Towards a rapid diagnostic method to identify bacteria associated with AOD

Oak trees native to Britain, Quercus robur and Quercus petraea, are under the threat from a disease called Acute Oak Decline (AOD). AOD was first reported in Britain about thirty years ago and is significantly increasing in incidence in the last several years. Affected trees have also been reported in several other countries in Europe such as Spain, France and Germany. The symptoms of infection are necrotic, longitudinal, bleeding cracks in the bark, from which emanates dark fluid. Severe cases are usually lethal within 4 to 5 years. Larval galleries of the buprestid beetle Agrilus biguttatus are regularly associated with the necrotic tissue.It is believed that the syndrome has a polymicrobial origin. Over the past decade, many strains have been isolated and identified. The predominant pathogens belong to the novel species Gibbsiella quercinecans and Brenneria goodwinii but also undescribed species included in the family Pseudomonadaceae have been isolated, although their role in the disease is not yet clear.Before identification and diagnostic methods for the bacteria isolated from symptomatic oak can be optimised, it is necessary to know in detail the species involved in the infection, and their roles. To determine the phylogenetic position of the undescribed Pseudomonads, 16S rRNA sequencing of the strains was performed on strains from symptomatic tissue. Three protein-encoding genes (rpoB, rpoD and gyrB) were also sequenced as part of a multilocus sequence analysis (MLSA) study for a more robust taxonomic classification. The phylogenetic analyses indicate that several possible novel species of Pseudomonas are associated with AOD in Britain. Further work, including phenotypic, genotypic and hypersensitivity response assays, will be performed to formally classify these strains in the genus Pseudomonas.