Antibacterial mode of action of thyme white (Thymus vulgaris L.) essential oil and its constituents, thymol and carvacrol against Agrobacterium tumefaciens via down-regulation of manganese transport genes, sitABCD and mntH.

In this study, we evaluated the antibacterial activities of plant essential oils (EOs) from the Lamiaceae family against Agrobacterium tumefaciens to find new eco-friendly antimicrobials. Thymus vulgaris L. (thyme white) EO demonstrated the most potent fumigant antibacterial activity among these. Key compounds identified in thyme white EO, including thymol, carvacrol, S-(-)-α-pinene, R-(+)-α-pinene, and (-)-limonene, also exhibited strong fumigant antibacterial activity against A. tumefaciens. The inhibition zone diameters observed for thymol, carvacrol, R-(+)-α-pinene, S-(-)-α-pinene, thyme white EO, and S-(-)-limonene were 2.85, 2.74, 1.64, 1.55, 1.15, and 0.86 cm, respectively, at a concentration of 5 mg per paper disc. Only thyme white EO, thymol, and carvacrol exhibited contact antibacterial activity against A. tumefaciens. The minimum inhibition concentrations (MIC) of thyme white EO, thymol, carvacrol, and streptomycin were 800 μg/mL, 350 μg/mL, 350 μg/mL, and 3.125 μg/mL, respectively. Using confocal laser scanning microscopy, we observed intracellular reactive oxygen species (ROS) production and membrane damage in A. tumefaciens treated with thyme white EO, thymol, and carvacrol. Additionally, we analyzed the differential gene expression level in A. tumefaciens treated with thyme white EO, thymol, and carvacrol, comparing them to untreated cells to elucidate the mode of action. In particular, the expression of manganese transport-related genes (sitABCD and mntH) was significantly downregulated, which impaired the function of sod. As a result, the system responsible for detoxifying ROS was disrupted. This led to excessive accumulation of ROS, causing damage to the cell membrane.