [∑ (disease score × number of leaves with that score)/(total number of leaves × highest score)] × 100.

2.2. Treatment of Chili Seedlings with Plant Resistance Inducers (PRIs) Under Greenhouse Conditions

The ability of PRIs to control bacterial leaf spot disease in chili seedlings was evaluated following the methodology of Safaie Farahani and Taghavi [17] using two pepper varieties, California bell pepper (susceptible variety) and Tavee 60 chili pepper (tolerant variety). The experiment followed a completely randomized design (CRD) with five replicates. The chili pepper leaves were sprayed with the abiotic PRIs as SA (Sigma-Aldrich, St. Louis, MO, USA), acibenzolar-S-methyl (Merck, Switzerland), and β-aminobutyric acid (Sigma-Aldrich, St. Louis, MO, USA) at concentrations of 0.25, 0.5, and 1 mM and chitosan (KTF, Kanchanaburi, Thailand) at concentrations of 500, 1000, and 1500 µL/L. To examine biotic plant resistance induction, Bacillus subtilis B01 and B. velezensis CH6 (107 CFU/mL) were applied by spraying and soil drenching, whereas water spraying was used as a control. Twenty-four or forty-eight hours after applying the PRIs, the plants were inoculated with the Xee and Xep bacteria, and disease assessments were conducted at days 14 and 21 after inoculation, as above-mentioned.

2.3. Efficacy of PRIs for Controlling Bacterial Leaf Spot Disease During the Flowering Stage Under Greenhouse Conditions

Susceptible California bell pepper plants at the flowering stage were sprayed with selected concentrations of each PRI based on the results of the seedling stage experiments. The experiment followed a CRD with five replications. The plants were inoculated with a bacterial pathogen at specific times after PRI treatment on the basis of the results of the seedling stage experiments, and symptoms were subsequently evaluated according to the visual DSI scale described by Kousik and Ritchie [16]. Mock samples were inoculated with the pathogen but were not treated with PRIs.

2.4. Plant Resistance-Associated Gene Expression and Metabolite Abundance in PRI-Treated Plant

One-month-old chili pepper plants were treated via foliar spraying or soil drenching with PRIs at optimized concentrations (see results and discussion sections), and control plants were treated with water. Chili leaves were inoculated with Xee XCVKK-C-1 48 h after the PRI applications, and leaf samples were collected 24, 48, 72, and 96 h after PRI application [17]. The samples were ground with liquid nitrogen and separated for gene expression and metabolomic analyses. The total RNA was extracted with the RNeasy® Plant Mini Kit (Qiagen, Hilden, Germany) according to the recommended procedures. Next, cDNA was synthesized using the UltraScript cDNA Synthesis Kit (PCR Biosystem, London, UK), and gene expression was evaluated via quantitative PCR (qPCR) with the qPCRBIO SyGreen Mix Lo-ROX (PCR Biosystem, London, UK). The PCR primer sets used were as follows: PR-1 (5′-CTTTTGCTATATTTCACTCAACACAAGCCC-3′ and 5′-TGCTGGATTATTTTCCTTTTAACACATGA-3′; [18], PR-2 (5′-ACAGGCACATCTTCACTTACC-3′ and 5′-CGAGCAAAGGCGAATTTATCC-3′; [19], PR-4 (5′-AACTGGGATTTGAGAACTGCCAGC-3′ and 5′-TCCAAGGTACATATAGAGCTTCC-3′; [20], CAT (5′-GTCCATGAGCGTGGAAGCCCCGAAT-3′ and 5′-CGCGATGCATGAAGTTCATGGCACC-3′; [18], and actin (5′-ATCCCTCCACCTCTTCACTCTC-3′ and 5′-GCCTTAACCATTCCTGTTCCATTATC-3′) [21]. Relative gene expression was calculated using the 2−ΔΔCt method [21].
For the metabolomics analysis, the GC-MS data were collected at 72 and 96 h after PRI application (24 and 48 h after inoculation with Xee XCVKK-C-1). One hundred milligrams of each leaf sample was extracted with 700 µL of methanol. The filtrate solvent was then evaporated to dryness under nitrogen gas. Forty microliters of methoxyamine hydrochloride (20 mg/mL in pyridine) was added for methoximation, followed with incubation at 37 °C for 120 min. Afterward, 70 µL of N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) was added before incubation at 37 °C for an additional 30 min, followed by gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis (GC 7890B; Agilent Technology, USA); the injection volume was 1.0 µL (split ratio 10:1) and the injection temperature was 230 °C. Separation was performed with an HP 5ms column (30 m × 0.25 mm; Agilent Technology, Santa Clara, CA, USA) with helium as the carrier gas at a flow rate of 1.0 mL/min. The column temperature program was as follows: initial temperature of 60 °C, where it was held for 3 min; then, the temperature was increased at a rate of 10 °C/min until it reached 325 °C, where it was held for 2 min. The mass spectrometer (7000D Triple Quad detector, Agilent Technology, Santa Clara, CA, USA) was directly coupled to the gas chromatograph and operated with an ion source temperature of 230 °C in electron ionization (EI) mode at 70 eV. Each total ion chromatogram (TIC) was recorded in scan mode with the mass range of 40 to 600 atomic mass unit (AMU). The data were processed using Agilent Mass Hunter Unknowns Analysis by comparing the obtained fragmentation patterns with those in the National Institute of Standards and Technology (NIST) mass spectrometry database. The identified compounds were further examined for fragmentation to select the most distinct ions for each test set.

2.5. Greenhouse Conditions

The greenhouse experiment was conducted at Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom, Thailand. The temperature ranged from 25 to 35 °C with a relative humidity of 70–90%. The seedlings were grown in plastic pots containing sterilized soil:sand:compost (2:1:1). Each treatment included ten seedlings per replicate, and five replicates were used.

