Towards a rapid diagnostic method to identify bacteria associated with AOD

British oaks, Quercus robur and Q. petraea are threatened by Acute Oak Decline (AOD). Severe cases are lethal within five years. Symptoms include bleeding, longitudinal, necrotic cracks in the bark, from which a dark fluid emanates. AOD is caused by the interaction of several bacterial species, with Brenneria goodwinii and Gibbsiella quercinecans being the predominant pathogens. Species belonging to the Enterobacterales and Pseudomonadaceae are also routinely isolated from symptomatic trees. To further investigate the bacteria associated with AOD, several novel species of Pseudomonas have been identified and described following a polyphasic approach. Their potential role in AOD was investigated through the genomic analysis of virulence factors, hypersensitive response tests in bean pods and tobacco leaves, and cell attachment assays. The study of the taxonomic relationships between the novel Pseudomonas and its neighbours led to a taxonomic reassessment of this polyphyletic genus. The polyphasic approach and phylogenetic analysis suggested that the phylogenetic boundaries within Pseudomonas and between Pseudomonas and neighbouring genera: Azomonas, Azotobacter and Azorhizophilus (collectively known as the “Azotobacter group”) were not well defined.Three novel AOD-associated Pseudomonas were described in this project: Pseudomonas daroniae sp. nov. Pseudomonas dryadis sp. nov., and Pseudomonas kirkiae sp. nov. These novel Pseudomonas do not present a typical phytopathogenic set of genomic or phenotypic features. The phylogenetic study of the Azotobacter group suggested that Pseudomonas, Azotobacter and Azomonas should remain separated; and in turn, the polyphyletic Pseudomonas should be divided into several novel genera, conserving as Pseudomonas only those belonging to the P. aeruginosa lineage. Many field samples of oaks affected by AOD must be processed on a daily basis, requiring a fast and cost-effective method of detection and identification. A multiplex high-resolution melt (HRM) analysis has been developed for the identification of AOD indicators: B. goodwinii, G. quercinecans, Rahnella victoriana and Lonsdalea britannica. HRM analysis is a real-time PCR-based technique in which single nucleotide polymorphisms can be identified in amplicons, without DNA sequencing. The multiplex HRM assay can identify up to four targets from field samples, in a single tube, in just 40 minutes per HRM run. For optimal detection, the sample must be incubated anaerobically in solid media to stimulate the growth of the targets, which are facultative anaerobes. The colonies can be used directly as a template for HRM without DNA extraction. Additionally, several tools designed to work with pure cultures were developed, including: a duplex-HRM assay of the genes atpD and rpoD for the identification of AOD-associated bacteria including the novel AOD-associated Pseudomonas; and three SNP-based HRM assays for the differentiation of species and subspecies of the AOD-associated genera Gibbsiella, Brenneria and Rahnella. The HRM-based diagnostic methods will potentially be useful tools for research institutions working with AOD and will save time and resources in the fight against the disease