Mechanistic Understanding of Antibiofilm Strategies of the Bioinspired Nanopesticide CuO NPs toward <i>Ralstonia solanacearum</i>

As novel nanopesticides, cupric oxide nanoparticles (CuO NPs) have emerged as a cost-effective, ecofriendly, and sustainable alternative for controlling plant pathogens. However, additional research effort is still needed to elucidate the antibacterial mechanism involved. In this study, bioinspired CuO NPs were synthesized, and their antibiofilm strategies against R. solanacearum were systematically investigated. CuO NPs effectively inhibited the biofilm formation of R. solanacearum at various stages of maturity (24, 48, and 72 h) by damaging the cellular morphology and reducing the extracellular polysaccharide (EPS) and protein content of bacteria within biofilms. The motility activities of R. solanacearum, including swimming, swarming, and twitching, were significantly inhibited upon exposure to CuO NPs. Furthermore, we confirmed that both two-dimensional and three-dimensional structures of mature biofilms (at 24 h) were disrupted, as determined by confocal laser scanning microscopy (CLSM), atomic force microscopy (AFM), and scanning electron microscopy (SEM), revealing a scattered morphology and a disrupted surface topology pattern. Importantly, the quantitative reverse transcription polymerase chain reaction (qRT-PCR) was employed to assess the transcriptional levels of genes related to biofilm formation and virulence in R. solanacearum stems following treatment with CuO NPs. Notably downregulated genes included those involved in chemotaxis (cheA and cheW), EPS-related genes (xpsR and epsE), swimming activity (flgC, fliA), the quorum-sensing (QS) system (solR, phcB, phcS), the type III system (T3SS) (prhI and hrpG), and the two-component system (pehS and pehR). These findings provide insight into the antibiofilm properties of CuO NPs and hold promise regarding their potential as nanoenabled strategies for combating pathogens and sustainable management of crop diseases.