2.6. Statistical Analysis

Data on disease severity and gene expression were analyzed using ANOVA, and treatment means were compared using Duncan’s multiple range test (DMRT) at p < 0.05. Statistical analyses were conducted with the RStudio program version 2024.04.2.

3. Results and Discussion

3.1. Severity of Xanthomonas Bacterial Infection in Chili Plants

The pathogenicity of Xee and Xep was evaluated in two pepper varieties—California and Tavee 60. Fourteen days after inoculation, both Xee and Xep caused disease in both pepper varieties. Xee XCVKK-C-1 was the most aggressive, inducing symptoms in both the California and Tavee 60 pepper varieties, with incidences of 80.67 and 32.67, respectively (Table 1, Figure 1). The California variety showed a higher disease severity index in both Xee and Xep than the Tavee 60 variety. This may indicate that the California variety is more susceptible than the Tavee 60 variety. The symptoms of infection included water-soaked lesions on the leaf that progressed to the necrotic spots shown in Figure 1, resulting in a distorted leaf shape and eventual leaf drop (Figure 2). Subsequent re-isolation of the bacteria from the diseased tissues on NA medium and Gram staining revealed characteristics consistent with those of the tested isolates. Therefore, Xee XCVKK-C-1 was selected for subsequent studies.

3.2. Treatment of Pepper Seedlings with Bacterial Leaf Spot Disease with PRIs Under Greenhouse Conditions

The efficacy of the foliar spray of SA, acibenzolar-S-methyl, β-aminobutyric acid, and chitosan at different concentrations as well as the application of B. subtilis B01 and B. velezensis CH6 in controlling bacterial leaf spot caused by Xee XCVKK-C-1 was evaluated in California and Tavee 60. The results revealed that disease severity was lowest upon spraying with 0.5 mM SA 48 h before inoculation (Table 2) and 0.5 mM acibenzolar-S-methyl 48 h before inoculation in both pepper varieties (Table 3), although there was no significant effect compared with the lower concentrations applied earlier. Applying 0.5 mM β-aminobutyric acid (Table 4) 48 h before inoculation minimized disease in California peppers at 14 and 21 days post-inoculation, whereas the best result in Tavee 60 was achieved with 0.5 mM β-aminobutyric acid applied 24 h before inoculation; however, these results were not significantly different from those with 0.25 mM β-aminobutyric acid. For chitosan (Table 5), 1000 µL/L applied 24 h before inoculation resulted in the lowest disease severity in both pepper varieties, but these results were not significantly different from those obtained after spraying 1500 µL/L on California or after spraying all of the tested concentrations on Tavee 60. B. subtilis B01 (Table 6) showed the greatest efficacy when used as a soil drench 24 h before inoculation in both pepper varieties; however, soil drenching 48 h before inoculation or a foliar spray 24 h before inoculation were equally effective in California, but there were no differences with Tavee 60. Treatment with B. velezensis CH6 (Table 7) yielded similar results, as soil drenching 24 h before inoculation minimized disease severity in both peppers; in California, the disease severity did not differ significantly from that after soil drenching for 48 h, and there were no differences among all of the tested methods in Tavee 60.

3.3. Efficacy of PRIs for Controlling Bacterial Leaf Spot Disease During the Flowering Stage Under Greenhouse Conditions

Further experiments were conducted on susceptible California bell pepper plants at the flowering stage to evaluate the efficacy of the application of the PRIs SA, acibenzolar-S-methyl, and β-aminobutyric acid (0.5 mM each) and chitosan (1000 µL/L) applied via spraying. Additionally, the beneficial bacteria B. subtilis B01 and B. velezensis CH6 were applied via a soil drench. Forty-eight hours after treatment with the inducers or beneficial bacteria, the plants were inoculated with Xee XCVKK-C-1, and the disease severity was assessed at 14 and 21 days. In the flowering stage, soil drenching with B. subtilis B01 was the most effective at reducing disease severity caused by Xee, exhibiting the lowest disease severity at all time points (14 and 21 days). The next most effective treatments were SA and B. velezensis CH6, which both significantly reduced the disease severity compared with the control. β-Aminobutyric acid showed moderate efficacy, especially at 21 days, whereas chitosan and acibenzolar-S-methyl led to increasing disease severity over time but were still more effective than the control treatment, which had the highest disease severity at all time points (Table 8).
Bacillus sp. have been well-documented for their ability to induce resistance in plants via the induced systemic resistance (ISR) mechanism. This process enhances the plant’s own defense system, making it more resilient to a range of pathogens including viruses, bacteria, and fungi [22,23,24]. ISR triggered by Bacillus sp. involves important hormone pathways, primarily the jasmonic acid/ethylene (JA/ET) pathway. The SA pathway may also be involved, especially in the case of B. cereus AR156, which triggers both the SA and JA/ET pathways in an NPR1-dependent manner [24].
Salicylic acid is an endogenous hormone that plants utilize to regulate the development of systemic acquired resistance (SAR). Salicylic acid also plays a crucial role in inducing the expression of pathogenesis-related (PR) genes, thereby increasing plant resistance to a variety of pathogens [25,26]. Acibenzolar-S-methyl is a synthetic compound that mimics the actions of salicylic acid and can similarly induce PR gene expression. However, its effectiveness in disease control may vary depending on the plant species and the type of pathogen involved [8].
β-Aminobutyric acid is a small molecule that triggers the priming process in plants, which prepares the plants to respond quickly when attacked by pathogens by rapidly producing defensive enzymes or antimicrobial compounds [27]. The efficacy of β-aminobutyric acid is typically more pronounced in the later stages, as plants need time to alter their metabolic processes. In contrast, chitosan, a polysaccharide extracted from shellfish (such as shrimp or crab shells), strengthens the plant cell walls, making it more difficult for pathogens to invade. Additionally, chitosan can activate certain defense-related enzymes [28]. However, the effectiveness of chitosan depends on several factors including the molecular weight, degree of deacetylation, concentration, and environmental conditions during application [29]. In some cases, adjustments to the formulation or application frequency may be necessary.
The results clearly revealed that B. subtilis B01 was the most effective at reducing the severity of disease caused by Xee in peppers. Following B. subtilis B01 in terms of efficacy, both salicylic acid and B. velezensis CH6 significantly reduced the disease severity compared with the control. β-Aminobutyric acid showed moderate efficacy, especially on day 21, whereas chitosan and acibenzolar-S-methyl led to increasing disease severity over time but were still more effective than the control treatment, which had the highest disease severity at all time points (Table 8). The efficacy of plant resistance inducers such as acibenzolar-S-methyl, β-aminobutyric acid, chitosan, and Bacillus spp. was typically observed for up to 21 days after application under controlled conditions, with the strongest effects occurring during the first to second weeks and a gradual decline occurring after three weeks [30], whereas Bacillus-based treatments have similarly been shown to trigger long-lasting systemic protection [31].
The use of Bacillus spp. in combination with other resistance inducers has shown variable results. Abo-Elyousr, et al. [32] reported a disease reduction of 88.2% when B. subtilis and benzothiadiazole combinations that had synergistic effects were used, whereas Mejdoub-Trabelsi and Chérif [33] reported no improvement when combining B. cereus with β-aminobutyric acid. Therefore, the careful optimization of timing, concentration, and application methods is crucial when developing combined treatment strategies [34].

