A Systems Approach to Investigating Acute Oak Decline in Britain

Sandra Denman

Oak Decline is considered a complex disease syndrome of native oak, which has received particular, episodic attention in Europe from the 1900s onwards. In the 1980s in Britain, native oak trees with distinctive stem weeping symptoms were first noticed and increasing reports over time raised concerns about cause and effects on these iconic trees. Symptomology studies revealed bark necrosis and vascular cambial tissue degradation underlying the weeping patches suggesting a microbial component, as well as >90% co-occurrence of larval galleries of the bark boring buprestid, Agrilus biguttatus (Coleoptera: Buprestidae) implicating it in the condition. The study confirmed that it is a distinctive disease now called Acute Oak Decline (AOD), with four diagnostic symptoms. 

To investigate possible causes of AOD spatial epidemiology studies were initiated to determine spatiotemporal patterns of outbreaks, and conventional isolation and 454 NGS targeted metagenomic studies on healthy and symptomatic trees were carried out to detect putative microbial elements. The spatial studies recorded important epidemiological information and demonstrated local clustering of affected trees. Overall results of microbial studies demonstrated corroboration of the different approaches and highlighted two novel species of bacteria, Brenneria goodwinii (Bg) and Gibbsiella quercinecans (Gq) consistently associated with necrotic tissue, suggesting a role in lesion formation. Recognition of the possible involvement of multiple agents led to the hypotheses that (i) Bg and Gq have roles in causing necrosis and degradation of oak phloem and sapwood, (ii) interaction between A. biguttatus and these bacteria will lead to typical AOD symptoms, (iii) AOD is a complex Decline disease (as opposed to a primary disease) dependent upon the interaction of multiple factors for disease establishment.   

To address these hypotheses Systems research approaches are being adopted at different scales from molecule to landscape, cross-linking disciplines and results to produce integrated knowledge and contribute to deciphering key elements, their function and effects of interactions in this complex disease.  The overall research plan will be outlined demonstrating that as far as finances allow, a holistic view of the problem and how to tackle it is being followed.

Microbiome analysis of UK oak; towards an understanding of Acute Oak Decline in the UK

James E. McDonald, Emma Ransom-Jones, James Doonan, Justin I. Pachebat, Peter N. Golyshin, Sandra Denman.

Acute Oak Decline (AOD) is a complex Decline-disease affecting both native oak species (Quercus robur and Q. petraea) in the UK. The presence of visible stem bleeds from cracks in the outer bark plates that overlie areas of necrotic tissue in the inner bark are the primary symptoms of AOD, and the predominance of bacteria in necrotic tissue implicates them as one of several putative contributors to the Decline. Two novel bacterial species, Gibbsiella quercinecans and Brenneria goodwinii, are consistently isolated from these necrotic lesions, revealing a suite of potential pathogenicity factors, including secretion systems and plant cell wall degrading enzymes. However, microbiome studies are also necessary to gain a greater understanding of the structure and function of microbial communities associated with healthy and AOD-affected trees. To investigate the role of the total oak microbiome in AOD, we developed methodologies for the extraction and enrichment of microbial nucleic acids from oak tissue and applied a combination of Illumina 16S rRNA gene amplicon sequencing and shotgun metagenomics to healthy oaks and AOD-affected trees at different stages of the syndrome (early, mid and late stage). In addition, we sequenced the total metatranscriptome (rRNA and mRNA) of the microbiome associated with necrotic lesions on AOD affected trees. These data provide important insights into the taxonomic and function composition of the oak microbiome, and ultimately, their role in AOD.

Genomic analysis of bacteria associated with Acute Oak Decline

James Doonan, Sandra Denman, Justin A Pachebat, Peter N Golyshin and James E McDonald

Both species of native oak trees in the UK (Quercus robur and Quercus petraea) are affected by a Decline syndrome termed Acute Oak Decline (AOD). Black weeping patches on the stems of mature trees are the primary symptom of AOD and indicate the presence of necrotic lesions in the underlying tissue. Two newly described bacterial species, Gibbsiella quercinecans and Brenneria goodwinii are consistently isolated from necrotic lesions. It has been suggested that these bacteria have a central role in tissue necrosis, possibly via genome encoded pathogenicity islands, plasmids or phage encoded enzymes.  The aim of this investigation is to identify the presence of genes associated with bacterial pathogenicity. Contemporary sequencing analysis allows high-throughput in silico analysis of novel pathogens providing large volumes of data and the starting point for the generation of hypotheses. This study used the Illumina MiSeq second generation and Pacific Bioscience third generation sequencing platforms, for the de novo assembly of Gibbsiella quercinecans and Brenneria goodwinii. Resultant assemblies were annotated using the RAST web server, PROKKA, a prokaryote specific annotation tool and the CAZy database. This genomic analysis has revealed a number of potential virulence factors. These include Plant Cell Wall Degrading Enzymes (PCWDE), numerous iron-acquisition siderophores such as enterobactin, virulence mediating quorum sensing systems and assorted secretion systems. Similar studies of bacterial associated plant pathogens have indicated that the synchronous relationship of these genes in for example Pectobacterium carotovorum leads to maceration of host cell tissue. The annotated genes encompass a suite of virulence factors providing G. quercinecans and B. goodwinii with the tools to compromise oak tree health. To ensure the accuracy of the genome annotation, and analyse differential gene expression at various time points of infection, complete mRNA expression analysis (transcriptomics) and laboratory based testing of putative pathogenic genes and operons is currently underway. A combination of second and third generation sequencing analysis has provided substantial evidence implicating Gibbsiella quercinecans and Brenneria goodwinii as putative pathogenic contributors to the Acute Oak Decline syndrome.