Antifungal activity of biosynthesized copper oxide nanoparticles (CuONPs) against three different formae speciales of Fusarium oxysporum.

Fusarium oxysporum is a major pathogen of economically important crops, including strawberries, blackberries, tomatoes, and many others. Conventional fungicides are often ineffective, and their excessive use poses risks to health and the environment. As an alternative, copper oxide nanoparticles (CuONPs) synthesized through eco-friendly methods have shown promising antifungal activity. To identify the optimal reducing agent for nanoparticle synthesis, this study evaluated supernatants from Trichoderma harzianum cultured at different temperatures against F. oxysporum. The supernatant obtained at 30 °C exhibited the highest intrinsic inhibitory activity (15.5%), followed by 33 °C (11.6%) and 25 °C (5%). The extract produced at 30 °C was selected and used for CuONPs biosynthesis to obtain a synergistic effect. CuONPs formation was confirmed by a UV-vis absorption peak at 290 nm. Dynamic light scattering analysis revealed a hydrodynamic diameter of 743.9 ± 150.2 nm, a Zeta potential of - 31 ± 4.48 mV, and a polydispersity index of 0.544. Using an automated machine-learning segmentation approach, transmission electron microscopy combined with machine-learning segmentation, showed nanoparticles with a mean Feret diameter of 2.73 ± 0.90 nm and were predominantly quasi-spherical in shape with a mean circularity of 0.80 ± 0.13. The biosynthesized CuONPs showed concentration-dependent antifungal activity across the tested range (0.357-0.716 mg/ml), with the highest inhibition (100%) observed at 0.716 mg/mL against F. oxysporum f. sp. fragariae (Fof), 90.98% against F. oxysporum f. sp. mori (Fom), and 93.52% against F. oxysporum f. sp. lycopersici (Fol). These findings demonstrate that CuONPs biosynthesized using temperature-optimized T. harzianum supernatants act as a highly effective synergistic nanobiopesticide (achieving 90.98%-100% inhibition at 0.716 mg/mL) across multiple F. oxysporum formae speciales, serving as a novel and sustainable alternative to chemical fungicides.