Copper tolerance in Pseudomonas syringae pv. tomato isolates from tomato crops in Eastern Australia

Pseudomonas syringae pv. tomato causing bacterial speck disease in tomatoes is a significant threat to commercial field tomato production in most growing regions of Australia and globally. Infection of crops with this pathogen can cause significant reductions in fruit quality and yields. There are limited pesticide control options available for bacterial diseases in tomato, with copper-based bactericides currently one of the few registered products globally. The state of Queensland (QLD) in Australia produces approximately 69% of Australia’s fresh market outdoor tomatoes estimated at a value of AUD$122 billion. P. syringae pv. tomato consistently threatens tomato production in QLD and other Eastern Australian states and many producers report copper products fail to adequately control disease progression. To date no studies have tested for copper tolerance in P. syringae pv. tomato in QLD Australia, despite reports of tolerance in many other countries. This study found that 100% of the P. syringae pv. tomato isolates collected were tolerant to copper and this tolerance was linked to the presence of cop genes in their genetic profiles. This is the first systematic study of copper tolerance prevalence in Eastern Australia, particularly QLD, and the first study analysing the genetic basis of copper tolerance in Australian P. syringae pv. tomato. Published copper tolerance and copper efficacy studies on bacterial disease control report a wide range of response data, generated under varying field and laboratory conditions, making it difficult to draw strong conclusions from individual studies. Therefore, a systematic literature review was completed, investigating the prevalence of copper tolerance, the relative efficacy of copper for the control of disease and the identification of key emerging alternative products to copper for disease control. Results highlighted that copper tolerance is a global issue, which is affecting the usefulness of copper-based products for the control of bacterial diseases. A large range of alternative products for disease control were identified and the efficacy of eight key products were evaluated. However, there was a limited volume of published efficacy data available of for these alternative products, particularly for the control of disease caused by P. syringae pv. tomato. The systematic literature review also identified inconsistencies with in vitro copper tolerance screening methodology for P. syringae pv. tomato in current literature, particularly in relation to the appropriate media to use, copper tolerance thresholds and inadequate reporting of media pH and/or pH adjustment steps. The effect of media and pH on copper tolerance results was therefore investigated, including the use of a pH buffering agent. Copper tolerance thresholds with different media were found to vary significantly and outside of a specific pH range, copper tolerance data was unreliable. A recommended methodology for copper tolerance screening was developed and published. This refined methodology was used to screen P. syringae pv. tomato isolates from a number of geographically distinct regions of QLD, New South Wales (NSW) and Victoria (VIC). To date, no published studies are available on copper tolerance stability in P. syringae pv. tomato. Understanding the stability or biological fitness of copper tolerance in P. syringae pv. tomato can provide valuable insights into how copper-based disease control programs could be modified to mediate or even reduce the prevalence copper tolerance. Therefore, the stability of copper tolerance in study isolates was investigated through in vivo experiments. Findings suggested that copper tolerance may not be stable in all isolates when copper selection pressure is removed in vivo. Despite a general consensus that plasmid cop genes are essential for copper tolerance in P. syringae pv. tomato, the gene and protein characterisation work undertaken to form these conclusions is solely based on isolates collected in America. Additionally, Australian P. syringae pv. tomato are yet to be genetically characterised. Polymerase Chain Reaction (PCR) assays and genomic analysis were used to explore the genetic basis of copper tolerance in Australian isolates, with a particular focus on the cop genes. Genetic analysis identified putative Cop-protein coding regions on a Cop operon and a CopA/B complex in Australian isolates. The analysis also suggested that the Cop operons may be located on either plasmid or chromosomal DNA, depending on the isolate studied. This study is the first detailed investigation of the genetic basis of tolerance in this species outside of America. This study presents a range of novel findings which are of significance to both the scientific community and the agricultural industry. The presence of widespread copper tolerance has serious implications for commercial tomato producers. Bacterial disease management programs need to be revised to mediate resistance development and provide a more environmentally sustainable approach to crop production.