Nanoformulated thymol loaded chitosan conjugates as potent antibacterials against Xanthomonas oryzae pv. oryzae

Nanomaterial-based antibacterial systems offer new opportunities for the control of phytopathogenic bacteria through targeted, multi-level cellular disruption. Herein, this study synthesizes and characterizes the thymol-loaded chitosan nanoparticles (TCNPs) using a variety of physicochemical and antimicrobial efficacy techniques against Xanthomonas oryzae pv. oryzae (Xoo), the causative agent of bacterial leaf blight in rice.

Physicochemical characterization confirmed efficient thymol encapsulation, nanoscale particle size, stable surface properties and morphology suitable for bacterial interaction. TCNPs exhibited strong antibacterial activity, reflected in the concentration-dependent reduction of bacterial growth coupled with metabolic viability and its decrease to ~ 60% at sub-lethal concentration (1/2 MIC). Further trypan blue staining revealed a massive increase in membrane-compromised Xoo cells upon TCNPs treatment, an indication of early loss of membrane integrity. Further, the exposure to TCNPs induced oxidative stress as evidenced by the elevated intracellular reactive oxygen species (ROS) and significantly increased lipid peroxidation, as shown by higher malondialdehyde (MDA) levels (44%) at 532 nm. Moreover, FTIR analysis demonstrated clear alterations in membrane lipid vibrations, protein secondary structures, and cell-wall carbohydrate regions that confirmed the structural destabilization of Xoo cells. The untargeted LC-MS profiling supported these spectral findings through the loss of intact phospholipids, appearance of oxidized lipid fragments, and depletion of some amino-acid signatures in treated samples. These molecular and biochemical changes together suggest that TCNPs disrupt the bacterial membrane and induce ROS-mediated oxidative damage leading to metabolic imbalance and loss of cell viability.

The overall study depicts the multifaceted antibacterial mechanism of TCNPs via membrane destabilization, oxidative lipid damage, and metabolic disruption in Xoo. These findings unravel the potential of TCNPs as a novel nanobiotechnological approach for the sustainable management of rice bacterial leaf blight.