Eneral enquiries on this form should be made to

6. It is Defra’s intention to publish this form.

Please confirm your agreement to do so. YES  NO 

(a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.

Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.

In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary

7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.

There are five main aphid species that cause infestations on apple both nationally and internationally. Only one, the rosy apple aphid (RAA, Dysaphis plantaginea) causes severe economic loss to UK growers, through fruit damage. Current control strategies for managing RAA is by spraying chemical pesticides. Because the economic damage threshold is so low for this aphid, with only a few aphids required to cause extensive fruit deformation, spraying is routine. As a consequence, pesticide resistant varieties of RAA are becoming apparent, with reports from the continent of ineffective carbamate based aphicides. We are therefore faced with the challenge of developing new, stringent, durable and preferably non-chemical based alternatives for aphid control. This project has set about investigating apple-RAA interactions at the genetic level as a means to seek new control strategies. During the course of evolution, plants have evolved an array of defence strategies to counteract aphid attack, such as physical and chemical deterrents and toxins. Insects have in response armed themselves with counter-defence strategies to overcome plant defences, such as evading detection and expressing detoxification enzymes. The breeding of pest and disease-resistant varieties of apple has been a major driver in apple cultivation. Apple breeders have been able to identify some natural aphid resistance material in the apple (Malus) gene pool, and have used it to breed varieties resistant to two apple aphid species (rosy leaf curling aphid and woolly aphid). However, for the RAA it has proven difficult to identify usable sources of natural resistance for commercial apple breeding. Currently, there is only one commercially available apple cultivar that is sold as RAA resistant; a French dessert-apple called Florina. This project has gathered information from the literature and from people who have worked with apples to produce a list of candidate cultivars of diverse origin that may hold some degree of resistance to RAA. Most resistance claims came from observations in the field and are therefore prone to error due to the random and patchy nature of natural RAA infestations. It was therefore decided to graft the plants and test their resistance credentials in the greenhouse via controlled artificial inoculation. Graftwood was collected from 26 candidates from the East Malling Research (EMR) apple collection and from the Brogdale national fruit collection of over 2,300 different varieties of apple, to produce testable potted plants. Resistance discoveries of particular interest are; Florina (the French cultivar sold as resistant to RAA) is not resistant to any of the UK RAA clones used; indeed it is one of the most susceptible. This may show that some natural resistances are not very durable. McIntosh is not resistant to RAA, but interestingly, on plants that produced flowers, the aphids did not feed near the fruit stems. This possibly means that the aphid does not cause fruit damage, despite infesting the cultivar’s leaves. This may indicate a tolerance mechanism could be developed. Ontario is particularly resistant to RAA. EMR’s apple breeders have now set about studying the inheritance of this resistance using a small parental cross of this cultivar. with a susceptible cultivar, Telemon. Having identified good natural resistance for potential breeding, a study was undertaken to see how RAA responds genetically to these defence mechanisms when exposed to the plants. This would provide some insight into the nature of the defence and provide information on target systems, biomarkers and the likely durability of any resistance. Gene expression profiles (a snapshot of genes turned on in an organism at a particular point in time) were produced using differential display reverse transcription (DDRT)-PCR, a genomic profiling technique suitable for a lab equipped with standard equipment. Genes found to be differentially expressed are involved in a number of biological systems, mainly protein synthesis and energy metabolism. A major global metabolic change seems to occur on aphids living on the resistant apples. Disruption of the energy metabolism and protein synthesis pathways would naturally have a dramatic effect on the aphid’s survival. Also found to be differentially expressed was a stress protein from the aphid’s bacterial symbiont (dissimilar organism associated with the aphid). This may indicate that novel aphid controls could be targeted at the symbiont. Gene expression was also compared during the aphid’s period on apple leading up to the aphid leaving for its secondary host and then living on its secondary host plantain. It has been shown that RAA may be particularly vulnerable to the nitrogen content in apple, forcing it to respond genetically by upregulating tranporters of nitrates and nitrites and eventually switch host. A closer look at aphid-nitrogen interactions could yield some exploitable control possibilities. Two interesting genes were also found expressed in aphids on the secondary host. They appear to be versions of two amino acid synthesis genes found missing when the aphid’s primary symbiont (Buchnera) was sequenced. Further analysis is required to ascertain if this means the isoleucine and methionine biosynthetic pathways are intact in RAA, and whether these genes are aphid or bacterial. An unexpected discovery during this project was the identification of two viruses infecting stressed RAA, which themselves may offer a possible future control opportunity after further investigation. This project has taken the first steps in identifying natural resistance in the apple-RAA system, and characterised some genes disrupted in aphids during the interaction. Many of the genes discovered have the potential to become robust RAA stress markers which can be used to rapidly quantify and qualify the level of underlying resistance in a plant, and so dramatically reduce the timescale involved in developing aphid resistant plants. As these genes highlight stressed systems in the aphid, they also flag potentially vulnerable targets for disruption studies.

