Optimising protocols to assess the efficacy of ZnO nanoparticles as antifungal agents

Several types of fungal growth inhibition assays have been developed in the past for synthetic antifungal compounds. However, most of the traditional antifungals have now been reviewed or banned in postharvest disease management. A class of materials which have recently received considerable attention as antifungals are metallic nanoparticles. Antifungal tests, therefore, need to be optimised in order to be suitable for this new category of compounds. The properties of Zinc Oxide nanoparticles (ZnO NPs) at concentrations ranging from 0 mM up to 18 mM, and in some cases up to 30 mM, were assessed against selected fungal isolates, i.e., Aspergillus niger and Aspergillus terreus, by: i) agar disk-diffusion test, ii) mycelium growth inhibition, and iii) turbidimetry techniques. All the techniques were investigated by making the proper adjustments to the protocols and to the experimental design, in order to obtain the most reproducible and consistent results. Potato Dextrose Agar obtained from raw potatoes was used for all the techniques, fungal spore suspensions were prepared starting from 10⁵ spores mL^‑1. The binary response (growth/no growth) and the radial growth against time were recorded for the disk-diffusion and the mycelium inhibition test respectively; while, for the turbidimetric assay, plots of optical density (O.D.) against time were produced. The diameter growth rate and the O.D. were analysed by mathematical prediction models. This was performed by nonlinear regression analysis. The obtained results demonstrate that methods such as the disk-diffusion assay may be applied to nanoparticles as a preliminary in situ screening the inhibitory effect of NPs. Techniques such as mycelium growth inhibition and turbidimetric assay can be considered and complemented by predictive mycology tools to quantitatively compare the efficacy of NPs against different fungi.