Aphids are among the world's most damaging group of insect pests, causing billions of US dollars of yield loss per annum to a wide range of food and commodity crops. The control of pest aphids has relied heavily on the use of chemical insecticides, however, the evolution of resistance poses a growing threat to their sustainable control. Aphids are in part so successful because of their remarkable capacity to rapidly increase in number under favourable conditions, indeed, it has been estimated that without predation or disease a single aphid could produce 600 billion descendants in just one season. This incredible reproductive output is achieved by asexual reproduction (parthenogenesis) - with adult female aphids giving birth to daughters that are clones of themselves for most of the growing season. For most aphid species this mode of reproduction alternates with a yearly cycle of sexual reproduction, however, in some countries such as the UK certain species, including important pests such as Myzus persicae, reproduce exclusively by parthenogenesis. Because aphid clonal lineages have been assumed to be genetically identical they have been referred to as 'evolutionary dead ends' with little or no capacity to evolve. However, we have recently shown that aphid clonal populations derived from a single female aphid can evolve heritable resistance to insecticides. Although this clearly demonstrates that aphid clonal lineages have adaptive potential the mechanisms by which aphid clones evolve are completely unknown. The aim of this project is to understand the adaptive potential of aphid clonal lineages and the molecular mechanisms underpinning clonal evolution in aphids. We will use the global aphid pest M. persicae as a model and exploit recent advances in genomics, transcriptomics and epigenetics to address a range of key knowledge gaps on this topic. These include understanding how quickly mutations accumulate in aphid clonal lineages, how this is affected by pesticide stress, and how clones adapt at the phenotypic and molecular level in the absence of sex. In the first objective of the proposal we will calculate the frequency and spectrum of mutations that spontaneously arise in clones of M. persicae using an experimental approach called mutation accumulation (which overcomes the difficulty of directly studying how rare mutations arise by allowing them to build up over several generations in lines propagated by single progeny descent). In this objective we will also test the hypothesis that sub-lethal insecticide exposure increases mutation rate by comparing how mutations accumulate in lines exposed to insecticide with insecticide unexposed lines. In the second objective we will use an approach called experimental evolution (the use of experimental populations exposed to specific conditions in the laboratory to study evolutionary processes) to examine the response of large clonal populations of M. persicae to insecticide selection. We will combine this approach with three different sequencing approaches to explore the molecular mechanisms underpinning evolved resistance, which will be validated in Objective 3 using functional approaches. The data from these experiments will fundamentally advance our understanding of how insects evolve in the absence of sex. Furthermore, the knowledge generated will be of considerable applied importance in relation to the sustainable control of aphid pests. For example, it will allow us to anticipate resistance risk development in countries where aphids reproduce asexually and optimise pesticide dosing strategies to avoid accelerating the evolution of mutations that might lead to resistance. Finally, understanding how clonal pest populations evolve resistance at the molecular level may provide opportunities to exploit and counter this process by developing strategies against the underpinning mechanisms involved.

Blackleg disease of potato caused by P. atrosepticum (Pba) is the most damaging bacterial plant pathogen in the UK, costing £50M p.a. in losses for the potato industry. Current knowledge assumes that disease is caused through Pba-infected seed tubers. However, our recent unpublished data have shown that under high soil moisture following irrigation, disease appears in plants grown from pathogen free seed (minitubers). The most likely explanation is that bacteria enter the plant and cause disease directly from the soil; something not previously considered. We have also shown that Pba is able to colonise roots of other plant species (including crops), possibly as natural rhizosphere-dwelling saprophytes in the soil. In pot trials with Pba alone, we showed there was no movement of Pba from soil into the plant. However, when free-living nematodes (FLN) were added to soil, a 100-fold increase in Pba in stems occurred. Through these and other findings we now have the potential to make a step change in how we manage blackleg. We will address knowledge gaps firstly by using Light Sheet and Confocal Laser Scanning microscopy, transparent soils and mesocosm studies to assess the role of FLN as vectors of Pba and how infection occurs. We will also examine how changes in standard irrigation regimes can help to reduce levels of blackleg in ware crops (where irrigation is often over-applied to avoid common scab disease that occurs in dry conditions), and how it might change FLN communities around potato root systems. Similarly, we will identify cover crops that limit natural Pba colonisation on their roots