General enquiries on this form should be made to:Defra, Science Directorate, Management Support and Finance Team,Telephone No. 020 7238 1612E-mail: research.competitions@defra.gsi.gov.uk

SID 5 Research Project Final Report

SID 5 (Rev. 3/06) Page 1 of 39

NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

This form is in Word format and the boxes may be expanded or reduced, as appropriate.

ACCESS TO INFORMATIONThe information collected on this form will be stored electronically and may be sent to any part of Defra, or to individual researchers or organisations outside Defra for the purposes of reviewing the project. Defra may also disclose the information to any outside organisation acting as an agent authorised by Defra to process final research reports on its behalf. Defra intends to publish this form on its website, unless there are strong reasons not to, which fully comply with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.Defra may be required to release information, including personal data and commercial information, on request under the Environmental Information Regulations or the Freedom of Information Act 2000. However, Defra will not permit any unwarranted breach of confidentiality or act in contravention of its obligations under the Data Protection Act 1998. Defra or its appointed agents may use the name, address or other details on your form to contact you in connection with occasional customer research aimed at improving the processes through which Defra works with its contractors.

Project identification

1. Defra Project code PS2114

2. Project title

Identification, provision and delivery of semiochemical tools for use within plant-pest-natural enemy systems in integrated crop management.

3. Contractororganisation(s)

Prof. J. A. PickettRothamsted ResearchWest CommonHarpendenHertfordshireAL5 2JQ

54. Total Defra project costs £ 486,000(agreed fixed price)

5. Project: start date................ 01 April 2006

end date................. 31 March 2007

SID 5 (Rev. 3/06) Page 2 of 39

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 Summary7. 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.

The main purpose of this continuation project was to pursue and develop key areas from previous projects PS2101 and PS2105. This project included the identification, provision and delivery of semiochemicals, i.e. signalling chemicals that control pest or natural enemy behaviour and development or act as signals to switch on defence effects in plants, as alternatives to conventional pesticides. By influencing the colonisation of crop plants and subsequent pest population dynamics, semiochemicals can thereby be used to disrupt or direct pests away from the crop and attract them to areas where they can be controlled (e.g. the “push-pull” strategy). Semiochemicals act through behavioural mechanisms rather than by toxicity and thus offer environmentally benign means of crop protection with which to minimise, supplement, or in the long-term replace, use of broad-spectrum pesticides in Integrated Pest Management (IPM). Chemically-based interactions between plants and other plants or microorganisms can similarly suppress weeds or diseases. In lower input systems, including organic farming, the use of semiochemicals complements the greater exploitation of biological control agents, selective natural pesticides and pest-resistant cultivars.

Objective 1. Focus on the identification of new plant stress signals to extend the practical range already obtained with cis-jasmone. These will act as plant activators for which optimal delivery systems will be (E)-ocimene has been shown to induce defence in healthy barley against pathogen and aphid attack. It is also a key compound that increases in the volatile profile of many crop plants when they are damaged by pests and pathogens or exposed to the plant activator cis-jasmone. In addition, it is a key component mediating host location by the aphid parasitoid, Aphidius ervi. Thus, (E)-ocimene has a dual role as a plant stress indicator, mediating tritrophic interactions and as a plant activator. Since it is an unstable chemical and its wider use would require large scale and expensive synthesis an alternative, natural source was identified from the fractionation of the essential oil of the catmint plant Nepeta cataria. In this essential oil (E)-ocimene is stabilised, and when exposed to the (E)-ocimene fraction of the oil, the defence mechanisms of healthy barley plants were induced making them less attractive for colonisation by the cereal aphid, Rhopalosiphum padi. In addition, the development and fecundity of the brassica specialist aphid, Lipaphis erysimi, was significantly reduced on turnip rape and oilseed rape plants that had been exposed to the ocimene fraction. This indicates that (E)-ocimene has a wider role as a plant signal and that it can be delivered in a stabilised form as an essential oil. The effects of cis-jasmone as a plant activator, eliciting defence chemistry, and as a semiochemical, directly repellent to pests and attractive to predators, have been demonstrated for a range of plant/pest/natural enemy complexes. A new, slow release formulation of cis-jasmone, compatible with conventional spray application techniques, has been developed to deliver both the plant activation and the direct semiochemical effects in the field. This formulation is also suitable for the controlled release delivery of other semiochemicals.

