The ROS-triggered antibacterial mechanism of zinc oxide nanoparticles against Xanthomonas citri subsp. citri for citrus canker management

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), severely impacts citrus production. This study developed a novel zinc oxide nanoparticles ROS-triggered generation system (ZnO NPs) as an effective bactericidal agent against Xcc. The ZnO NPs exhibited higher antibacterial activity than the commercially available pesticide zinc thiazole. The minimum inhibitory concentration (MIC) of ZnO NPs (104.5 μg/mL) is 7.42 times higher than that of 104.5 μg/mL zinc thiazole. Additionally, 1 MIC of ZnO NPs and 1 MIC of zinc thiazole (690.5 μg/mL) exhibited similar therapeutic efficiency, with disease incidences of 3.75 ± 2.04% and 3.33 ± 0.59%, respectively. As the concentration of ZnO NPs increased from 0 to 104.5 μg/mL, Xcc fluorescence density increased from 4377 ± 266.48 to 20,158.67 ± 3133.39 a.u. The ZnO NPs significantly reduced the activity of two key citrate cycle (TCA cycle) enzymes, malate dehydrogenase and succinate dehydrogenase, and disturbed multiple ROS-related metabolic pathways, such as TCA cycle, glutathione metabolism, and ABC transporters. Metabolites associated with ROS-scavenging and cell membrane synthesis, such as carnosine, biotin, valeric acid, and adrenic acid, were significantly downregulated. Pantothenate and CoA biosynthesis, tryptophan metabolism, arginine and proline metabolism, and arginine biosynthesis were identified as potential target pathways. These results suggest that the antibacterial activity of ZnO NPs is mediated by enhanced ROS accumulation, metabolic disruption, and cell membrane damage, leading to leakage of intracellular contents. Moreover, ZnO NPs enhanced citrus disease resistance. This study demonstrates the ZnO NPs as a promising antibacterial agent against Xcc and elucidates its antibacterial mechanisms.