as a possible way to reduce Pba numbers in soil prior to planting potato. Little is known about the microbiome on potato roots and how these might be influenced to favour or reduce colonisation by Pba. We will therefore characterise the potato microbiome prior to and following irrigation using shotgun metagenomics sequencing and the latest bioinformatics tools, with a focus on the Pectobacteriaceae and wider Gamma-proteobacteria. We will also use GC-MS to examine how changes in root architecture and the constituents of root exudates influence the composition of these bacterial groups, to assess whether the use irrigation and cover crops alter the balance between beneficial and harmful bacteria associated with potato. Finally, we will determine whether novel antimicrobials (bacteriocins) in closely related non-pathogenic bacteria in the microbiome could act as a management option against Pba. Our recent modelling research using the Scottish Government's in-house potato inspections database (SPUDS), shows that blackleg incidence on a national scale does not occur randomly but in clusters. Reason(s) for this remain unclear but could be due to several things that, when identified, may assist growers in managing their crops, e.g. potato crop distribution, weather, soil type, soil moisture, leaf wetness, FLN distribution, crop type and rotation prior to planting. Using data generated from this project, an extensive array of data from other recent and historical investigations and the latest data from government and industry we will model, using innovative machine learning methods, at national scale these data to identify trends and drivers of Pba incidence in both space and time and, through this, produce predictive models to support development of a set of decision support tools for evaluation by stakeholders during the project and early adoption thereafter. Further, through scenario testing, we will quantify the predicted effects of climate change on future blackleg incidence in association with FLN presence thus providing the industry with robust and novel data to underpin sector resilience planning.

Although oilseed rape (OSR; Brassica napus) has traditionally been grown as the most profitable break crop, a loss of controls for cabbage stem flea beetle (CSFB; Psylliodes chrysocephala), has resulted in UK cropping area declining by 35% between 2012 and 2019. The National Farmers Union (NFU) estimate the removal of neonicotinoid seed treatments has cost farmers ~£94 million/year in lost opportunity and crop loss. Additional costs have been absorbed by the UK crushing industry because of the need to import OSR. Collectively, this poses a serious risk to the viability of the UK OSR industry and current farm crop rotation practices. With the withdrawal of chemical controls, resistant cultivars are central to supporting Integrated Pest Management (IPM) strategies. However, unlike plant-pathogen interactions, our understanding of the interactions between host plants and chewing insects is limited. CSFBs are attracted to glucosinolates, chemicals used by Brassica species to deter non-specialist insect pests. Even if B. napus has defence or resistance mechanisms that deter CSFB feeding, the genetic control of such mechanisms and whether they can be exploited to breed for resistance has remained an open question. There are no known examples of resistance in B. napus and little knowledge of resistance mechanisms within our UK crop species. No resistant cultivars are currently available for any insect pest of OSR. This proposal builds on preliminary data using controlled feeding studies and field trials, which shows that variation for reduced adult CSFB feeding is present within a diverse panel of B. napus. The panel, comprising historical varieties of winter and spring OSR, Chinese OSR, swede and kale, contains genetic diversity which is unlikely to be present in elite cultivars. This has enabled us to identify loci associated with CSFB feeding damage. Controlled larval infestation studies within this population have also identified variation in the numbers of emerging adult CSFB, demonstrating the presence of resistance to CSFB larvae. Together these observations indicate that some varieties of B. napus carry genes which can 1) deter adult CSFB feeding and 2) confer resistance against larval infestation or reduce larval fecundity. If identified, this variation can be exploited to breed OSR resistant to both damaging stages of CSFB. This proposal developed by two research institutes (JIC and Rothamsted Research), seven major plant breeders and the OSR growers' Levy board (AHDB), aims to discover loci associated with adult CSFB feeding and larval resistance in B. napus. In parallel, we aim to develop an understanding of crop adaptations which affect OSR-CSFB interactions. This will be coupled with larval development studies, gene expression analysis and metabolite profiling to further elucidate key mechanisms by which Brassica plants identify and defend themselves against beetle attack. Key genes implicated in resistance and defence responses will be investigated using candidate gene studies in model plants, including candidate genes which underlie two loci implicated in the supporting data. Collectively, this knowledge, combined with germplasm and markers that will be used by participating breeders to integrate resistant alleles into commercial breeding pipelines, will facilitate the introduction of tolerant varieties into the UK OSR market.