Abstract
Alternaria leaf spot disease is a major constraint in mustard cultivation, causing substantial reductions in crop yield. To control Alternaria leaf spot caused by Alternaria brassicicola, novel silver nanoparticles (AgNPs) and copper nanoparticles (CuNPs) were synthesized using nontoxic, biodegradable guava leaf extracts, which serve as both capping/stabilizing and reducing agent. The green-synthesized CuNPs and AgNPs were characterized and confirmed using ultraviolet–visible spectroscopy, fourier transform infrared spectroscopy, scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy, and X-ray diffraction. Six concentrations of AgNPs (10, 25, 50, 100, 500, and 1000 mg/L) and four concentrations of CuNPs (20, 40, 80, and 100 mg/L) were applied as seed priming treatments. The experiments were arranged in a randomized complete block design with three replications. The in vitro assays assessed antifungal activity against A. brassicicola, while in vivo seed-priming experiments evaluated growth promotion and phytotoxic thresholds. The in vitro study showed that AgNPs (1000 mg/L) and CuNPs (100 mg/L) exhibited significantly higher pathogen-inhibitory activity (99.71% and 95.76%) and induced greater oxidative stress compared with the control group. However, considering plant-nanoparticle interactions, the optimum concentrations of AgNPs and CuNPs were 100 mg/L AgNPs and 40 mg/L CuNPs, which significantly inhibited the growth of A. brassicicola (97.11% and 88.90%). Microscopic observations and biochemical assays confirmed fungal inhibition, as evidenced by hyphal deformation, reduced conidia production, and elevated oxidative stress relative to the control. An in vivo study revealed that AgNPs at 100 mg/L and CuNPs at 40 mg/L exhibited the most pronounced nonphytotoxic effects, significantly improving germination percentage (by 49.51% and 54.66%) compared with the untreated control. Moreover, seed priming with 100 mg/L AgNPs and 40 mg/L CuNPs markedly increased shoot length (37.93% and 35.34%), root length (105% and 40%), dry weight (67.80% and 59.15%), total chlorophyll content (93.81% and 81.45%), protein content (28.46% and 36.15%), and total soluble sugar (18.40% and 9.91%) relative to the control. Furthermore, seed priming with 100 mg/L AgNPs and 40 mg/L CuNPs was identified as the optimal treatment, effectively modulating reactive oxygen species (hydrogen peroxide and malondialdehyde) while enhancing antioxidant enzyme activities (ascorbate peroxidase, peroxidase, catalase, and 2,2-diphenyl-1-picrylhydrazyl radical scavenging capacity). These findings suggest that seed priming with CuNPs and AgNPs provides a promising strategy for managing Alternaria leaf spot in mustard while promoting healthy seedling growth.