The taxonomic and genomic investigation of soil microorganisms associated with Acute Oak Decline

Cases of the current episode of Acute Oak Decline (AOD) were first described in the UK in 2008 on native British oaks, but currently this disease is seen to affect most species of oak on a global scale. The disease has become a focus of forest research, due to the large number of abiotic and biotic components that contribute to AOD. Some of the most recent discoveries have shown the rhizosphere microbiome may play a significant role in the disease.As such the work presented here utilised ten healthy and ten AOD symptomatic trees spread over the woodland and parkland areas of Hatchlands Park, Guilford, UK to look for functional differences within their rhziosphere. The first aim was to investigate if the rhizosphere soil could function as a reservoir of infection for the four bacteria currently associated with AOD, by using microbiological and rapid diagnostic molecular methods to detect their presence. The second aim was to isolate and classify potentially novel members of the Enterobacteriaceae isolated from the rhizosphere of both healthy and diseased oak. The final aim was to use 16S rRNA gene sequence analysis to identify differences in the microbiome community composition of the oak rhizosphere.Using a modified Enterobacteriaceae enrichment method followed by undiluted spread plating on a range of media, HRM was used to test for AOD bacteria from both healthy and diseased oak rhizosphere samples. Gibbsiella quercinecans and Rahnella victoriana were identified in the rhizosphere microbiome of healthy and diseased oak using high resolution melt analysis, but Brenneria goodwinii and Lonsdalea britannica were not. However, B. goodwinii and L. britannica were identified in acorns from the same site, indicating that they may be endophytic members of the seed microbiome which is inherited between generations of oak.Using the same enrichment method, 100’s of isolates were collected from the same rhizosphere samples taken from Hatchlands and the use of polyphasic taxonomy allowed for the identification of two novel genera of bacteria and nine new species. These novel species appear to be potential plant pathogens and growth-promoting bacteria based on the genomic comparisons. This highlights the untapped potential that the oak rhizosphere microbiome offers in relation to bacterial taxonomy.Finally, single gene community analysis via synthetic long read 16S rRNA gene sequencing was performed on DNA isolated from the Hatchlands rhizosphere samples. The analysis of sequencing data revealed important distinctions between samples, with the largest effect being seen between woodland and parkland samples. Parkland healthy and diseased trees presenting the second largest effect with different relative abundance and microbiome composition. However, this effect was not seen between single healthy and diseased trees with minimised sampling distances, though a difference between the exorhizosphere and endorhizosphere was recorded for both diseased and healthy trees. These differences indicate that the composition of the rhizosphere may be linked to health status of oak, and that the difference seen between the endosphere of diseased and healthy oak may be influenced by which bacteria are recruited from the rhizosphere