Advanced bifunctional nanotherapeutics display multi-tiered defense against Magnaporthe oryzae through targeted fungal inhibition and rice immunity enhancement

Rice blast disease, caused by Magnaporthe oryzae (Mo), severely threatens global rice production. In this study, biogenic copper nanoparticles (bio-CuNPs) were synthesized extracellularly using Bacillus amyloliquefaciens Q1 and characterized for antifungal activity and plant defense induction. Bio-CuNPs (16-62 nm, average 37 nm) exhibited potent antifungal effects by significantly inhibiting Mo mycelial growth, conidial germination, and appressorium formation in a dose-dependent manner. Microscopic observations revealed that bio-CuNPs disrupted Mo hyphal integrity, caused intracellular leakage, and induced DNA damage. Transcriptomic profiling identified key regulatory genes in Mo upon CuNP exposure, with key disruptions in cell wall biosynthesis (MoCHS-A, MoCHS-B, MoCHS-C, and MoCHS-D) and membrane transport pathways (MoMFS-1 MoMFS-2, and MoMSC-2). Deletion mutants for these genes demonstrated heightened sensitivity to bio-CuNPs, indicating that these genes are critical for helping Mo to withstand the antifungal effects of bio-CuNPs. However, bio-CuNPs disrupted their functions in Mo, confirming these genes as one of the molecular targets to suppress Mo growth and virulence. In planta assays revealed that bio-CuNP foliar application reduced disease severity, improved plant growth, and activated antioxidant enzymes, while suppressing oxidative stressors (i.e., super oxide radicle and hygrogen peroxide). Metabolomic analysis revealed significant alterations in defense-related pathways, including phenylpropanoid and amino acid metabolism. Additionally, CuNPs enhanced salicylic acid and methyl jasmonate levels, which subsequently upregulated defense gene expression. Cytotoxicity assays revealed that bio-CuNPs were non-toxic to AML12 cells at effective concentrations, highlighting their potential as an eco-friendly strategy for sustainable rice blast management.