Objective 2. Identify semiochemicals associated with host plant location and avoidance of unsuitable potential hosts by representative pest species. Determine methods for exploitation of host plant recognition and

SID 5 (Rev. 3/06) Page 3 of 39

avoidance cues by pests in novel crop protection strategies.Two model crop/pest complexes were used to explore the hypothesis that modification of the specific ratio, by augmentation of key members of the attractive semiochemical mixture, will interfere with the host location abilities of the whole pest complex for a particular crop. In the cereal model, key attractive volatile semiochemicals produced by wheat were identified for the orange wheat blossom midge, Sitodiplosis mosellana, and the grain aphid Sitobion avenae. Female S. mosellana were attracted to a 6 component synthetic blend of volatiles, collected at ear emergence and presented in the natural ratio and concentration. The blend was as attractive as the natural sample of the wheat variety and remained as attractive when reduced to three components, acetophenone, (Z)-3-hexenyl acetate and 3-carene. However, entrainments of other midge susceptible wheat varieties showed that production of these three components varied greatly as did the composition of minor components. 6-Methyl-5-hepten-2-one was found in the volatile profile of only a few varieties, but when presented to female S. mosellana in an unnatural ratio the blend was not attractive. Thus, for this insect there is clear evidence that host recognition is conferred by ubiquitous compounds, some of which must be present in specific ratios. However, a synthetic blend of volatiles, which was attractive to S. mosellana, was significantly repellent to S. avenae. Two components of this blend, α-pinene and 6-methyl-5-hepten-2-one, were repellent when tested on their own. These data indicate that S. mosellana and S. avenae are using similar chemical cues from the host plant, but further study is necessary to understand how these cues can be manipulated to affect both pest species.In the legume model, GC coupled electrophysiological studies with the pea and bean weevil, Sitona lineatus, and the black bean aphid, Aphis fabae, identified key semiochemicals from the legume host. For S. lineatus many host volatiles are perceived by specialist olfactory cells, a number of which are paired with cells for the aggregation pheromone, 4-methyl-3,5-heptanedione, indicating that the weevil can discriminate between ratios of these compounds. Field trapping studies with the pheromone, plus mixtures of host plant volatiles, showed that some mixtures enhanced the attraction of the weevil to the trap whilst other mixtures reduced attraction. Of the 12 electrophysiologically active compounds identified for A. fabae, only three, methyl salicylate, (-)-linalool and (Z)-3-hexenyl acetate, were perceived by S. lineatus. The twelve compounds were significantly attractive to A. fabae when presented in the natural ratio. Three of them, methyl salicylate, 6-methyl-5-hepten-2-one and 4,8,12-trimethyl-1,3-7,11 tridecatetraene are associated with plant stress, particularly through pest or pathogen damage, and have been shown to contribute to reduced attraction of other aphid species to their host plants. However, the behaviour of A. fabae was found to be unusual since it was significantly attracted to 4,8,12-trimethyl-1,3-7,11 tridecatetraene and to 6-methyl-5-hepten-2-one and was unaffected by methyl salicylate. We suspect that, since A. fabae occurs in very large colonies, it is capable of dealing with the stress related chemistry produced by the plant and has evolved to utilise it in host location and acceptance.

Objective 3. Determine and evaluate the potential of exploiting rhizosphere allelopathy, in controlling pest/plant interactions, and suppressing competitive weeds.The genes (Bx genes) in the hydroxamic acid biosynthetic pathway in wheat have recently been cloned and sequenced and to confirm the role of this pathway in defence against pests in cereals, and to provide the basis for a breeding program to develop resistance in wheat, a detailed spatial and temporal gene expression analysis was undertaken. Expression of four biosynthetic genes, encoding cytochrome P450s (Bx genes 2 -5) were measured in the root, coloeptile and leaf of 12 day old seedlings by real time PCR. Varieties from the UK, South America and Australia were selected, and in all except one the highest level of expression was in the coleoptile and Bx 5 was the most highly expressed of the genes tested. Two Chilean varieties, Alifen and Taluen and the Australian variety, Tasman had up to 9 fold higher expression levels of Bx 5 in the coleoptile than the UK varieties. Since analytical studies indicate that the hydroxamic acid DIMBOA is present in the leaves, it is hypothesised that the compound is biosynthesised in the coleoptile and transported to the leaf, and also possibly the root, via the phloem.To determine if wheat varieties differ in levels of induced defence after treatment with cis-jasmone, the development of the grain aphid, Sitobion avenae, was investigated in laboratory bioassays and field trials on winter wheat varieties. Aphid development and population increase varied on the different varieties indicating that there may be differences in the natural constitutive levels of defence. There were also differences in the induced effects between varieties. In laboratory trials the intrinsic rate of population increase was reduced on all the cis-jasmone treated varieties and field populations of aphids were also lower in the treated plots.Root exudates were collected from wheat, the common weed black grass, Alopecurus myosuroides, and white clover, Trifolium repens, which is often sown under cereals in organic farming. The exudates and a solution of the hydroxamic acid DIMBOA were tested for potential allelopathic effects, such as the suppression or stimulation of seed germination, against a number of weed species. The exudates of both wheat, and to a greater extent white clover, slowed the germination of black grass although the data were not significant due to variation across the replicates. DIMBOA caused a greater suppression of germination and most of the weed seeds that did germinate died or had stunted development. These results suggest that wheat varieties with high inducible or constitutive levels of hydroxamic acids would have a dramatic effect on competitive weeds. Identification of the chemical composition of the root exudates, particularly from the clover, could provide an alternative approach to the management of weeds, many of which, e.g. black grass and wild oat, have developed resistance to a range of herbicides.

