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

Rice blast disease, caused by <i>Magnaporthe oryzae</i> (<i>Mo</i>), severely threatens global rice production. In this study, biogenic copper nanoparticles (bio-CuNPs) were synthesized extracellularly using <i>Bacillus amyloliquefaciens</i> 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 <i>Mo</i> mycelial growth, conidial germination, and appressorium formation in a dose-dependent manner. Microscopic observations revealed that bio-CuNPs disrupted <i>Mo</i> hyphal integrity, caused intracellular leakage, and induced DNA damage. Transcriptomic profiling identified key regulatory genes in <i>Mo</i> upon CuNP exposure, with key disruptions in cell wall biosynthesis (<i>MoCHS-A</i>, <i>MoCHS-B</i>, <i>MoCHS-C</i>, and <i>MoCHS-D</i>) and membrane transport pathways (<i>MoMFS-1 MoMFS-2</i>, and <i>MoMSC-2</i>). Deletion mutants for these genes demonstrated heightened sensitivity to bio-CuNPs, indicating that these genes are critical for helping <i>Mo</i> to withstand the antifungal effects of bio-CuNPs. However, bio-CuNPs disrupted their functions in <i>Mo</i>, confirming these genes as one of the molecular targets to suppress <i>Mo</i> growth and virulence. <i>In planta</i> assays revealed that bio-CuNP foliar application reduced disease severity, improved plant growth, and activated antioxidant enzymes, while suppressing oxidative stressors (<i>i.e.</i>, 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.