3.4. Analyses of the Expression Levels of Genes and Abundances of Metabolites Related to Plant Resistance After Treatment with PRIs

In this study, the expression levels of four genes associated with plant resistance after the application of various resistance inducers, PR-1, PR-2, PR-4, and CAT, were investigated 24, 48, 72, and 96 h after PRI application. The pepper plants were treated with the inducers 48 h prior to inoculation with Xee XCVKK-C-1. These results indicate that soil drenching with B. subtilis B01 activates the SA pathway, which is integral for SAR. The PR-1 gene is associated with the production of PR proteins [35], and the findings here were similar to those of Kloepper, Ryu and Zhang [13], who reported that lipopeptides produced by Bacillus spp., such as surfactin, iturin, and fengycin, act as elicitors to increase plant resistance by stimulating the production of SA—a key signal in SAR. This leads to the protein synthesis of PR-1, which has antimicrobial properties that inhibit bacterial growth and works in conjunction with PR-2, which further reinforces SAR.
Twenty-four hours after PRI application, the highest expression of the PR-1 gene was detected in the SA (2.87 ± 0.02) and B. subtilis B01 soil drenching treatments (2.61 ± 0.17) compared with that in the control group (Figure 3A). Conversely, the lowest PR-1 expression was detected in plants treated with acibenzolar-S-methyl (0.52 ± 0.02). Forty-eight hours after PRI application, soil drenching with B. subtilis B01 resulted in the highest PR-1 gene expression (51.34 ± 1.59), followed by acibenzolar-S-methyl (33.09 ± 3.03). Treatment with B. velezensis CH6 and foliar spraying with chitosan resulted in moderate PR-1 gene expression levels (23.74 ± 0.88 and 22.82 ± 3.43, respectively). At 72 h after PRI application (24 h after bacterial inoculation), PR-1 expression significantly decreased in all the treatment groups, with the lowest expression observed after soil drenching with B. subtilis B01 and foliar spraying with β-aminobutyric acid. Most PRIs did not induce PR-1 gene expression at 96 h after PRI application (48 h after bacterial inoculation), except for soil drenching with B. subtilis B01 (9.76 ± 2.72) and B. velezensis CH6.
In terms of the expression of the PR-2 gene 24 h after PRI application, compared with the control treatment, soil drenching with B. subtilis B01 resulted in the highest PR-2 gene expression (2.58 ± 0.35) (Figure 3B). In contrast, treatment with chitosan and acibenzolar-S-methyl led to the lowest PR-2 expression levels (0.43 ± 0.03 and 0.46 ± 0.11, respectively). Forty-eight hours after PRI application, SA significantly induced PR-2 expression (72.78 ± 2.00), followed by acibenzolar-S-methyl (17.95 ± 4.58) and soil drenching with B. subtilis B01 (12.90 ± 4.08). At 72 h after PRI application (24 h after bacterial inoculation), the highest PR-2 expression was detected in the presence of chitosan (6.53 ± 2.38), potentially reflecting the role of chitosan in the long-term pathogen response [28]. Conversely, soil drenching with B. subtilis B01 and foliar spraying with β-aminobutyric acid led to the lowest PR-2 expression (0.10 ± 0.10 and 0.27 ± 0.06, respectively) 72 h after PRI application. Furthermore, soil drenching with B. subtilis B01 maintained high PR-2 expression levels (97.95 ± 19.83), which significantly increased compared with those at earlier stages. Finally, soil drenching with B. velezensis CH6 resulted in the significant upregulation of only PR-2 (4.51 ± 0.31) at 48 h after application.
PR-4 encodes a specific type of PR protein critical for the plant immune system that operates independently of the SA pathway. PR-4 expression is primarily induced by jasmonic acid (JA) and ethylene (ET) signaling [34,36]. In our experiment, the induction of PR-4 was not as high as that of PR-1 and PR-2. Twenty-four hours after the application of the PRIs, compared with the control, chitosan induced the highest PR-4 expression (5.93 ± 0.64), followed by acibenzolar-S-methyl (3.10 ± 0.06) and β-aminobutyric acid (2.96 ± 0.04) (Figure 3C). Forty-eight hours after PRI application, SA elicited the highest PR-4 expression (2.16 ± 0.12), although this expression was lower than the chitosan-induced expression at 24 h. Chitosan plays a pivotal role in the early (24 h) induction of PR-4, aligning with its function as an elicitor that activates resistance through the JA/ET pathways. Given that chitosan is a polysaccharide similar to chitin found in fungi, it rapidly induces PR-4 gene expression and the production of defense proteins, such as hevein-like proteins, which combat fungi and bacteria [37]. However, the response decreased in the long-term, which is consistent with the results of Amborabé, et al. [38], with protection lasting only 3 days, suggesting the need for repeated applications every 3–5 days or in combination with longer-lasting inducers such as B. subtilis B01.
The CAT gene is involved in the production of the enzyme catalase, which plays a critical role in reducing the reactive oxygen species (ROS) levels during the plant’s immune response to pathogens. Twenty-four hours after the application of the PRIs, β-aminobutyric acid induced the highest CAT expression (5.08 ± 0.31), whereas soil drenching with B. subtilis B01 resulted in the lowest expression (0.55 ± 0.06) (Figure 3D). At 48 h after the application of the PRIs, β-aminobutyric acid treatment again led to the highest CAT expression (1.91 ± 0.20), whereas SA and acibenzolar-S-methyl resulted in moderate expression of this gene (1.27 ± 0.08 and 1.27 ± 0.02, respectively). Seventy-two hours after the application of the PRIs (24 h after bacterial inoculation), compared with the control treatment, the β-aminobutyric acid treatment maintained the highest CAT expression (15.57 ± 3.32), followed by soil drenching with B. subtilis B01 (7.86 ± 0.28). The elevated CAT expression at 72 h following β-aminobutyric acid application is likely due to the role of β-aminobutyric acid as a potent immune stimulator that triggers priming, enabling rapid and robust plant responses to infection by facilitating ROS management. β-Aminobutyric acid promotes CAT gene expression to control ROS within the ISR process, which is linked to the JA and ET signaling pathways [39].
