Inhibitory Effects and Mechanisms of Juglone Against Walnut Blight Pathogens

This study aimed to determine the antibacterial activity of juglone against the major pathogens causing walnut blight, Xanthomonas arboricola pv. juglandis (Xaj) and Pantoea agglomerans (Pa), and to investigate its mechanism of action, so as to provide a theoretical basis for the green control of walnut blight.

The antibacterial activity of juglone against Xaj and Pa was evaluated in vitro by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Growth curves and resazurin staining were used to characterize the dynamic changes in the viability of Xaj and Pa after juglone treatment. Scanning electron microscopy (SEM) was used to observe alterations in the cell surface and morphology of the two pathogens. Relative electrical conductivity and cell surface hydrophobicity were measured to reveal the effects of juglone on membrane integrity and surface properties. Changes in pathogen protein and nucleic acid electrophoretic profiles were analyzed to assess the interference of juglone with nucleic acids, proteins, and related biological processes. UV absorption spectroscopy and molecular docking were applied to clarify the interaction mode between juglone and pathogen DNA. Leaf inoculation assay was conducted to evaluate the protective effect of juglone against walnut blight on leaves.

Juglone showed obvious antibacterial activity against the major pathogens causing walnut blight, and Xaj was more sensitive than Pa. The MIC values of juglone against Xaj and Pa were 0.1 and 0.4 mg·mL^-1, respectively, while the corresponding MBC values were 0.2 and 0.8 mg·mL^-1. Growth curve analysis showed that juglone inhibited the growth of both pathogens in a concentration-dependent manner. After treatment with 0.5×MIC juglone for 30 min, the cell viability of Xaj and Pa decreased by 35.25% and 37.43%, respectively. SEM observations showed that juglone treatment caused obvious morphological abnormalities in bacterial cells, including shrinkage, depression, and even rupture. After treatment with 0.5×MIC juglone, the relative electrical conductivity and cell surface hydrophobicity of both pathogens increased, accompanied by increased leakage of intracellular nucleic acids, proteins, and other substances, indicating that juglone disrupted cell membrane integrity. Ultraviolet absorption spectroscopy and molecular docking suggested that juglone might interact with bacterial DNA through groove binding mediated by non-covalent interactions such as hydrogen bonding. The detached-leaf assay showed that application of 1×MIC juglone significantly reduced the lesion area on infected walnut leaves, exhibiting good leaf control efficacy.

Juglone showed obvious antibacterial activity against the pathogens causing walnut blight. Its antibacterial effect was mainly achieved by disrupting cell membrane integrity, increasing membrane permeability, and further affecting nucleic acid-and protein-related biological processes. In addition, juglone showed control potential against walnut blight on leaves.