Mechanism study of jasmonic acid and salicylic acid enhances the cabbage black rot resistance by improving oxidative clearance capacity and synthesis of secondary metabolites

Technical Summary: Mechanism of Jasmonic Acid and Salicylic Acid in Enhancing Cabbage Black Rot Resistance

Problem Statement
Black rot, caused by the bacterium Xanthomonas campestris pv. campestris (Xcc), represents a severe threat to cabbage (Brassica oleracea L. var. capitata L.) production globally. The pathogen invades through wounds or natural openings, colonizing vascular tissues and causing characteristic "V"-shaped yellow lesions, vein blackening, and eventual plant wilting. Infection disrupts reactive oxygen species (ROS) metabolism, leading to oxidative stress, membrane lipid peroxidation, and reduced cell membrane stability. While traditional copper-based control measures are common, there is a growing need for green prevention strategies. Although Jasmonic acid (JA) and Salicylic acid (SA) are known defense hormones, the specific physiological and biochemical mechanisms by which they enhance resistance to Xcc in cabbage—particularly regarding endogenous hormone regulation, oxidative clearance, and secondary metabolite synthesis—remained unclear.

Methodology
The study utilized the susceptible cabbage variety "Zhonggan 11" and the highly pathogenic Xcc race 1. Seedlings at the "three leaves and one heart" stage were subjected to four treatments: a control (CK, distilled water), exogenous JA (100 μM), exogenous SA (500 μM), and a combined JA + SA treatment. Plants were inoculated with Xcc (10⁸ CFU) via spray inoculation and maintained in a high-humidity greenhouse.

Samples were collected at 0, 1, 3, and 5 days post-inoculation to analyze:

• Disease Severity: Disease index and incidence rates were calculated based on lesion size and leaf coverage.
• Endogenous Hormones: Levels of JA, SA, ABA, GA, IAA, and BR were quantified using HPLC.
• Antioxidant Defense: Activities of SOD, POD, and CAT enzymes were measured, alongside ROS content (O₂⁻ and H₂O₂) via quantitative assays and histochemical staining (NBT and DAB).
• Osmotic Regulation & Membrane Stability: Contents of soluble sugars, soluble proteins, and proline were determined. Relative electrical conductivity (REC) and malondialdehyde (MDA) levels were measured to assess membrane permeability and lipid peroxidation.
• Secondary Metabolites: Glucosinolate profiles (9 components), phenolic acid compounds, and lignin content were analyzed using HPLC and histochemical staining (toluidine blue).
• Statistical Analysis: Data were analyzed using ANOVA and Duncan's test (p<0.05), with correlation matrices generated for hormonal and metabolic interactions.

Key Results

1. Disease Resistance: Both exogenous SA and JA treatments significantly reduced the disease index compared to the control. SA treatment demonstrated the highest efficacy (Disease Index: 29.78, 32.32% reduction), followed by JA (31.56, 28.27% reduction). Notably, the combined JA + SA treatment (Disease Index: 37.78) showed no synergistic effect; its efficacy was significantly lower than either single treatment and closer to the control.
2. Endogenous Hormone Regulation: SA treatment was most effective at maintaining high endogenous SA and BR levels while suppressing ABA accumulation. JA treatment primarily promoted JA accumulation. The combined treatment generally exhibited a weaker regulatory effect on endogenous hormones compared to single treatments.
3. Oxidative Clearance: SA treatment most effectively inhibited the accumulation of O₂⁻ and H₂O₂ and maintained the highest activities of antioxidant enzymes (SOD, POD, CAT). JA treatment was also effective but less so than SA. The combined treatment showed significantly weaker ROS scavenging capabilities than single treatments, particularly in the early stages.
4. Membrane Stability and Osmotic Regulation: SA and JA alone significantly increased soluble proteins, sugars, and proline while reducing MDA and REC, thereby stabilizing cell membranes. The combined treatment failed to significantly enhance soluble protein and sugar accumulation, showing only a weak effect on proline, suggesting antagonism at the osmotic regulation level.
5. Secondary Metabolite Synthesis:
   • Glucosinolates: SA treatment resulted in the highest accumulation of total glucosinolates and specific components (e.g., IBE), followed by JA. The combined treatment was less effective than single treatments.
   • Phenolics and Lignin: SA treatment most strongly promoted the accumulation of phenolic acids (e.g., ferulic, chlorogenic, gallic acids) and enhanced lignin deposition in the xylem. The combined treatment showed significantly weaker induction of these defense metabolites compared to SA alone.

Key Contributions
The study elucidates the physiological mechanisms by which exogenous JA and SA mitigate black rot in cabbage. It confirms that both hormones enhance resistance by regulating endogenous hormone balance, improving oxidative clearance capacity, stabilizing cell membranes, and promoting the synthesis of secondary metabolites (glucosinolates, phenolics, and lignin). A critical contribution is the identification of an antagonistic interaction between JA and SA in this specific context; the combined application did not yield synergistic benefits and was often less effective than single applications, particularly regarding antioxidant enzyme activity and secondary metabolite accumulation.

Significance
The paper concludes that exogenous SA is the more effective agent for enhancing cabbage resistance to black rot under the tested conditions, outperforming JA in regulating chemical and structural defenses. The findings suggest that for green prevention and control strategies, applying SA or JA alone is more effective than combining them. These results provide a theoretical basis and data support for optimizing exogenous hormone regulation strategies to manage cabbage black rot, moving away from chemical pesticides toward induced resistance mechanisms.