Chitosan and carboxylic acids, impact on membrane permeability and DNA integrity of Pseudomonas syringae: interest for biocontrol applications

Abstract Background Pseudomonas syringae causes major damage to a wide variety of plants and is a good model for antibacterial activity assessment. It is important to understand to the fullest extent the mechanisms underlying biobased antimicrobials so as to finely tune them as biocontrol treatments. Chitosan and carboxylic acids (solubilizing agents) are known to be active against phytopathogenic bacteria but the underlying mechanisms are not fully understood. The objective of this study was to assess the impact of pH, concentration, time and the association of chitosan and two types of carboxylic acids—acetic and tartaric acids—on membrane permeability and DNA alteration and the impact on cell inhibition. Results A standard method using 1-N-phenylnaphthalen-1-amine (NPN) and propidium iodide (PI) was used to measure membrane permeability and to assess DNA integrity. Chitosan exhibited a protective effect on permeabilization at higher concentration but was still effective in inhibiting bacteria. The study of pH and concentration showed a wider pH range of permeabilization for acetic acid than pH effect alone, but tartaric acid exhibited a faster DNA alteration at pH3 (30 vs. 90 min for acetic acid, visualized by a decrease in PI fluorescence). The combination of the different molecules showed an additive effect with a decrease of chitosan protection and DNA alteration by tartaric acid even at pH 4. The DNA alteration from the contact with tartaric acid revealed itself to be pH reversible in just an hour. Furthermore, the use of DAPI (4′,6-diamidino-2-phenylindole) showed an opposite effect than with PI in marking DNA of cells exposed to tartaric acid. Conclusions Tartaric and acetic acids showed different kinetics of action on P. syringae. Their mix, together or with chitosan, further highlights the importance of combining different bioactive molecules to have a large range of pH and concentration and limit protective effects due to hindrance of chitosan in high concentration solutions. They also exhibited pH reversible DNA alteration, most likely correlated with the change in conformation, that could lead to a biostatic effect. Graphical Abstract