Objective 4. Identify new strategies for exploiting insect predators and parasitoids via semiochemical tools.Ladybird footprint components comprise a mixture of “generic” straight-chain hydrocarbon compounds, specifically n-tricosane, n-pentacosane and n-heptacosane and species-specific, branched hydrocarbons. For the seven spot

SID 5 (Rev. 3/06) Page 4 of 39

ladybird, Coccinella septempunctata, major specific components include methyl, di- and trimethylalkanes, and for the two spot ladybird, Adalia bipunctata, these include 7-methyltricosane and 9-methyltricosane.Whilst the n-alkanes have been shown to elicit the oviposition avoidance response by aphid parasitoids the branched alkanes have been implicated in mediating the intraspecific interactions. To confirm this latter aspect, samples of ‘footprints’ were obtained by solvent extraction of surfaces on which larval ladybird species had walked. GC-MS analysis suggested that there were 39 alkanes in the tracks of larvae of the 2-spot ladybird, Adalia bipunctata. There were also fatty acid methyl esters. The extract included two putative oviposition deterrent pheromone (ODP) compounds, 11, 19 diMeC31 and 13, 17 diMeC31. A new botanical resource for the ladybird footprint compounds n-tricosane, n-pentacosane and n-heptacosane was investigated from the wax-type material which is generated from extraction of wheat straw, Triticum aestivum, using liquid/supercritical carbon dioxide. A whole straw extract and a hydrocarbon fraction of the extract, collected by liquid chromatography, have been shown by GC analysis to contain these footprint compounds, and were tested in an aphid parasitoid foraging bioassay. In replicated trials, single female Aphidius ervi spent significantly less time on wheat seedlings sprayed with the wax extracts compared to control plants. We envisage a use for these compounds in repelling natural enemies of pests from insecticide treated fields.

Project Report to Defra8. 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).

BackgroundSemiochemicals are signalling chemicals that control pest or natural enemy behaviour and development, or act as signals to switch on defence effects in plants. These effects mean they have potential for use as alternatives to conventional pesticides. By influencing the colonisation of crop plants and subsequent pest and natural enemy population dynamics, semiochemicals can thereby be used to disrupt or direct pests away from the crop and attract them to areas where they can be controlled (e.g. the “push-pull” strategy). Semiochemicals act by manipulating insect behaviour rather than by toxicity and thus offer environmentally benign means of crop protection with which to minimise, supplement, or in the long-term replace, use of broad-spectrum pesticides in Integrated Pest Management (IPM). Chemically-based interactions between plants and other plants or microorganisms can similarly suppress weeds or diseases. In lower input systems, including organic farming, the use of semiochemicals would complement the greater exploitation of biological control agents, selective natural pesticides and pest-resistant cultivars. This continuation project was to combine and focus the output from projects PS2101‘Identification and provision of potential semiochemical tools for use in integrated crop management’, the main purpose of which was to identify and provide semiochemicals and PS2105 ‘Delivery of semiochemicals within

SID 5 (Rev. 3/06) Page 5 of 39

plant-pest-natural enemy systems’, which was to develop the most appropriate methods for delivering semiochemicals, identified in a previous Defra programme (PI03) and in project PS2101, to the crop environment and to achieve the intended effect on representative plant-pest-natural enemy complexes within simplified cropping systems. This project amalgamated the Objectives from PS2101 and 2105 and condensed the key areas into four new Objectives, continuing to investigate new semiochemicals and plant stress chemicals as alternative plant protection technology, provide all semiochemical materials and develop methods for delivering semiochemicals to the crop environment to achieve the intended effect on target species. The research was focussed on the identification and development of natural plant stress signals that can act as plant activators, and the investigation of the role of mixtures of volatile semiochemicals in host plant location and avoidance of unsuitable potential hosts, for pests and their natural enemies. The potential value of exploiting allelopathy in the rhizosphere (interplant interactions either beneficial e.g. to crop plants or detrimental e.g. to weeds) to develop a broader semiochemical approach to crop protection, including suppression of diseases, weeds and pests was also investigated.

Objectives1. Focus on the identification of new plant stress signals to extend the practical range already obtained with cis-jasmone. These will act as plant activators for which optimal delivery systems will be devised and impact on representative crop/pest scenarios, determining effects up to the third trophic level, will be assessed.

2. Identify semiochemicals associated with host plant location and avoidance of unsuitable potential hosts by representative pest species. Determine methods for exploitation of host plant recognition and avoidance cues by pests in novel crop protection strategies.

3. Determine and evaluate the potential of exploiting rhizosphere allelopathy, in controlling pest/plant interactions, and suppressing competitive weeds.

4. Identify new strategies for exploiting insect predators and parasitoids via semiochemical tools.

Where there is significant overlap in results between different Objectives, these have been cross referenced. The Figures are appended at the end of the report.

