Overview

Ash dieback, caused by the invasive fungus Hymenoscyphus fraxineus (Hf), has been a destructive disease of European ash (Fraxinus excelsior) since 1992 and was first seen in the UK in 2012. This epidemic has serious implications for the ecological role of ash as a keystone species in nature, its economic value as timber, and its service value in landscaping and ecosystem functions such as restriction of flooding, soil erosion and collapse of river and canal banks. While most trees in heavily affected areas are severely damaged, a small minority are clearly less susceptible, raising the question of why, in evolutionary terms, most ash is so susceptible to this alien pathogen and why, by contrast, a small proportion is not.

 

Whole genome sequencing has revealed extensive genetic heterogeneity between UK ash, making genetic approaches challenging. Since small molecular are the key players in pathological outcomes, our group has pioneered targeted and untargeted metabolite profiling of ash trees. Untargeted metabolite profiling unexpectedly revealed a specific class of secondary metabolites known as iridoid glycosides (IG), associated with susceptibility to dieback in Danish ash trees (Sollars et al. 2017 Nature). IG are well-known as compounds which deter insect herbivory in diverse plants, thus it is possible that if selection for dieback-resistance were to reduce levels of IG, susceptibility to herbivores such as insects could be enhanced, so replacing one destructive agent by another and thwarting attempts to restore ash populations. An insect of special concern is another invasive alien species, the emerald ash borer (Agrilus planipennis), which has devastated millions of ash trees in the USA, Canada and Russia. Thus it is critical to move this knowledge forward. This project will address (i) the diversity of IGs in UK ash resistant and tolerant to ADB, (ii) whether, and if so how, low IG levels confer resistance to Fraxinus species and (iii) whether low IG levels render ash susceptible to insect herbivory (unfortunately plant health regulations prevent working with Agrilus planipennis.

Methodology

We have verified low IGs in putative “resistant” trees from a unique collection of tolerant and resistant UK ash provided by our collaborators at the John Innes. A NERC quantitative skills REP recipient, Jack Parker has developed an extraction method to characterise IGs, and we have expanded our 6 putative IGs identified in Danish ash to more than 25 in susceptible trees. We (LS) are developing a targeted profiling method (single ion reaction monitoring) to discriminate and individually quantify these IG on a newly acquired triple quadrapole mass spectrometer with sub-pmol sensitivity.

The candidate will undertake detailed characterisation, and then purify selected IG fractions. These will be subject to insect feeding (DC) and pathology (MG) assays to determine their anti-herbivory and impact on phytopathogenic fungi/bacteria.

Mass spectrometry methods will be refined to enable high throughput screening of environmental ash samples, as a future conservation method.

 

Training and Skills

This truly multidisciplinary project involves field sampling, sample extraction and metabolomics, which itself will lead to bespoke training in large data manipulation, visualisation and interpretation.  The candidate will be trained in a broad range analytical science skills, while at the same time  developing skills for working with insects and pathogens. We will ultimately develop an assay, for potential use in the nursey industry, or bodies interested in monitoring their amenity resources (e.g. country councils, road authorities, railtrack). Thus, the candidate will learn communication skills, as well as delivering outreach to the general public, who are particularly interested in ADB.

Timeline

Year 1:  Training on mass spectrometry, operation, sample preparation, data analysis – targeted and untargeted pipelines. Validate method,  profile and quantify iridoid glycosides in UK ash leaves sampled from different geographical locations.

Year 2: Complete iridoid characterisation, target 3-4 most abundant samples of unknown structure for preparative HPLC analysis. Milestone: Robust IG profiling method and definitive catalogue of IGs (paper 1).  Develop HPLC methods for reproducible separation of IGs for (i) NMR and (ii) pathogen assays.

Year 3: Assay ash IG activity for pro- anti-fungal activity and anti-feeding deterrant (paper 2). Roll out rapid screening method for IGs that can be used by relevant authorities, nursery trade. Apply IG method to (i) ash species from other countries (particularly asia where both the Hf fungus and Emerald Ash borer emerged, (ii) profile ash leaves from bud burst to senesence to determine development changes in irodiods.

 

Partners and collaboration (including CASE)

The project will involve 3 supervisors who each bring in their specific skills. MG has more than 25 years experience in molecular plant pathology and was an expert member of the “Tree Health” White Paper (2011) and served as a DEFRA Tree Health & Biosecurity Initiative expert. He brings expertise in plant biology, metabolomics, pathogen assays. LS has been collaborating with MG for 2 year and provides the analytical expertise, while DC brings entomology expertise, both in feeding behaviour, but also chemical biology.  The project has John Innes and East Malling Research as collaborative partners.

Further Details

Potential impact - This project will contribute essential information for informed selection and replanting of ash with lower susceptibility to dieback, for the benefit of public bodies, charities and forestry companies concerned with tree-planting. It will provide evidence of the risk of trade-offs between dieback-resistance and deterrence of herbivores mediated by secondary metabolites. In particular, it will help greatly to predict the likely damage by emerald ash borer, should that highly destructive beetle ever become established in the UK.

 

https://www2.warwick.ac.uk/fac/sci/lifesci/people/mgrant/