Melatonin and copper oxide nanoparticles synergistically mitigate clubroot disease and enhance growth dynamics in Brassica rapa

Clubroot, a devastating soil borne disease affecting 30%similar to 50% of Brassicaceae crops worldwide, lacks effective control measures. In the present study, we explored the potential of melatonin (MT) and copper oxide nano- particle (CuO-NPs) in mitigating clubroot severity in the Brassica rapa ssp. pekinensis. Following 18 h priming with MT, CuO-NPs, or both seeds were grown in controlled environment using synthetic potting mix. Inoculated with Plasmodiophora brassicae spores on 5th day, followed by a soil drench phyto-nano treatment with a week interval. Plants were assessed for various health and growth indices including disease, biometrics, photosynthesis, reactive oxygen species (ROS), antioxidant enzyme activity, hormones and genes expression at onset of secondary clubroot infection using established protocols. Statistical analysis employed ANOVA with Fisher's LSD for significance assessment (P < 0.05). Our results revealed that seed priming with both MT (50 mu Mol/L) and CuO-NPs (200 mg/L), followed by soil drenching significantly reduced clubroot incidence (38%) and disease index (57%), compared to control treatments. This synergistic effect was associated with enhanced plant growth (shoots: 48% and roots: 59%). Plants treated with both MT and CuO-NPs showed robust antioxidant defenses, significantly increased superoxide dismutase (SOD (25/29%)), catalase (CAT (83/55%)), and ascorbate peroxidase (APX (83/46%)) activity in both shoots/roots, respectively, compared to infected control. Notably, salicylic acid and jasmonic acid levels doubled in treated plants, while stress hormone abscisic acid (ABA) decreased by 80% in roots and 21% in shoots. Gene expression analysis corroborated these findings, showing that the combined treatment activated antioxidant defense genes (SOD, APX and CAT) by 1.9-7.2-fold and upregulated hormone signaling genes JAZ1 (7.8-fold), MYC2 (3.9-fold) and SABP2 (36-fold). Conversely, ABA biosynthesis genes (ABA1 and NCED1) were downregulated up to 7.2-fold, while plant resistance genes NPR1, PRB1 and PDF1.2 were dramatically increased by up to 6.3-fold compared to infected plants. Overall, our combined treatment approach significantly reduces clubroot severity in B. rapa via enhanced antioxidant defenses, improved ROS scavenging, coordinated hormonal regulation and increased pathogen response genes. This study offers promising strategy for developing effective control measures against clubroot in susceptible cruciferous crops.