Objective 1.In Objective 1, the work has concentrated on investigating and confirming the potential of the plant derived isoprenoid hydrocarbon (E)-ocimene as a plant activator and testing the viability of delivering this semiochemical via a natural plant extract. In addition, we have extended studies on the previously identified plant activators, cis-jasmone (i.e. at Rothamsted; Birkett et. al., 2000), methyl jasmonate and salicyclic acid for use in Defra funded project PS2113 “A framework for the practical application of semiochemicals in field crops” and the RELU Programme (Rural Economy and Land Use) funded project ‘Re-bugging the System: Promoting Adoption of Alternative Pest Management Strategies in Field Crop Systems’. Milestone 1. Investigate the role of (E)-ocimene as a plant activator Previous laboratory studies at Rothamsted have shown that barley plants exposed to the volatiles from uninfested thistle plants, Cirsium spp., are less attractive to the cereal aphid Rhopalosiphum padi, than unexposed plants and that settling by both R. padi and the grain aphid, Sitobion avenae, is significantly reduced on Cirsium-exposed plants (Glinwood et. al., 2004; and see final report for PS2101, 2006). Preliminary studies implicated the isoprenoid hydrocarbon (E)-ocimene, which we have also shown to induce defence in barley against a pathogen and to mediate interactions at higher trophic levels, particularly for aphid parasitoids, as the plant activator (see final report for PS2101, 2006). However, the instability of synthetic material precluded confirmation of biological activity at physiologically

SID 5 (Rev. 3/06) Page 6 of 39

relevant levels. A number of essential oils, including material derived from Nepeta cataria in the LINK programme, Competitive Industrial Materials from Non-Food Crops, “New semiochemical opportunities from Nepeta spp. as a non-food crop”, contain high levels of (E)-ocimene. In contrast to the situation with synthetic material, these essential oils are highly stable in relation to their (E)-ocimene content. Nepeta cataria essential oil was fractionated using a combination of chromatographic and distillation techniques, and samples of the fractions were analysed by GC and GC-MS. Enriched fractions of N. cataria essential oil were found to contain both the (Z) and (E)-ocimene isomers in a more stable form. In glass house trials, replicated pots of 6 barley seedlings, enclosed in chimney cages with a constant throughput of air, were exposed to a 1mg/ml solution of the fraction of the essential oil containing the (Z) and (E)-ocimene isomers for 24 and 48h. Control pots were exposed to clean air for 24 or 48h. Three days after the end of the period of exposure, ten apterous R. padi were introduced into each cage. Significantly fewer R. padi settled on the treated plants with both 24hrs and 48hrs of exposure compared to the controls (Figures 1 & 2). These results suggest that the ocimene fraction of N. cataria essential oil is inducing defence mechanisms in the exposed barley plants, which are detected by the aphids.

In addition, the earlier work with Cirsium showed that there is a diel periodicity in the induction of the barley. Only plants exposed to Cirsium volatiles during the scotophase showed a significant reduction in aphid attraction and settling. To study this effect further, samples of volatiles were collected by air entrainment from intact Cirsium vulgare plants during the photophase and scotophase to determine any differences in the volatile profiles produced under light and dark conditions. The GC traces (Figure 3) show that the major peaks, with one exception, are more or less the same during light or dark. The major difference is in the background peaks, which in the night profile are much simpler, and the main substances are thus present in greater proportion. It is likely that the diel periodicity observed in the induction of barley relates to a differential sensitivity of the receiving plant. Confirmation of the identification of Peak 4 could be investigated in future studies.

To determine any generic effects of (E)-ocimene as a plant activator, the N. cataria fraction containing (E)-ocimene was tested against oilseed and turnip rapes as an alternative crop plant/aphid complex compatible with our push-pull strategy in PS2113 (and see Milestone 3 below). Thirty small spring turnip rape, cv Agena, and spring oilseed rape, cv Topic, plants were exposed to a 30mg/ml solution of the (E)-ocimene fraction in a large (90litre) sealed chamber for 24h and the effects on the development of the brassica specialist turnip aphid, Lipaphis erysimi, was determined. Control plants were exposed to the solvent, diethyl ether, alone for 24h. Neonate aphids were weighed on day 1 and after 8 days development on plants of each treatment to obtain the mean relative growth rate (MRGR, van Emden, 1969). The aphids continued development on the same treatment and the time taken to produce their first nymph and the total number of nymphs produced over an equivalent period were recorded and used to calculate the intrinsic rate of population increase, rm (Wyatt and White 1977; see Milestone 6 for a full description of this technique).As observed in other trials (see final report for PS2105, 2006 and Milestone 3 below) the aphids were significantly heavier at 8 days on turnip rape compared to oilseed rape (Figure 4) and developed faster on the turnip rape compared to the oilseed rape. Development of the pest was slower on the plants treated with the N. cataria (E)-ocimene fraction compared to the controls (Figure 4, unpaired t test). For the turnip rape this was only significant for the 8 day weights, but for the oilseed rape both 8 day weight and MRGR were significantly reduced on the (E)-ocimene treatment. The number of nymphs produced and the overall rate of population increase was also significantly reduced on the (E)-ocimene treatment for both plant types (Figure 4). These results indicate that (E)-ocimene has a wider role as a plant signal and that delivery can be made in a protected form or via a plant, for example a food or industrial crop in the same family as Cirsium, i.e. Asteraceae (Compositae). In addition to its action as a plant a plant activator, (E)-ocimene was shown to act directly on insect behaviour in work in a previous Defra funded project, PI0341. Bean plants, damaged by feeding bean seed beetles Bruchus rufimanus, produced elevated amounts of (E)-ocimene and (Z)-3-hexenol. Both compounds were shown to reduce the attractiveness of undamaged flowering bean plants to colonising bruchid beetles, however, (E)-ocimene was difficult to release from standard slow release dispensers. Its