The experimental results further demonstrated that the PRIs acibenzolar-S-methyl and SA effectively stimulated the initial defense responses of the plants before pathogen challenge (within 48 h of treatment). The notable exception was CAT gene expression, which increased consistently post-inoculation, particularly under β-aminobutyric acid treatment. Additionally, chitosan spraying and soil drenching with the B. subtilis isolate B01 and B. velezensis CH6 induced the expression of the PR-2 and PR-4 genes during the middle to late stages of infection due to the associations between microorganisms and biological inducers [40,41].
For GC-MS analysis, a total of 524 and 584 compounds were identified with log2-fold change compared with the mock test (Tables S1 and S2). A total of 157 compounds were found in both the 72 and 96 h timepoints. The top 50 compounds with a maximum value of log2-fold change values after treatment with PRIs for 72 and 96 h are shown in Figure 4. Six compounds, namely, 1,3-dioxolane-2-methanol, 1H-benzotriazole, 4-amino-1-methyl-, 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole, DL-norvaline, ethyl ester, methyl 3-isothiocyanatopropionate, and pyridine, were found in both time points.
Salicylic acid treatment resulted in strong but targeted responses, particularly in the activation of alkaloid compounds including pyridine derivatives. Despite being the archetypal plant defense hormone, compared with its synthetic analogue acibenzolar-S-methyl, salicylic acid induced more limited metabolic activation, possibly because of rapid metabolism, compartmentalization, or feedback regulation [42,43]. The observed response pattern observed reflects the role of salicylic acid as the core SAR signaling molecule, with effects primarily mediated through NPR1-dependent pathways that lead to PR protein induction [44,45]. Recent studies have demonstrated that SA levels significantly influence metabolic responses, with SA-deficient and SA-overaccumulating plants showing distinct volatile organic compound profiles and defense gene expression patterns [42]. The accumulation of proline derivatives following SA treatment aligns with reports of SA-induced changes in amino acid metabolism, particularly under stress conditions, where proline serves as both an osmolyte and a ROS scavenger [46].
Acibenzolar-S-methyl demonstrated the most comprehensive metabolic response, activating 30 compounds. This synthetic salicylic acid analogue effectively triggered systemic acquired resistance (SAR), consistent with previous findings that acibenzolar-S-methyl activates stomatal-based defense mechanisms and induces pathogenesis-related (PR) protein expression including PR-1, PR-2, and PR-5 [47]. The broad-spectrum activity observed aligns with reports demonstrating acibenzolar-S-methyl effectiveness against diverse pathogens in cucumber, Japanese radish, and cabbage through the generation of systemic mobile signals [47,48] . Acibenzolar-S-methyl treatment enhanced nitrogenous compounds and silyl derivatives, indicating extensive secondary metabolite production. This metabolic breadth reflects acibenzolar-S-methyl function as a SAR inducer, activating multiple defense pathways simultaneously through salicylic acid-dependent signaling cascades [49]. The high specificity observed, with 20 compounds responding exclusively to ASM treatment, demonstrates unique metabolic signatures associated with this synthetic elicitor, supporting its distinct mechanism compared with natural SA.
β-Aminobutyric acid treatment had the greatest methyl 3-isothiocyanatopropionate response. This pattern supports β-aminobutyric acid’s established role as a priming agent rather than a direct defense activator [50,51]. The treatment particularly activated organic acids and amino acid derivatives, consistent with the β-aminobutyric acid mechanism of enhancing metabolic pathways involved in stress resistance without constitutively activating defense responses [52]. The high-intensity but selective response pattern observed in our study supports the use of β-aminobutyric acid’s reputation as a priming agent that enhances plant responsiveness to subsequent stress through metabolic pathway sensitization [51]. The involvement of isothiocyanate compounds, such as methyl 3-isothiocyanatopropionate, is consistent with the documented effects of β-aminobutyric acid on glucosinolate metabolism and callose deposition priming [51]. These compounds serve as antimicrobial agents and contribute to the broad-spectrum protection of β-aminobutyric acid against biotic and abiotic stresses.
Chitosan treatment demonstrated selective but potent activation, particularly of alkaloid compounds and unique metabolites such as 2,4-hexadiyne. As a pathogen-associated molecular pattern (PAMP), chitosan triggers plant defense responses through pattern recognition receptors, leading to extensive metabolic changes [53,54,55]. The ability of the polysaccharide to induce quinoline derivatives reflects its role in activating the octadecanoid pathway and nitric oxide signaling. This activity is consistent with reports showing that chitosan enhances the jasmonic acid (JA) content and activates phenylpropanoid metabolism, leading to increased phenolic compound accumulation [54,56]. The effect of chitosan on TCA cycle intermediates and sugars observed in our study are consistent with previous metabolomic analyses showing elevated sucrose, malate, and oxaloacetate levels following chitosan treatment [53]. This metabolic shift supports energy-demanding defense processes including lignification, ROS bursts, and phenolic synthesis [54].
A variety of responses were observed when testing the biological plant inducers from the Bacillus genus. B. subtilis B01 demonstrated limited activity but effectively induced alkaloid-like compounds, particularly pyridine derivatives. Differences between bacterial strains highlight the importance of strain selection in biocontrol applications, as plant–bacteria interactions involve distinct mechanisms [57]. B. velezensis CH6 showed a notable effectiveness among bacterial treatments, particularly in inducing indole compounds including indol-2(3H)-one derivatives. These finding are consistent with reports that B. velezensis strains produce indole-3-acetic acid and other bioactive indole derivatives that trigger plant defense responses [58,59,60]. The effectiveness of B. subtilis and B. velezensis in producing cyclic lipopeptides, including surfactin, iturin, and fengycin families, contributes to both direct antimicrobial activity and induced systemic resistance (ISR) activation [59,60]. These compounds can penetrate plant cell membranes and trigger the expression of the defense gene, which explains the robust metabolic responses observed [59].