Project Report to Defra

8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include:

 the scientific objectives as set out in the contract;

 the extent to which the objectives set out in the contract have been met;

 details of methods used and the results obtained, including statistical analysis (if appropriate);

 a discussion of the results and their reliability;

 the main implications of the findings;

 possible future work; and

 any action resulting from the research (e.g. IP, Knowledge Transfer).

1 INTRODUCTION

1.1 The rosy apple aphid

Aphids are parasites of plants and, in an agricultural and horticultural context, are pests that reduce the quality and yield of produce. Sustainable control of UK pests is essential for a competitive UK industry. Of the five common aphids infesting apple, the rosy apple aphid (RAA, Dysaphis plantaginea) is the most serious as it causes severe economic loss to UK growers, through extensive fruit damage.

The current control strategy for this aphid is by spraying insecticides as soon, or even before, the pest is detected in the orchard. This is because the economic damage threshold is very low for this aphid, with few aphids causing extensive fruit deformation. Due to the damaging nature of this pest, the limited number of aphidicides compatible with IPM, and the high risk of resistance to such pesticides (a strain of RAA has already appeared in the south of France that is resistant to many aphidicides, including the main one pirimicarb), it is becoming necessary to focus on developing alternative techniques to control this aphid.

RAA has a host alternating holocyclic life cycle. It emerges from an egg in spring and spends early summer on apple, forming dense colonies and causing fruit and leaf malformation. Around mid-summer winged aphids are produced that fly to a herbaceous secondary host, plantain. The aphid remains on plantain for a few generations then migrates back to the primary host in autumn to sexually reproduce and lay overwintering eggs. It is unclear why some aphids host alternate and yet others, such as D. devecta (also an apple pest) do not. Various cues have been put forward as potential inducers of host alternation in the summer, including a range of abiotic factors (temperature; photoperiod) as well as biotic factors (crowding; host decline; host defence; maternal effects; predator/pathogen build-up). This migration provides two windows of opportunity to disrupt the aphid’s life cycle.

1.2 Plant-aphid interactions

During the course of evolution, plants have evolved a vast array of defence strategies to counteract aphid attack. These adaptations include surveillance genes, toxic secondary metabolites, and defence proteins which affect aphid host preference, longevity, development rate, fecundity and egg viability. Aphids have in response armed themselves with counter-defence strategies to overcome plant defences. These include evading detection, modifying toxin target sites, or by qualitative and or quantitative production of enzymatic detoxification systems.

Breeders have been able to identify some aphid resistance material in the apple (Malus) gene pool and have used it to breed varieties resistant to two apple aphid species (rosy leaf curling aphid and woolly aphid). However, there is only one commercially available apple cultivar that is sold as containing RAA resistance genes; a French dessert–apple called Florina (Rat-Morris, 1999). So far, it has proven impossible to identify sources of natural resistance to RAA that could be used for future commercial apple breeding.

This project attempts to seek out natural RAA resistance in the Malus gene pool and then study how aphids respond physically and genetically to this defence. Understanding the genetic responses will provide valuable data on the potential durability of any defence mechanism by helping make predictions about how the pest’s response might evolve. Information on sustainability would be invaluable when breeding difficult-to-replace perennial plants.

The vast majority of plant-aphid genetic interaction studies have focussed exclusively on the effect of the aphid on the host. (Qubbaj et al., 2005; Zhu-Salzman et al., 2004, 2008; Kehr, 2006; Smith and Boyko, 2007). These show that aphid feeding induces (possibly via elicitors in their saliva) a defence response characterised by a strong cellular salicylic acid rise and induction of pathogenesis-related (PR) proteins. This is similar to defences to pathogens and suggests aphids avoid triggering damage based defences. Chewing insects, on the other hand, have a defence characterised by the jasmonic acid (JA) pathway and release of protease inhibitors. There is, however, substantial variation in the extent of antagonistic and synergistic cross-talk between these phytohormone networks among published host-pest analyses. Thus, for each plant-insect system, assumptions regarding the important molecular events when developing control solutions should be avoided. JA and SA foliar treatments have already been proposed and tested as novel elicitors of plant resistance in pest management compatible with biocontrol by natural, with variable results on aphids (Cooper and Goggin, 2005; Boughton et al., 2006).