SID 5 (Rev. 3/06) Page 7 of 39

delivery, via the ocimene fraction of N. cataria essential oil, will be field tested in a new Defra LINK “Integrated control of the bean seed beetle, Bruchus rufimanus”.The ability to provide stabilised material, that is suitable for long-term storage, will greatly facilitate the potential for its commercial development for use in crop protection strategies, particularly as an activator of defence. Further funding opportunities will be sought to investigate the scientific basis of this stability, which is most likely due to the presence of powerful antioxidants.

Milestone 2. Devise an optimal formulation for the delivery of cis-jasmoneThe effects of cis-jasmone as a plant activator eliciting defence chemistry have been demonstrated against pest aphid species and their parasitoids and predators on a number of plants including cereals, (Bruce et. al., 2003a,b) legumes and a brassica (see also final report for PS2105, 2006). In addition to the effect as a plant activator, cis-jasmone also has direct effects as a semiochemical, particularly in attraction of highly polyphagous predators such as ladybirds and lacewings. However, it must be noted that a spray application of cis-jasmone in a standard formulation is effective in the field as a plant activator, but the life of the formulation is too short (less than 1 hour) to exploit the direct effect on these predators. In addition, the timing of the application is crucial to maximise the effect against aphids. Thus, a new slow release point source formulation was developed to provide increased longevity (several weeks) and to alleviate the problems with timing of application. However, new formulations that have increased longevity, and are more compatible with application by conventional sprayers, are still required, particularly for application to the crop in the field studies conducted in the related Defra project PS2113 and in the RELU Programme (Rural Economy and Land Use) funded project ‘Re-bugging the System: Promoting Adoption of Alternative Pest Management Strategies in Field Crop Systems’.

Collaborative studies were undertaken with a number of SMEs who produced prototype, slow release formulations, principally based on microencapsulation. The test formulations were applied to oilseed rape plants, all at the same growth stage, using a track sprayer and an application rate equivalent to 50g cis-jasmone/ha. A single leaf of an equivalent size from each treated plant was then entrained (Agelopoulos et. al., 1999) for periods of 30 minutes, 2h after treatment then at 24h intervals, until no cis-jasmone was detectable by GC analysis in the collected samples. Typical results are presented in Figure 5. Formulation 2 released high levels of cis-jasmone in the first few hours after application, but showed no detectable release at 48h. Formulation 1 released less initially and cis-jasmone was just detectable at 48h. However, formulation 3, which did not use microencapsulation, was more promising. This released the cis-jasmone at a much lower rate to begin with and detectable levels were still being produced up to 6 days after application. This enhanced life will overcome the problem with timing of application and provide direct attraction to predators at the key stage of early colonisation by the pest. In a no choice behavioural trial, replicated groups of 10 alate grain aphids, S. avenae, were put into cages with wheat seedlings, either sprayed with cis-jasmone formulation 3 or with a blank formulation, and settlement and nymph production were recorded. Aphid settlement was significantly reduced and fewer nymphs were produced on wheat plants treated with the cis-jasmone formulation (Figure 6).

This formulation of cis-jasmone will be field tested in PS2113 and in the RELU programme. It will also be investigated for suitability for the slow release delivery of other semiochemicals.

Milestone 3. Determine the effects of plant activators on spring oilseed rape and turnip rapePrevious work at Rothamsted (in Defra projects in PI03) and elsewhere in Rothamsted has shown that application of methyl jasmonate (MeJa) to brassica plants induces the production of indolylglucosinolates, defence compounds which help protect brassicas from generalist herbivores. In addition, application of salicylic acid (SA) induces production of alkyenylglucosinolates, defence compounds that break down upon plant damage to produce volatile isothiocyanates, which are highly attractive to brassica specialist pests such as the pollen beetle, Meligethes aeneus. In field trials in the continuation Defra Project 2113, MeJa and SA were applied respectively to spring oilseed rape (SOSR) and the trap crop turnip rape as part of the development of a pest management strategy for the mainly

