Media pH and media type can significantly affect the reliability of in vitro copper tolerance assessments of Pseudomonas syringae pv. tomato

Aims: There are inconsistencies with in vitro copper tolerance screening methodology for Pseudomonas syringae pv. tomato in the current literature, particularly in relation to the appropriate medium to use, copper tolerance thresholds and reporting medium pH and/or pH adjustment steps. This study investigates the effect of medium and pH on copper tolerance results, including the potential use of 2-(N-morpholino)ethanesulfonic acid (MES) buffer to stabilize medium pH. Methods and Results: Copper tolerance methodology was investigated through in vitro and in vivo testing of P. syringae pv. tomato. Four different media were tested, nutrient agar, Casitone yeast extract glycerol agar, King's B medium and potato dextrose agar. Highly variable copper tolerance profiles were observed for different isolates on the media tested. A pH range of 5·8–7·0 produced consistent copper tolerance data; outside of this range the data were unreliable. Addition of MES to media buffered the pH to within the acceptable levels. Conclusions: Copper tolerance thresholds with different media can vary significantly and the lowering effect of copper sulphate on media pH must be considered in media preparation. Significance and Impact of the Study: Methodology presented in the study can be extrapolated to copper tolerance testing for other pathogenic plant bacteria, particularly other pseudomonads. © 2018 The Society for Applied Microbiology