Towards protecting the UK landscape; a novel method to screen for resistance to ash dieback while mitigating herbivory tradeoffs.

Lead Research Organisation: University of Warwick
Department Name: Biological Sciences


The UK' s track record in dealing with alien invasive pests and pathogens affecting our trees is poor, particularly when considering (i) the enormous volume of trade in plants and plant products and (ii) the environmental and societal impacts of tree pathogens. No case of tree diseases has a higher profile than ash dieback - the only plant disease that has resulted in the Government's emergency COBRA group meeting twice! Recent spates of tree diseases have highlighted and exposed some of the deficiencies in past biosecurity measures and funding for tree health (Woodward & Boa 2013). For example in his excellent book "The Ash Tree" Oliver Rackham (1939-2015) called for a radical change in our attitude towards trees and a revival in 'the science of pathology' which 'has been scandalously neglected in Britain'. But it is not just the need to improve plant pathology skills but to embrace state-of-the-art molecular technologies to make valuable contributions to tree biology in the UK and worldwide. Indeed has molecular technologies been applied to ADB earlier it may not have taken nearly 20 years to identify the causal agent of ADB as Hymenoscyphus fraxineus and not Hymenoscyphus albidus which was known to have exist in Europe for more than 150 years. As a consequence there was no possibility under EU law to restrict imports of ash trees to Britain.
This proposal uses "metabolite profiling", a cutting edge molecular technology to help address ADB. We initially looked at the "metabolic profile" of tolerant and susceptible Danish ash, as the Danes have been battling ADB for more than 20 years. Using sophisticated equipment that can measure the "weight" of these small molecules we can make of list of their presence/absence and relative abundance in in tolerant and susceptible ash. Our work has looked at nearly ~20,000 such small molecules and identified several hundred that discriminate tolerant from susceptible ash. Of these at least 50-60 are really "discriminatory" and can be used to develop a method to look at British ash trees.
However our work also discovered something totally unexpected. Some compounds that we know are involved in stopping insects eating plants were present in very low levels in tolerant ash compared to susceptible ash. Now this could be critically important. Why? We are currently committing millions of pounds towards selecting British ash with tolerance to ADB. However, any strategy for breeding British ash trees resistant to ADB will need to consider that they will be suitable food sources for emerald ash borer (Agrilus planipennis), a destructive jewel beetle native to China. Emerald ash borer has killed tens of millions of ash trees since its arrival in North America 20 years ago and has recently been reported 250 km west of Moscow. It is important to further investigate our preliminary data that suggest trees selected for resistance to ADB have very low levels these anti-feeding compounds, technically known as iridoid glycosides we may be unwittingly be selecting for enhanced susceptibility to emerald ash borer.
This application is designed to build on these very recent findings that identified features which could discriminate between tolerant and susceptible Danish ash trees. We now seek funding to develop this methodology further and establish the necessary tools to: (i) discriminate tolerant and susceptible British ash germplasm by screening for specific small molecule signatures; (ii) determine whether low levels of these anti-herbivory compounds are intimately associated with tolerance to ash dieback (ADB) and; (iii) generate a detailed metabolome (small molecules existing in samples) of tolerant and susceptible British ash leaves. The ability to monitor metabolomics signatures of selected British ash dieback resistant germplasm can ensure selection programmes are not inadvertently selecting for susceptibility to Emerald Ash Borer.

Technical Summary

This proposal will use both targeted and untargeted metabolite profiling to screen British ash for markers associated with tolerance to ash dieback. These markers, originally identified by unbiased metabolite profiling of tolerant and susceptible Danish ash by accurate mass LC-QToF will be trialled on British ash genotypes identified as tolerant to ADB derived from a variety of diversity trials. We will undertake two targeted approaches. The first involves quantitative QQQ analysis of 50-60 highly significant features that clearly discriminant tolerant from susceptible Danish ash from geographically separated genotypes.
The second involves a more detailed investigation of the finding that tolerant trees have very low levels of iridoid glycosides, known anti-feeding deterrents. This is important as the most devastating known pest of ash is emerald ash borer and it is highly likely this beetle will arrive in the UK in the future. By accurate mass LC-QToF profiling using precursor ion scanning we will characterise the suite of iridoid glycosides in British ash focussing on levels in trees identified as tolerant.

Finally we will undertake unbiased global metabolite profiling of tolerant and susceptible British ash emerging from the established ash diversity screens. This will use established methods and pipelines developed in Exeter. Briefly, freeze dried ash leaf samples are extracted in methanol and features separated by reverse phase (Polaris C18) HPLC coupled to a QToF 6520 mass spectrometer (Agilent). Positive and negative ion data are converted into mzData and peak identification and alignment performed using the Bioconductor R package, XCMS. Features are detected using the centWave method and peaks matched across samples using the obiwarp algorithm. Missing peak data are filled and resulting peaklists annotated using the Bioconductor R package, CAMERA. Statistical analysis and modelling is performed using MetaboAnalyst v3.0.