SID 5 (Rev. 3/06) Page 8 of 39

coleopterous pests. The MeJa was applied to the SOSR to reduce attractiveness of the crop to the coleopterous brassica pests and provide protection for the crop from more generalist herbivores. SA was applied to the trap crop, to induce production of 2-phenylethylglucosinolate and consequently the volatile catabolite, 2-phenylethyl isothiocyanate, which was to maximise the attractiveness of the trap crop for the main coleopterous pests and their parasitoids. Air entrainment samples were taken from the turnip rape and from the SOSR in situ in the field to determine the volatile profiles, before and after treatment with plant activators. Single racemes were enclosed in a custom made glass vessel that had fitted around the stem at the bottom enabling volatile collections to be made with live rather than cut material. Purified air that had passed through a charcoal filter was pushed into the vessel at a rate of 500ml/min. Air was drawn from the vessel at a rate of 400ml/min passing through a Porapak Q filter which trapped the volatiles on exit. Samples were eluted with redistilled diethyl ether and analysed by GC. A typical trace is shown in Figure 7. There was upregulated emission of some early eluting compounds which require further investigation. In addition, experiments were conducted to assess the effect of SA and MeJa, applied at a range of concentrations to turnip rape and oilseed rape respectively, on host plant selection by the specialist pests, pollen beetles, Meligethes aeneus, and the aphid Brevicoryne brassicae, and by the generalist aphid, Myzus persicae. Glasshouse-grown plants were sprayed with 0.1 mM, 1 mM, 10 mM, or 100 mM solutions of the chemicals formulated with the non-ionic surfactant ethylan BV. Control plants were sprayed with ethylan BV blank formulation. The treated plants were kept in the glasshouse for 48 h before use, to allow induction of glucosinolates to take place. Control plants were kept in a separate glasshouse from treated plants to avoid any signalling effects from treated plants.

Effect on pollen beetle colonizationTwo semi-field arena bioassays were conducted in a modified polytunnel (Cook, et. al., 2007) to assess the effect of the concentration of SA and MeJa, applied to turnip rape and oilseed rape respectively, on host plant selection by pollen beetles. One plant of each treatment was placed across the span of the polytunnel and 1,000 pollen beetles, collected from the field and starved for 24h, were released down wind. After 3 h, the numbers of beetles colonizing each plant was recorded. Five replicates were conducted of each test in a Latin square design. The results were analysed by ANOVA. Approximately 5-10% of the beetles released were recovered in each experiment. The numbers of pollen beetles colonizing turnip rape plants treated with different concentrations of SA did not differ significantly from the control (F = 0.66; df = 4, 12; P = 0.634) (Figure 8). Similarly, the numbers of beetles colonizing oilseed rape plants treated with MeJa did not differ significantly from the control (F = 0.38; df = 4, 12; P = 0.821) (Figure 9).

Effect of plant defence-inducing chemicals on the growth rate of aphidsTreated plants were placed in controlled environment rooms and generalist, M. persicae, and specialist, B. brassicae, aphid nymphs (1 day old) were caged in groups of three on the plants. The aphids were weighed before the experiment and after 6 days. Mean aphid weights were compared by ANOVA. There was no significant difference in the birth weight of the aphids, or in the weight of the aphids on the oilseed rape treated with MeJa after 6 days (Figure 10). However, the weight of both of the aphid species on the turnip rape treated with SA after 6 days was significantly less than the weight of the aphids on the control plants (Figure 11).

There is no evidence from the experiments conducted here with a narrow range of elite varieties that oilseed rape can be made less attractive to pests following treatment with MeJa or that application of SA to turnip rape could improve the performance as a trap crop. However, the negative effect of SA on the growth rate of aphids is interesting and in the opinion of the researcher requires further investigation. Furthermore, as has been observed with cis-jasmone induced defence in elite wheat varieties, the response to cis-jasmone is highly dependant on variety, opening the opportunity for further improvements by selection of varieties and directed breeding programmes.

Objective 2.

SID 5 (Rev. 3/06) Page 9 of 39

This objective extended our previous studies on the underlying mechanisms mediating host location by insects, which have shown that specific ratios of ubiquitous plant-derived compounds can provide positive information relating to potential hosts (Bruce et. al., 2005). Also, by the same argument, the unsuitability of a plant as a host can be denoted by incorrect ratios of these ubiquitous plant semiochemicals. We used two model, but economically relevant crop/pest complexes (wheat/grain aphid/orange wheat blossom midge and beans/black bean aphid/pea and bean weevil), to explore the hypothesis that for each crop, modification of the specific ratio, by augmentation of one or a limited number, of key members of the attractive semiochemical mixture will interfere with the host location abilities of the whole pest complex for that particular crop.

Milestone 4. Investigate key semiochemicals for pests of cerealsKey attractive volatile semiochemicals, produced by wheat at the growth stage susceptible to the orange wheat blossom midge, Sitodiplosis mosellana, have already been obtained by air entrainment techniques, identified using coupled GC-electrophysiology and their behavioural activity determined in an olfactometer bioassay (Birkett et.al., 2004 and see final report for PS2101, 2006). Female midges were attracted to a 6 component synthetic blend of volatiles, when they were presented in the natural ratio and concentration, and the blend was as attractive as the natural air entrainment sample of the ears. In subsequent assays, the blend was reduced to three components, acetophenone, (Z)-3-hexenyl acetate and 3-carene, and yet remained as attractive as the natural sample. However, entrainments of a range of midge susceptible wheat varieties showed that production of these three components varied greatly (Figure 12) and that the composition of minor components was also very variable. One component, 6-methyl-5-hepten-2-one, was found in the volatile profile of only a few varieties, but when it was presented to female midges in an unnatural ratio, (15ng instead of 5ng) the blend was not attractive. Thus, for this insect, these data provide clear evidence that host recognition is conferred by ubiquitous compounds, some of which must be present in specific ratios. Further investigation of the potential for disrupting these ratios, for example by selective plant breeding, could provide new varieties with reduced susceptibility to pest colonisation.

