Comparative efficacy of methylated and WPC-microencapsulated OligoDNAs for controlling Verticillium dahliae through gene silencing

The practical application of synthetic oligonucleotides (OligoDNAs) for fungal disease control is limited by inefficient delivery into fungal cells. This study evaluated two delivery-enhancement strategies-chemical methylation and whey protein concentrate (WPC) microencapsulation-to improve the antifungal efficacy of OligoDNAs targeting the Verticillium dahliae genes Clp-1 and HiC-15, which are naturally regulated by plant miRNAs miR166 and miR159. Physicochemical analyses confirmed the formation of stable, spherical WPC microcapsules (≈ 0.2-0.36 μm) encapsulating OligoDNAs. Fluorescence microscopy showed that Cy3-labeled oligoDNA was associated with fungal hyphae, generating discrete punctate fluorescence signals that may indicate localized association with fungal structures. However, due to the limitations of the imaging method, definitive evidence of intracellular localization or uptake cannot be concluded, and higher-resolution imaging techniques are required for confirmation. Both delivery strategies significantly suppressed target gene expression in vitro, with the encapsulated formulation inducing stronger downregulation than the methylated one (p < 0.05). Correspondingly, the capped formulation caused the greatest reduction in fungal colony growth. In tomato bioassays, both treatments reduced Verticillium wilt severity, but the capped formulation provided superior protection, significantly lowering AUDPC compared with the methylated treatment and pathogen control (p < 0.001), while WPC alone had no effect. These findings suggest that delivery strategy influences OligoDNA efficacy under the evaluated conditions. WPC microencapsulation showed enhanced antifungal activity compared with chemical methylation under the conditions tested by enhancing antifungal activity, highlighting its potential as a sustainable platform for nucleic acid-based management of soil-borne fungal pathogens.