4. Conclusions

This study successfully demonstrated the efficacy of various plant resistance inducers in controlling bacterial leaf spot caused by Xee in bell pepper, with significant implications for sustainable disease management strategies. Among the six PRIs tested, soil drenching with B. subtilis B01 delivered the most pronounced and durable protection, restricting disease severity compared with the control. B. subtilis B01 elicited a rapid SA-dependent systemic acquired-resistance response, as evidenced by the detection of PR-1 transcripts 48 h after treatment and an early peak in PR-2 expression. Foliar spraying of salicylic acid and its functional analogue acibenzolar-S-methyl also activated the SA signaling arm, with SA markedly increasing the PR-2 levels within 48 h. In contrast, β-aminobutyric acid principally primed the antioxidative defenses, maintaining the highest catalase expression through 72 h post-inoculation and thereby supporting reactive oxygen species homeostasis. Chitosan triggered an early jasmonate/ethylene-linked induction of PR-4, underscoring its role in reinforcing cell-wall and phenylpropanoid defenses. Metabolomic profiling corroborated these gene-level trends. Treatment with B. subtilis B01 resulted in preferential accumulation of pyridine-type alkaloids, whereas treatment with B. velezensis CH6 led to the enrichment of indole derivatives—both compound classes with documented antimicrobial or signaling activity. Together, these distinct metabolite signatures highlight the importance of strain and chemical selection in tailoring induced resistance strategies. Collectively, our data indicate that a single soil application of B. subtilis B01, complemented by strategic salicylic acid or acibenzolar-S-methyl foliar sprays, can provide season-long suppression of Xanthomonas with minimal reliance on conventional bactericides. This PRI-based program offers a pragmatic route toward the integrated, environmentally sustainable management of bacterial leaf spot in chili production systems.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture15171859/s1, Table S1: Log2-fold changes in metabolites at 72 h after treatment with PRIs; Table S2: Log2-fold changes in metabolites at 96 h after treatment with PRIs.

Author Contributions

Conceptualization, P.K., R.P., S.P., and C.R.; Methodology, P.K., S.P., J.W., and C.R.; Software, P.K. and C.R.; Validation, R.P. and C.R.; Formal analysis, P.K. and C.R.; Investigation, P.K., S.B., and C.R.; Resources, P.K., S.P., J.W., and C.R.; Data curation, P.K.; Writing—original draft preparation, P.K.; Writing—review and editing, R.P., S.P., J.W., S.B., S.C., and C.R.; Visualization, P.K., C.R., and S.C.; Supervision, P.K. and C.R.; Project administration, C.R.; Funding acquisition, P.K., C.R., and S.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Kasetsart University Research and Development Institute, KURDI (Grant No. YF(KU)33.66) and the International Station for Tropical Agricultural Sciences, Nagoya University.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

Data are available upon request; please contact the corresponding author.

Acknowledgments

This research was supported by the Kasetsart University Research and Development Institute, KURDI (Grant No. YF(KU)33.66). The authors would like to thank Niphone Thaveechai, Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand for generously provided B. velezensis CH6. We would like to thank the members of the Physiology of Plant Disease Laboratory, Department of Plant Pathology, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Nakhon Pathom, Thailand, for provided B. subtilis B01 and their kind support.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study.