In collaboration with an industrial company, a sophisticated electronic emission device was evaluated as a delivery mechanism for the simplified attractive blend of orange wheat blossom midge compounds, acetophenone, (Z)-3-hexenyl acetate and 3-carene, in a trap under field conditions. This device provided fine control of the ratio of compounds emitted. Nevertheless this trap did not perform well presumably because of competition with natural sources of kairomones under field conditions. When evaluated with the female midge sex pheromone, the device was as effective in capturing male midges as the standard field trap. This device has great potential for other practical field applications.

Semiochemicals mediating host plant attraction were also investigated for the grain aphid, Sitobion avenae. As for the midge, wheat volatiles were collected by air entrainment and aphids were exposed to samples of these volatiles in an olfactometer bioassay. This bioassay compares the amount of time spent in treated and control arms of an arena where aphids are exposed to discrete odour fields. There was no significant attraction to most samples of wheat volatiles collected under glasshouse conditions, however, a field collected sample of wheat volatiles at the milk grain growth stage was significantly attractive. This is surprising, since the volatile profiles of wheat varieties grown under glass or in the field were very similar. The field samples were analysed and chemicals identified from them (4-isopropylbenzaldehyde, 1,4 diethyl benzene, 4-isopropyl-benzyl alcohol, 2-tridecanone) were also tested in the olfactometer bioassay. There was significant attraction to 4-isopropylbenzaldehdye (P = 0.03, paired t test). A synthetic blend of volatiles identified from wheat variety ECO22 (Advanta) at the ear emergence growth stage was also tested with S. avenae. Interestingly, this blend, which was attractive to the orange wheat blossom midge, was significantly repellent to S. avenae. Two components of this blend, α-pinene and 6-methyl-5-hepten-2-one, were repellent when tested on their own. α-Pinene has already been identified as the repellent component of cumin oil for S. avenae and 6-methyl-5-hepten-2-one as one of the key chemicals mediating host plant acceptance by the midge (see above and PS2101 final report, 2006). These data indicate that the aphid and the midge are using similar chemical cues

SID 5 (Rev. 3/06) Page 10 of 39

from the host plant, but further study is necessary to understand how these cues can be manipulated to affect both pest species.

The slow release formulation developed for cis-jasmone (see Milestone 2 above) would also be suitable for the formulation of these wheat semiochemicals to enable their use under field conditions. As alterations in ratios of host-plant semiochemicals have been shown to switch off attraction it is possible that field formulations targeted at disrupting host plant attraction by distorting ratios of compounds emitted can be developed.

Milestone 5. Investigate key semiochemicals for pests of legumesGC coupled electrophysiological studies, already carried out on the pea and bean weevil, Sitona lineatus, identified key semiochemicals from the legume host as (Z)-3-hexenyl acetate, (Z)-3-hexenol, 1-octen-3-ol, hexanal, linalool, methyl salicylate, benzyl alcohol, 2-phenylethanol, α-terpineol and β-caryophyllene. Many of the compounds are perceived by specialist olfactory cells and a number, including the green leaf volatiles (Z)-3-hexen-1-ol and (Z)-3-hexenyl acetate, are paired with cells for the aggregation pheromone, 4-methyl-3,5-heptanedione, indicating that the weevil can discriminate between ratios of these compounds. Field trapping studies with the pheromone, plus complex mixtures of the host plant volatiles, showed that some host-plant mixtures enhanced the attraction of the weevil to the trap whilst other mixtures resulted in reduced attraction.

In this milestone we have extended the study to include aphid pests of legumes. Key host plant compounds for the black bean aphid, Aphis fabae, have been obtained by air entrainment techniques and identified using coupled GC-electrophysiology (Figure 13). Only three of these compounds were perceived by S. lineatus. The twelve electrophysiologically active compounds for A. fabae, identified using GC-MS, were (-)-germacrene D, (E)-β-farnesene, undecanal, decanal, methyl salicylate, nonanal, (-)-linalool, 6-methyl-5-hepten-2-one, benzaldehyde, (E)-3-hexenal, (Z)-3-hexenyl acetate and 4,8,12-trimethyl-1,3-7,11 tridecatetraene. These compounds elicited significant attraction in an olfactometer bioassay when presented in the natural ratio. Three of these semiochemicals, methyl salicylate, 6-methyl-5-hepten-2-one and 4,8,12-trimethyl-1,3-7,11 tridecatetraene are associated with plant stress, particularly through pest or pathogen damage, and have been shown to contribute to reduced attraction of aphids, and increased attraction for aphid parasitoids, to host plants. In addition, 6-methyl-5-hepten-2-one and 4,8,12-trimethyl-1,3-7,11 tridecatetraene are released by a number of crop plants when they are induced by the plant activator cis-jasmone. When tested alone in an olfactometer assay, 4,8,12-trimethyl-1,3-7,11 tridecatetraene was shown to be repellent to a number of aphid species (see final reports for PS2101 and PS2105, 2006). However, in preliminary bioassays, the behaviour of A. fabae was found to be unusual since it was significantly attracted to 4,8,12-trimethyl-1,3-7,11 tridecatetraene and to 6-methyl-5-hepten-2-one and was unaffected by methyl salicylate. These results support the previous observation that this particular aphid is unaffected by cis-jasmone and actually benefits when the defences of bean plants are induced (see final reports for PS2105, 2006). We suspect that A. fabae behaves differently to other aphids because it occurs in very large colonies and is therefore somehow capable of dealing with the stress related chemistry produced by the plant. As part of a BBSRC funded PhD studentship, further work on A. fabae response to modified blends in unnatural ratios is planned once bioassays to identify a minimum component blend of attractants have been completed.

