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

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.

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.

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 (http://www.futuretrees.org/) 'Plus Trees' grown in clonal orchards (including East Malling) and the Living Ash Project partners (http://livingashproject.org.uk/) 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.