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Agriculture 15 01859 g001
Figure 1. Symptoms of bacterial leaf spot on bell pepper plants 14 days after inoculation with Xee XCVKK-C-1 by foliar spraying. Adaxial (A) and abaxial (B) sides of the inoculated leaf that exhibited water-soaked lesions.
Figure 1. Symptoms of bacterial leaf spot on bell pepper plants 14 days after inoculation with Xee XCVKK-C-1 by foliar spraying. Adaxial (A) and abaxial (B) sides of the inoculated leaf that exhibited water-soaked lesions.
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Figure 2. Disease severity in California bell and Tavee 60 chili peppers 14 days after inoculation with Xee and Xep. (A) Bell pepper California (control); (B) bell pepper California inoculated with Xee XCVKK-C-1; (C) bell pepper California inoculated with Xee 20HP1591; (D) bell pepper California inoculated with Xep 62XPKK2; (E) bell pepper California inoculated with Xep XCVKK-T-31; (F) Chili pepper Tavee 60 (control); (G) chili pepper Tavee 60 inoculated with Xee XCVKK-C-1; (H) chili pepper Tavee 60 inoculated with Xee 20HP1591; (I) chili pepper Tavee 60 inoculated with Xep 62XPKK2; (J) chili pepper Tavee 60 inoculated with Xep XCVKK-T-31.
Figure 2. Disease severity in California bell and Tavee 60 chili peppers 14 days after inoculation with Xee and Xep. (A) Bell pepper California (control); (B) bell pepper California inoculated with Xee XCVKK-C-1; (C) bell pepper California inoculated with Xee 20HP1591; (D) bell pepper California inoculated with Xep 62XPKK2; (E) bell pepper California inoculated with Xep XCVKK-T-31; (F) Chili pepper Tavee 60 (control); (G) chili pepper Tavee 60 inoculated with Xee XCVKK-C-1; (H) chili pepper Tavee 60 inoculated with Xee 20HP1591; (I) chili pepper Tavee 60 inoculated with Xep 62XPKK2; (J) chili pepper Tavee 60 inoculated with Xep XCVKK-T-31.
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Figure 3. The expression of the PR-1, PR-2, PR-4, and CAT genes at 24, 48, 72, and 96 h after the application of PRIs and inoculation with Xee XCVKK-C-1 at 48 h. Expression of (A) PR-1, (B) PR-2, (C) PR-4, and (D) CAT. SA, salicylic acid; ASM, acibenzolar-S-methyl; BABA, β-aminobutyric acid; CHI, chitosan; B01, B. subtilis B01; CH6, B. velezensis CH6; Control, not sprayed with a PRI. Bars represent the means ± standard deviation (SD). Same letters above bars indicate no statistically significant difference (p < 0.05) according to Duncan’s multiple range test (DMRT).
Figure 3. The expression of the PR-1, PR-2, PR-4, and CAT genes at 24, 48, 72, and 96 h after the application of PRIs and inoculation with Xee XCVKK-C-1 at 48 h. Expression of (A) PR-1, (B) PR-2, (C) PR-4, and (D) CAT. SA, salicylic acid; ASM, acibenzolar-S-methyl; BABA, β-aminobutyric acid; CHI, chitosan; B01, B. subtilis B01; CH6, B. velezensis CH6; Control, not sprayed with a PRI. Bars represent the means ± standard deviation (SD). Same letters above bars indicate no statistically significant difference (p < 0.05) according to Duncan’s multiple range test (DMRT).
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Figure 4. Heatmap of the top 50 maximum log2-fold change at 72 and 96 hours after treatment with PRI. (A) 72 h after treatment; (B) 96 h after treatment. SA, salicylic acid; ASM, acibenzolar-S-methyl; BABA, β-aminobutyric acid; CHI, chitosan; B01, B. subtilis B01; CH6, B. velezensis CH6; CON, control (not sprayed with a PRI).
Figure 4. Heatmap of the top 50 maximum log2-fold change at 72 and 96 hours after treatment with PRI. (A) 72 h after treatment; (B) 96 h after treatment. SA, salicylic acid; ASM, acibenzolar-S-methyl; BABA, β-aminobutyric acid; CHI, chitosan; B01, B. subtilis B01; CH6, B. velezensis CH6; CON, control (not sprayed with a PRI).
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Table 1. Disease severity in the leaves of both pepper varieties at 14 days after foliar inoculation with the Xanthomonas species.
Table 1. Disease severity in the leaves of both pepper varieties at 14 days after foliar inoculation with the Xanthomonas species.
IsolateDisease Severity Index 1/
Bell Pepper CaliforniaChili Pepper Tavee 60
Xee XCVKK-C-180.67 ± 4.04 a32.67 ± 2.51 a
Xee 20HP159158.67 ± 6.35 b29.33 ± 6.40 a
Xep 62XPKK257.23 ± 8.18 b12.30 ± 3.49 b
Xep XCVKK-T-3148.45 ± 12.65 b10.05 ± 2.17 b
F-test***
* Significantly different at the 95% confidence level (p < 0.05). ** Significantly different at the 99% confidence level (p < 0.01). 1/ Data are represented as the mean ± standard deviation (SD). Means within a column followed by different letters were significantly different (p < 0.05) according to Duncan’s multiple range test (DMRT).
Table 2. Disease severity of pepper leaves treated with salicylic acid and inoculated with Xee XCVKK-C-1 at 14 and 21 days post-inoculation.
Table 2. Disease severity of pepper leaves treated with salicylic acid and inoculated with Xee XCVKK-C-1 at 14 and 21 days post-inoculation.
Salicylic Acid
Concentration (mM)		Time Before Pathogen
Inoculation (h)		Disease Severity Index 1/
Bell Pepper CaliforniaChili Pepper Tavee 60
14 DAI 2/21 DAI14 DAI21 DAI
0.252422.00 ± 10.98 b40.28 ± 8.50 bc39.60 ± 7.21 a44.00 ± 6.51 a
4840.86 ± 9.55 ab58.57 ± 12.80 ab37.40 ± 6.80 a50.60 ± 7.20 a
0.52431.43 ± 12.01 ab39.29 ± 7.83 bc24.75 ± 12.92 ab33.00 ± 2.84 b
4816.50 ± 8.42 b23.83 ± 7.39 c8.25 ± 5.90 b8.25 ± 3.82 c
12428.29 ± 7.41 b36.14 ± 5.62 bc35.20 ± 7.58 a49.84 ± 7.51 a
4852.64 ± 9.50 a68.20 ± 8.47 a33.00 ± 6.50 a44.00 ± 6.80 a
02452.80 ± 9.84 a71.71 ± 8.17 a44.00 ± 7.85 a56.40 ± 7.90 a
F-test******
* Significantly different at the 95% confidence level (p < 0.05).** Significantly different at the 99% confidence level (p < 0.01). 1/ Data are represented as the mean ± standard deviation (SD). Means within a column followed by different letters were significantly different (p < 0.05) according to Duncan’s multiple range test (DMRT). 2/ DAI = days after inoculation.
Table 3. Disease severity of pepper leaves treated with acibenzolar-S-methyl and inoculated with Xee XCVKK-C-1 at 14 and 21 days post-inoculation.
Table 3. Disease severity of pepper leaves treated with acibenzolar-S-methyl and inoculated with Xee XCVKK-C-1 at 14 and 21 days post-inoculation.
Acibenzolar-S-methyl Concentration (mM)Time Before Pathogen
Inoculation (h)		Disease Severity Index 1/
Bell Pepper CaliforniaChili Pepper Tavee 60
14 DAI 2/21 DAI14 DAI21 DAI
0.25247.86 ± 4.20 c18.86 ± 5.50 b22.00 ± 13.51 b25.67 ± 6.58 b
4811.00 ± 8.50 bc23.57 ± 7.21 b8.80 ± 4.39 b15.40 ± 5.49 b