Planned Impact

There is a very clear, defined translation of these research outputs into practice. Successful development of a method that can be used to screen for Fraxinus excelsior resistance to the causal agent of ash dieback (ADB) Hymenoscyphus fraxineus - and/or corroborates phenotypes arising from diversity screens and wider-environment survivors - will have an enormous social and environmental impact. Most importantly, it will inform on the strategies and speed with which we can manage regeneration and replanting of UK woodlands to ensure F. excelsior remains a dominant aboral species in the UK landscape. We will generate knowledge and innovation with potential for wider application overseas. For example, this technology would be of interest to the European and Mediterranean Plant Protection Organization (EPPO; an intergovernmental organization responsible for European cooperation in plant health) members who are interested ADB resistance while maintaining their local genetic diversity within their member state. We would make this technology as widely available as possible while protecting BBSRC and Exeter's investment through our Research Knowledge Transfer intellectual property team.
An unexpected finding emerging from our initial profiling that has could have enormous impact on informing policy-making and management decision structures in DEFRA's ADB response is the realisation that selection for ADB tolerance may inadvertently result in selection for enhanced susceptibility to herbivory. With the imminent arrival for Emerald Ash Borer into the country, it would be remiss not to further investigate. If validated these data must be incorporated into any long-term breeding and deployment strategy to ensure we have ash resistant to Emerald Ash Borer, most likely when, rather than if it arrives in the UK.
Our results will be directly translated on the ground by working closely with organisations directly involved in environmental screening with established and newly generated ash diversity collections such as Future Trees Trust ( 'Plus Trees' grown in clonal orchards (including East Malling) and the Living Ash Project partners ( who are funded to identify ADB tolerant ash through a combination of half-sibling progeny trials of over 40,000 ash seedlings across three sites in Britain and a citizen sciences driven initiative to identify ADB resistance in the wider environment. Recently, an extension of the Nornex funding has recently initiated the establishment of a highly replicated collection of 300 British ash genotypes from a wide geographical region.
Societal impact of this work could be profound and we have already seen the social "amplification of risk" demonstrated in the public response to ABD (which led to the Government convening two emergency COBRA meetings. Our outputs will illustrate how cutting edge technologies can be applied to tree health issues. This is important given the past deficiencies in past biosecurity and funding for tree health. Indeed the House of Commons Science and Technology Committee Forest Research Inquiry of 2011 highlighted the limited investment in forestry-based research in UK Universities.
Translational opportunities will arise through impact on policy (DEFRA, Forest Commission) as well as silviculture industry and charities interested in preserving woodlands.
Description We identified a class of anti-feeding deterrents that were highly significantly associated with ash dieback (ADB) tolerant ash trees. If this is indeed that case then selecting for ADB tolerant trees may instead select trees that are highly susceptible to herbivory -- in particular emerald ash borer which is currently in Russia and spreading southwards.
Exploitation Route Need to further investigate extent of antifeeding deterrents in UK tolerant ash -- problem is in identifying these trees first - with confidence. The Danish had a decade head start.

We have develop a method for discriminating Danish ash trees, however due to major issues identifying and generating material from UK trees, we have not been able to apply this with an confidence to UK ash. however, we have secured further funding in collaboration with JIC and EMR to move this forward and further develop the method with more sensitive mass spectrometers and a new refined pipeline.
see "Retaining the Ashes: The potential for ash populations to be restored following the dieback epidemic".
Sectors Agriculture, Food and Drink,Environment,Government, Democracy and Justice

Description Public awareness of the challenges facing tree health and the need for enhanced biosecurity Lectures to public on ash dieback and the dual threat of emerald ash borer Public outreach on ash dieback and economic impact of mitigating against dead trees
First Year Of Impact 2015
Sector Agriculture, Food and Drink,Environment
Impact Types Societal,Policy & public services

Description DEFRA Ash Dieback and Emerald Ash Borer Evidence meeting
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
Impact Presented some of the limited fundamental research, and contributed to the discussion and policy document that helps underpin the evidence base for management of ash dieback, and the future of UK ash in the face of the emerging threat of emerald ash borer to the UK (and Europe).
Description UKPSF Growing the Future
Geographic Reach National 
Policy Influence Type Implementation circular/rapid advice/letter to e.g. Ministry of Health
Description Retaining the Ashes: The potential for ash populations to be restored following the dieback epidemic
Amount £274,693 (GBP)
Funding ID BB/R018944/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2018 
End 12/2020
Description MetaboLights is a database for Metabolomics experiments and derived information. The database is cross-species, cross-technique and covers metabolite structures and their reference spectra as well as their biological roles, locations and concentrations, and experimental data from metabolic experiments. 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? Yes  
Impact DEposited data at Metabolights under the following: Sambles, C. M. et al. MetaboLights MTBLS372 (2016). 
Description Accessing UK Ash resistant to ash dieback 
Organisation John Innes Centre
Country United Kingdom 
Sector Academic/University 
PI Contribution We are screening ash saplings derived from the wider environment to test if differences in iridoid glycoside profiles correlate to susceptibility or disease
Collaborator Contribution Provision of freeze dried ash leaves, replicated lines tested for tolerance and resistance to ash dieback.
Impact Initial mass spectral profiles (positive and negative mode LSC-Tof-MS) of putatively ash dieback resistant and susceptible ash.
Start Year 2017
Description Dynamic video display at Fascination of Plants Day, Warwick Crop Centre, Wellescourne April 2017 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Ran a dynamic stand addressing the impact and consequences of ash dieback - the role of the chloroplast in plant immunity and the importance of bananas to global plant security.
Year(s) Of Engagement Activity 2017
Description Kenilworth Agricultural Show 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Set up displays and shows in the Education Tent at the Kenilworth Show, looking at public perception of plant pathogens, how disease develops and how we are using state-of-the-art techniques to mitigate this. Used ash dieback as an exemplar which the public could readily engage and introduced the concept of mass spectrometry to understand how we could look at small molecules that might be causal to disease prevention or progression.
Year(s) Of Engagement Activity 2019
Description Public engagement evening - Food Security 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Public Engagement evening consisting of 4 talks and 1 hour of demonstrations, hands on events and technology displays
Year(s) Of Engagement Activity 2017
Description ash dieback publication publicity 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact A number of media outlets took up news stories relating to a Nature publication on Ashdieback.
Internaiton coverage as it was reported in Financial Times
Year(s) Of Engagement Activity 2016