Multimodal Impact of Bacillus subtilis-derived Zinc oxide and Copper Oxide Nanoparticles on Citrus Biochemistry, Horticultural Attributes and Greening Disease Management

Present research work explored the multimodal function of Bacillus subtilis -mediated zinc oxide and copper oxide nanoparticles towards citrus greening disease, with their impact on the kinnow biochemical response. UV-Vis spectroscopy confirmed nanoparticle formation with surface plasmon resonance peaks at 380 nm (ZnONPs) and 280 nm (CuONPs), while particle sizes averaged 60 nm and 85 nm, respectively. Zeta potentials were − 35 mV (ZnONPs) and − 20 mV (CuONPs), indicating stable colloidal dispersions. XRD analysis confirmed their FCC crystalline nature, and FTIR spectra revealed plant-derived functional groups (O–H, N–H, C = O, C–O) involved in nanoparticle capping. SEM imaging showed irregular spherical ZnONPs (45–55 nm) and rod-shaped CuONPs (70–80 nm). Greenhouse assays showed significant disease suppression, with ZnONPs + CuONPs reducing incidence to 16.95%, outperforming ZnONPs (19.61%) and CuONPs (28.76%). Concentration- and time-dependent reductions under ZnONPs + CuONPs reached 12.84% at 0.75% and 12.06% at 21 days. Field trials confirmed similar trends (25.90% incidence under ZnONPs + CuONPs). Biochemically, ZnONPs at 0.75% enhanced SOD (6.06 µg/g FW), POD (5.98), CAT (5.04), TPC (5.38), TSP (5.85), TSS (6.15), proline (4.99 µmol/g FW), H₂O₂ (4.73), and MDA (5.43). Agronomic traits were also improved at 100 mg/L ZnONPs: juice pH (3.50), juice content (46.46%), vitamin C (16.03 mg/100 mL), fruit weight (213.53 g), diameter (72.29 mm), peel thickness (9.29 mm), and yield (258.03 kg/tree). These findings underscore the promise of biogenic ZnONPs and CuONPs as sustainable, multifunctional tools for integrated disease management and crop health improvement.