0.5244.71 ± 2.42 c17.29 ± 5.24 b19.80 ± 11.20 b22.00 ± 4.10 b
482.11 ± 1.60 c11.00 ± 8.90 b4.40 ± 4.20 b11.00 ± 9.28 b
12428.29 ± 9.51 b59.71 ± 8.41 a17.60 ± 9.10 b26.40 ± 6.67 b
484.71 ± 2.42 c33.00 ± 13.43 b13.20 ± 4.62 b22.00 ± 3.37 b
02452.80 ± 9.84 a71.71 ± 8.17 a44.00 ± 7.85 a56.40 ± 7.90 a
F-test*******
* Significantly different at the 95% confidence level (p < 0.05).** Significantly different at the 99% confidence level (p < 0.01). 1/ Data are represented as the mean ± standard deviation (SD). Means within a column followed by different letters were significantly different (p < 0.05) according to Duncan’s multiple range test (DMRT). 2/ DAI = days after inoculation.
Table 4. Disease severity of pepper leaves treated with β-aminobutyric acid and inoculated with Xee XCVKK-C-1 at 14 and 21 days post-inoculation.
Table 4. Disease severity of pepper leaves treated with β-aminobutyric acid and inoculated with Xee XCVKK-C-1 at 14 and 21 days post-inoculation.
β-aminobutyric acid Concentration (mM)Time Before Pathogen
Inoculation (h)		Disease Severity Index 1/
Bell Pepper CaliforniaChili Pepper Tavee 60
14 DAI 2/21 DAI14 DAI21 DAI
0.252428.29 ± 11.84 bc34.57 ± 10.53 b14.67 ± 4.10 bc29.33 ± 5.51 b
4836.14 ± 7.39 ab56.57 ± 9.20 a15.40 ± 5.21 bc26.40 ± 4.90 b
0.52411.00 ± 4.90 c33.00 ± 9.50 b11.00 ± 3.49 c11.00 ± 3.52 c
4812.57 ± 4.34 c15.71 ± 10.01 b15.40 ± 4.60 bc19.80 ± 5.57 bc
12415.71 ± 5.04 bc31.43 ± 8.10 b22.00 ± 6.11 b22.00 ± 4.20 b
4814.14 ± 4.69 bc20.43 ± 9.00 b15.40 ± 5.50 bc24.20 ± 3.87 b
02452.80 ± 9.84 a71.71 ± 8.17 a44.00 ± 7.85 a56.40 ± 7.90 a
F-test******
* Significantly different at the 95% confidence level (p < 0.05). ** Significantly different at the 99% confidence level (p < 0.01). 1/ Data are represented as the mean ± standard deviation (SD). Means within a column followed by different letters were significantly different (p < 0.05) according to Duncan’s multiple range test (DMRT). 2/ DAI = days after inoculation.
Table 5. Disease severity of pepper leaves treated with chitosan and inoculated with Xee XCVKK-C-1 was at 14 and 21 days post-inoculation.
Table 5. Disease severity of pepper leaves treated with chitosan and inoculated with Xee XCVKK-C-1 was at 14 and 21 days post-inoculation.
Chitosan Concentration (µL/L)Time Before Pathogen
Inoculation (h)		Disease Severity Index 1/
Bell Pepper CaliforniaChili Pepper Tavee 60
14 DAI 2/21 DAI14 DAI21 DAI
5002411.00 ± 8.93 bc47.14 ± 15.86 b19.80 ± 3.61 b24.20 ± 5.46 b
4826.71 ± 11.21 b40.86 ± 9.50 b17.60 ± 3.84 b24.20 ± 7.70 b
1000243.14 ± 2.50 c18.86 ± 14.01 bc11.00 ± 7.38 b13.20 ± 1.86 c
487.86 ± 6.76 bc11.00 ± 6.50 c13.75 ± 4.74 b19.25 ± 3.57 b
15002414.67 ± 9.78 bc31.17 ± 14.11 bc11.00 ± 8.45 b11.00 ± 3.44 c
4822.00 ± 9.86 b40.86 ± 9.80 b26.40 ± 10.50 ab33.00 ± 10.77 b
02452.80 ± 9.84 a71.71 ± 8.17 a44.00 ± 7.85 a56.40 ± 7.90 a
F-test*******
* Significantly different at the 95% confidence level (p < 0.05). ** Significantly different at the 99% confidence level (p < 0.01). 1/ Data are represented as the mean ± standard deviation (SD). Means within a column followed by different letters were significantly different (p < 0.05) according to Duncan’s multiple range test (DMRT). 2/ DAI = days after inoculation.
Table 6. Disease severity of pepper leaves treated with B. subtilis B01 and inoculated with Xee XCVKK-C-1 at 14 and 21 days post-inoculation.
Table 6. Disease severity of pepper leaves treated with B. subtilis B01 and inoculated with Xee XCVKK-C-1 at 14 and 21 days post-inoculation.
Application MethodTime Before Pathogen
Inoculation (h)		Disease Severity Index 1/
Bell Pepper CaliforniaChili Pepper Tavee 60
14 DAI 2/21 DAI14 DAI21 DAI
Spray2420.43 ± 7.75 b40.86 ± 9.58 bc22.00 ± 4.98 b30.80 ± 5.28 b
4844.00 ± 8.51 a51.86 ± 13.20 ab22.00 ± 5.21 b29.33 ± 6.09 b
Soil drench249.43 ± 4.35 b28.29 ± 12.84 c5.50 ± 2.12 c11.00 ± 4.11 c
4815.71 ± 5.99 b39.29 ± 11.50 bc2.75 ± 1.30 c24.75 ± 4.76 b
Spray with water2452.80 ± 9.84 a71.71 ± 8.17 a44.00 ± 7.85 a56.40 ± 7.90 a
F-test********
** Significantly different at the 99% confidence level (p < 0.01). 1/ Data are represented as the mean ± standard deviation (SD). Means within a column followed by different letters were significantly different (p < 0.05) according to Duncan’s multiple range test (DMRT). 2/ DAI = days after inoculation.
Table 7. Disease severity of pepper leaves treated with B. velezensis CH6 and inoculated with Xee XCVKK-C-1 at 14 and 21 days post-inoculation.
Table 7. Disease severity of pepper leaves treated with B. velezensis CH6 and inoculated with Xee XCVKK-C-1 at 14 and 21 days post-inoculation.
Application MethodTime Before Pathogen
Inoculation (h)		Disease Severity Index 1/
Bell Pepper CaliforniaChili Pepper Tavee 60
14 DAI 2/21 DAI14 DAI21 DAI
Spray2456.57 ± 7.43 a78.57 ± 8.92 a19.54 ± 6.47 b26.40 ± 8.67 b
4823.57 ± 9.84 b45.57 ± 7.86 b19.80 ± 7.63 b27.40 ± 6.82 b
Soil drench247.86 ± 6.76 b25.14 ± 6.35 c11.00 ± 5.21 b19.83 ± 5.38 b
4822.00 ± 7.50 b31.43 ± 6.92 bc8.80 ± 4.55 b8.92 ± 3.12 c
Spray with water052.80 ± 9.84 a71.71 ± 8.17 a44.00 ± 7.85 a56.40 ± 7.90 a
F-test*******
* Significantly different at the 95% confidence level (p < 0.05). ** Significantly different at the 99% confidence level (p < 0.01). 1/ Data are represented as the mean ± standard deviation (SD). Means within a column followed by different letters were significantly different (p < 0.05) according to Duncan’s multiple range test (DMRT). 2/ DAI = days after inoculation.
Table 8. Disease severity of bell pepper California leaves at the flowering stage treated with resistance inducers and inoculated with Xee XCVKK-C-1 at 14 and 21 days after inoculation.
Table 8. Disease severity of bell pepper California leaves at the flowering stage treated with resistance inducers and inoculated with Xee XCVKK-C-1 at 14 and 21 days after inoculation.
Plant Resistance InducerDisease Severity 1/
14 DAI 2/21 DAI
Salicylic acid4.43 ± 2.94 c36.66 ± 8.56 ab
Acibenzolar-S-methyl14.67 ± 7.77 bc35.43 ± 9.79 ab
β-Aminobutyric acid11.00 ± 7.24 bc29.30 ± 5.18 b
Chitosan22.34 ± 4.20 b47.67 ± 2.45 a
B. subtilis B013.67 ± 1.85 c7.33 ± 4.23 c
B. velezensis CH611.44 ± 6.93 bc36.67 ± 8.50 ab
Mock29.33 ± 2.38 a51.54 ± 10.60 a
F-test****
** Significantly different at the 99% confidence level (p < 0.01) 1/ Data are represented as the mean ± standard deviation (SD). Means within a column followed by different letters were significantly different (p < 0.05) according to Duncan’s multiple range test (DMRT). 2/ DAI = days after inoculation.
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MDPI and ACS Style

