Integrated histochemical, biochemical, and ultrastructural analyses elucidate synergistic virulence mechanisms of Curvularia lunata in maize

Curvularia lunata is a destructive maize pathogen, but the interplay between its infection strategy and toxin production remains unresolved. We identified C. lunata using multilocus sequence analysis of ITS rDNA, GAPDH, and TEF1α regions and profiled its metabolites, revealing methyl-5-hydroxyfuran-2-carboxylate (M5HF2C) as the dominant toxin along with citrinin, endocrocin, and butyrolactone II. Maize plants (7-8 days old) exposed to pathogen inoculum, crude metabolite extract, or purified M5HF2C exhibited progressive necrotic and chlorotic lesions. Physiological assays demonstrated severe pigment loss (up to 79%), electrolyte leakage (88%), and lipid peroxidation (9.7-fold increase), while histochemical staining and ultrastructural imaging confirmed ROS accumulation and cellular collapse. Antioxidant and defense enzymes (SOD, CAT, APX, POX, GR, GPX, PPO, and PAL) were strongly induced but insufficient to prevent damage. Notably, the severity of effects followed the order pathogen > crude metabolite > purified toxin, underscoring the synergistic contribution of multiple virulence factors to pathogenesis. Together, these results establish oxidative stress and combined metabolite action as the central drivers of Curvularia virulence in maize.