Retaining the Ashes: The potential for ash populations to be restored following the dieback epidemic

Ash dieback has been a destructive disease of European ash since 1992 and was first seen in the UK in 2012. The fungus which causes this disease is native to East Asia and is thought to have arrived in north-east Europe during the 1980s. The dieback epidemic has serious implications for the UK given the ecological role of ash as a keystone species in nature, its economic value as timber, its amenity value in cities and parks, and its service value in landscaping and ecosystem functions, notably reduction of flooding by reducing water run-off from fields and consolidating river and canal banks. While most trees in heavily affected areas are severely damaged, a small minority are clearly less susceptible, raising a significant hope that this genetic variation might lead to the long-term recovery of ash in the UK and Europe generally.

This project aims to understand why this variation in dieback-resistance has evolved in European ash. This tree species is highly variable and the population in the UK has diverged substantially from that in continental Europe. Our hypothesis is that certain chemicals (secondary metabolites) confer resistance to damage by certain insects but increase the susceptibility of ash to dieback. As a resource for this project and for future research, we have collected 328 diverse ash genotypes from the UK, including numerous lines which have had low susceptibility to dieback even under intense disease pressure. This collection, known as JENNIFER, will be used to research the relationship between multiple traits to understand the ecological context of ash dieback.

Ash trees with low susceptibility to dieback may either have partial resistance to dieback, so their leaves are less easily infected by the fungus, or have a form of growth and development which leads to lower exposure to spores of the fungus, thus promoting disease escape. Objective 1 of the project is to characterise the diversity of low susceptibility to dieback in UK ash. We will measure disease severity in replicated field trials exposed to natural infection, investigate components of resistance, estimate contributions of resistance and escape to low susceptibility, and test if genetic resistance to dieback in UK trees has diverged from that studied previously in Denmark. Small-scale methods of studying infection by the dieback fungus, developed by the project partners, greatly increase the capacity to study variation in resistance.

In earlier research, low levels of iridoid glycosides, a class of secondary metabolite, were found to be associated with resistance to dieback but ash also contains many other secondary metabolites. Objective 2 is to test the hypothesis that diverse secondary metabolites in ash are associated with variation in dieback-resistance. We will use untargeted metabolite profiling to identify chemical features which most strongly discriminate infected and uninfected leaves of resistant and susceptible clones, combine metabolite and transcriptome analysis to determine the genetic basis of changes in secondary metabolites in response to infection, characterise temporal variation in levels of discriminant molecular features, and relate them to resistance in the JENNIFER panel.

Objective 3 is to test the hypothesis that secondary metabolites control contrasting responses of ash to dieback and to diverse herbivorous insects. This will be done by testing if variation in dieback-resistance and levels of key metabolites are associated with variation in insect feeding, growth and reproduction, and by assessing the susceptibility of the JENNIFER ash clones to attack by invertebrates in natural conditions.

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. While most trees are very susceptible to this alien pathogen, a small minority are less diseased. As ash in Europe was not previously exposed to Hf, this project aims to understand the evolutionary origin of this polymorphism in terms of the forces of natural selection which act on dieback-susceptibility.

We have assembled 328 diverse ash lines from the UK, including many which have low dieback-susceptibility even at heavily affected sites. In Objective 1, we will estimate the contributions of resistance and disease escape to low susceptibility in this collection in replicated field trials exposed to natural infection. We will use controlled inoculation in lab and greenhouse experiments to investigate components of resistance and assess the divergence of resistance in the UK from that in continental Europe.

In a limited sample of trees from Denmark, dieback-susceptibility was correlated with levels of iridoid glycosides in uninfected leaves. In Objective 2, we will combine untargeted metabolite analysis by LC-QToF-MS with transcriptome analysis to survey a very wide range of secondary metabolites (SM), both constitutive and induced on infection, and identify SM and biosynthetic pathways that discriminate responses of resistant and susceptible ash to Hf. We will then test the association between SM and responses to Hf across our panel of ash lines.

Diverse SM, including iridoid glycosides, reduce attack by invertebrate herbivores by acting as repellants, anti-feedants or toxins. In Objective 3, we will test the hypothesis that certain SM mediate a trade-off between responses of ash to dieback and to certain insects, by testing if dieback-resistance and levels of key SM in ash clones are correlated, positively or negatively, with insect feeding, growth and reproduction.

Ash dieback has caused severe damage to European ash (Fraxinus excelsior) since it was first observed in north-eastern Europe in 1992. It was first seen in the UK in 2012 but may have been present here since c.2000. Although the fungus kills juvenile shoots quickly, disease progress in mature trees is slow but relentless, causing death in 5-10 years. In the most severely affected counties of the UK, almost all trees are infected and some ashwoods have been devastated. It is striking, however, that a minority of ash trees are much less diseased than others of the same size and age nearby.

Ash is a keystone species in the natural environment which supports diverse wildlife, a source of high-quality timber for furniture, tools and sporting goods and an important tree for landscaping and shade in urban and rural settings including residential areas, car parks and industrial estates. Perhaps the most significant benefit of ash is for ecosystem services, including such as flood prevention by preventing run-off of rainwater from fields, soil erosion and collapse of river and canal banks.

We have assembled a panel of 328 ash genotypes, known as JENNIFER, for use in this project and as a resource for future research. An important impact of this project is that JENNIFER contains a substantial number of lines with strong partial resistance to dieback. Seed from trees such as these has the potential to re-establish ash for all the purposes mentioned above, although further selection will be needed to identify plants with good form for timber production. A previous project discovered genetic markers for resistance among trees from Denmark but they have limited power to predict resistance in ash from the UK. Moreover, reliance on a few markers should be avoided in plant breeding, to minimise the risk of pleiotropy or linkage drag with undesirable traits. Further understanding of the pathology, diversity and ecology of ash dieback in the UK is therefore required.

In a small sample of the Danish study population, certain secondary metabolites (SM), assigned as iridoid glycosides (IG), were associated with susceptibility to dieback. IG are well-known as compounds which deter insect herbivory in diverse plants but it is not yet known why low IG levels are associated with resistance to dieback. It is thus 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, which has been extremely destructive in the USA, Canada and Russia. Fraxinus species contain many diverse SM, however, so it may be possible to select for (or against) different metabolites which confer resistance (or susceptibility) to dieback and to herbivores, or even for metabolites which have positive effects on both types of bio-antagonist. Exciting advances in technology for identifying low levels of metabolites mean it will soon be possible to select F.excelsior breeding stock with a high probability of good resistance to dieback and to herbivores, based on sensing of multiple odours.

The project will also 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 reveal the diversity of dieback-resistance in ash from the UK and indicate 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. It will also identify traits which can be selected in order to promote disease escape.