Keawmanee, P.; Pongpisutta, R.; Patarapuwadol, S.; Watcharachaiyakup, J.; Chiba, S.; Bincader, S.; Rattanakreetakul, C. Sustainable Management of Bacterial Leaf Spot in Bell Pepper by Biological and Chemical Resistance Inducers. Agriculture 2025, 15, 1859. https://doi.org/10.3390/agriculture15171859

AMA Style

Keawmanee P, Pongpisutta R, Patarapuwadol S, Watcharachaiyakup J, Chiba S, Bincader S, Rattanakreetakul C. Sustainable Management of Bacterial Leaf Spot in Bell Pepper by Biological and Chemical Resistance Inducers. Agriculture. 2025; 15(17):1859. https://doi.org/10.3390/agriculture15171859

Chicago/Turabian Style

Keawmanee, Pisut, Ratiya Pongpisutta, Sujin Patarapuwadol, Jutatape Watcharachaiyakup, Sotaro Chiba, Santiti Bincader, and Chainarong Rattanakreetakul. 2025. "Sustainable Management of Bacterial Leaf Spot in Bell Pepper by Biological and Chemical Resistance Inducers" Agriculture 15, no. 17: 1859. https://doi.org/10.3390/agriculture15171859

APA Style

Keawmanee, P., Pongpisutta, R., Patarapuwadol, S., Watcharachaiyakup, J., Chiba, S., Bincader, S., & Rattanakreetakul, C. (2025). Sustainable Management of Bacterial Leaf Spot in Bell Pepper by Biological and Chemical Resistance Inducers. Agriculture, 15(17), 1859. https://doi.org/10.3390/agriculture15171859

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