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Controlling of biofilms formation in numerous pathogenic bacteria is one of the most difficult tasks in the control of bacterial diseases. Plasma has attracted extensive attention due to their potential applications for effective inhibiting of biofilm. Recently, plasma-activated water (PAW) has developed as an alternative method for bacterial inactivation and disinfection of foods owing to advantages of more convenient and efficient storage and transportation than direct plasma application. However, most previous studies about PAW have only focused on the improvement of its antibacterial effect instead of antibiofilm activity. Therefore, we report the development of N2 plasma treated buffer (NPB) and the super-antibiofilm effect of NPB against Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) as a plant pathogenic bacterium. Scavenger assays using various antioxidants revealed that reactive oxygen species were involved in the inhibitory cellular actions of NPB, with H2O2 and singlet oxygen proving essential for bacterial death. Intensive analysis of NPB, stored at different periods and temperatures, showed that the antimicrobial efficacy was well maintained for 3 months at − 80 °C. Importantly, further studies showed that NPB effectively inhibited not only the growth of planktonic Pst DC3000 but also biofilm formation. The remarkable inhibition on the biofilm was analyzed and visualized using LIVE/DEAD viability assays and confocal laser scanning microscopy (CLSM) imaging. The 3D CLSM imaging data revealed that the bactericidal activity of NPB was permeable enough to affect the cells embedded inside the biofilm. This prominent permeability could be a crucial feature of NPB contributing to effective super-antibiofilm. [ABSTRACT FROM AUTHOR]

Suppression of pathogenic bacterial growth to increase food and agricultural productivity is important. We previously developed novel hexapeptides (KCM12 and KCM21) with antimicrobial activities against various phytopathogenic bacteria and N 2 plasma-treated buffer (NPB) as an alternative method for bacterial inactivation and as an antibiofilm agent of crops. Here, we developed an enhanced antibiofilm method based on antimicrobial hexapeptides with N 2 plasma-treated buffer against plant pathogens. Our results demonstrated that hexapeptides effectively inhibited the growth of Pseudomonas syringae pv. tomato DC3000 ( Pst DC3000) and the biofilm it formed. Potent biofilm formation-inhibiting effects of hexapeptides were observed at concentrations of above 20 µM, and samples treated with hexapeptide above 100 µM reduced the ability of the bacteria to produce biofilm by 80%. 3D confocal laser scanning microscopy imaging data revealed that the antimicrobial activity of hexapeptides was enough to affect the cells embedded inside the biofilm. Finally, combination treatment with NPB and antimicrobial hexapeptides increased the antibiofilm effect compared with the effect of single processing against multilayered plant pathogen biofilms. These findings show that the combination of hexapeptides and NPB can be potentially applied for improving crop production.