1.3 Genomic techniques

Genomic techniques are now becoming accessible to small-scale projects studying non-model organisms. This provides an opportunity to study the molecular background to questions of relevance to horticultural pest management.

All genomic profiling techniques (proteomics, lipidomics, metabolomics, transcriptomics) have their advantages and disadvantages (Feder and Walser, 2005).

A number of larger institutions have taken the genomic approach of sequencing all the genes being expressed in an aphid (Hunter et al., 2003; Sabater-Muñoz et al., 2006; Figueroa et al., 2007; Ramsey et al ., 2007) and its obligate bacterial symbiont Buchnera aphidicula (Shigenobu et al., 2000). Although essentially a cataloguing exercise, these sequences now deposited in GenBank are a very power resource to more focused projects, such as this one. Over 4 million sequences now exist in the NCBI trace archive from the pea aphid genome sequencing project. At the time of writing the EST database contains 120000 Acyrthosiphon pisum (pea aphid) sequences and 29000 Myzus persicae (green peach aphid) sequences.

A range of techniques now exist to profile the transcriptome (the actual genes being expressed as mRNA at a given point in time) of an organism to investigate adaptive gene expression changes involved in biological phenomena.

Most recent studies on aphid-plant interactions have used microarray technology (Voelckel et al., 2004; Wilson et al., 2006; Ramsey et al., 2007). Microarrays require prior knowledge of the sequence to be detected, which is then spotted onto a glass surface and probed with mRNA from the organism of interest. If the gene is expressed its mRNA will bind to the matching sequence on the glass. Problems with this technique are lack of sensitivity, nonspecific- and cross-hybridization, plus the need to select genes.

This project has not used microarray, mainly because the technology to undertake the protocol is not available at EMR. Also, the international databases were deplete of aphid sequences in 2005, so a meaningful screen of the RAA genome was impossible.

The method employed in this project is differential display reverse transcription (DDRT)-PCR, a technique pioneered and made by the GenHunter Corporation (www.genhunter.com). The concept of differential display is to use a limited number of short arbitrary primers in combination with anchored oligo-dT primers (to match the poly-A tail at the end of all eukaryotic mRNAs) to systematically amplify and visualize most of the mRNA in a cell. The amplified mRNAs from different samples can then be run side by side on a gel, so that up- and down-regulated genes can be detected. Bands (genes) can then be cut out of the gel and sequenced for identification. The advantage of using differential display for this project is that the technique can be carried out easily in a lab equipped with standard bench apparatus. Also, the technique makes no assumption about which genes would be assayed, so novel genes could be detected. The main pitfall levelled at this technique are the production of false positives. We have tried to avoid false positives by running replicate samples (never less than 3) of each ‘treatment’ and only characterised bands differentially expressed in all the replicates. Also, as many bands as was possible were characterised to produce a broad range of candidate markers.

Now that databases are filling up with aphid data from bulk sequencing projects, the mining of candidate genes is becoming easier. This should allow the development of gene-specific primer sets that can then be tested in modern real-time PCR machines for differential expression. However, the danger is that, like microarrays, assumptions are made as to what genes might be important before the investigation. At the end of this project an attempt was made to mine the GenBank aphid sequences and design degenerate primer sets to target detoxification genes directly in the RAA genome.

Quantitative RT-PCR is often used to support the findings of transcription profiling. Congruence between the methods is often low. False positives and contamination of probes are both cited as possible reasons. For example, RT-PCR primers will bind similar templates produced by alternatively splicing, gene duplications, paralogues genes and alleles. Primers designed close to the more variable non-coding 3’ region may be expected to be less likely to be prone to these ambiguities.

It is also apparent that transcript abundance may not necessarily correlate with protein abundance and activity, as there are many regulatory steps between mRNA abundance and protein activity. Interestingly, results presented here correlate well with genes discovered using a protein profiling assay on aphids (Francis et al., 2006).

Also, having found a gene expressed consistently under a specific regime, does not mean it turns out to be essential. This is because of the extensive redundancy in the phenome of organisms, as organisms can commonly compensate for perturbations in other gene networks.

In short, trancriptomics is a very powerful first step in highlighting candidate genes related to a particular biological system, but the extent of these gene’s impact on a pest’s fitness needs to be analysed further for redundancy.