Objective 3. Determine and evaluate the potential of exploiting rhizosphere allelopathy, in controlling pest/plant interactions, and suppressing competitive weeds.Allelopathy is defined as ‘any direct or indirect harmful or beneficial effect by one plant (including micro-organisms) on another through production of chemical compounds that escape into the environment’. This Objective advanced studies in the previous PS2101 where it was shown that the plant activator, cis-jasmone, induces aphid resistance in some wheat cultivars and reduces cereal aphid populations in the field to an extent that would potentially maintain pest populations below economic threshold levels thus limiting the requirement for pesticide application (Pickett et. al., 2007). Preliminary laboratory studies showed that some of the genes involved in the biosynthesis of

SID 5 (Rev. 3/06) Page 11 of 39

hydroxamic acids (HAs), e.g. DIMBOA, are up regulated by cis-jasmone, suggesting that they may play a role in the induced resistance since DIMBOA, and a number of related compounds in its biosynthetic pathway, have been shown to be active against a range of insect pests, including aphids. In addition, production of HAs in roots can result in their release into the rhizosphere, giving some control of potentially competing plants.

Milestone 6. Investigate constituent and inducible levels of hydroxamic acids in UK wheat varieties. The genes (Bx genes) in the hydroxamic acid biosynthetic pathway in wheat have recently been cloned and sequenced (Figure 14), but no detailed analysis of their regulation in response to plant activators or pathogen infection, or in relation to product accumulation has yet been achieved. Consequently, in order to confirm the role of this pathway in defence in cereals and to provide the basis for a breeding program to develop disease resistance in wheat a detailed spatial and temporal gene expression analysis has been undertaken.

Initially, expression of four biosynthetic genes, encoding cytochrome P450s (Bx genes 2 -5, Figure 14) were measured in the root, coleoptile and leaf of 12 day old seedlings by real time PCR. Varieties from the UK, South America an Australia were selected for this analysis and the results are shown in Table 1. In all except one of the varieties (the UK midge resistant variety, Welford, which had high root expression) the highest level of expression was in the coleoptile and Bx 5 was the most highly expressed of the genes tested. A comparison between the varieties revealed that the two Chilean varieties, Alifen and Taluen and the Australian variety, Tasman had up to 9 fold higher expression levels of Bx 5 in the coleoptile than the UK varieties.

Table 1. Relative expression levels of the hydroxamic acid biosynthetic gene, Bx 5, in different tissues of 8 varieties of hexaploid wheat. Expression was measured by real time PCR, using actin as the endo gene and values are expressed relative to the levels detected in Alifen root tissue, set at a value of one.Plant tissue Alifen Axona Solstice Malacca Taluen Tasman Option WelfordRoot 1 1.6 3.5 2.2 1.7 2.8 2.73 13.5Inner coleoptile 67 28 29 15.4 128 60 16 8Outer coleoptile 43 24 8.75 17.4 137 52 25 20First leaf 1.2 2.6 2.9 3.4 14.4 2.7 1 0.6Second leaf 1.04 0.5 0.3 0.3 7.7 1.02 0 0

Expression in the first leaf and root was similar, but with the exception of Bx 3 in all varieties and Bx 5 in Taluen, at this stage of development the genes were hardly expressed in the second leaf. Since chemical analytical studies indicate that DIMBOA is present in the leaves, it seems most likely that the compound is biosynthesised in the coleoptile and transported to the leaf, and also possibly the root, via the phloem.

The concentration of DIMBOA is highest in young wheat seedlings and decreases rapidly during early development but the regulation of Bx gene expression during this time period has not been studied. To address this, changes in expression of these genes over the first 14 days after germination in each of the different tissues was analysed. In addition to the Bx genes, the expression of three glucosidase genes was also measured. The enzymes encoded by these genes have been shown to convert the inactive DIMBOA glucoside to the aglucone and this reaction represents an important step in the regulation of the activation of the pathway. Due to the large number of measurements required for this investigation, only a single hexaploid variety, Welford, was studied and the inner and outer coleoptile and first and second leaves were combined. The results obtained demonstrated that the expression of all of the Bx and glucosidase genes decreases dramatically over this time period, mostly following a similar patter to that shown in Figure 15 A, B and C for each of the respective tissues. The major exception to this is the behaviour of Bx 3 in the leaf (Figure15 D) where expression is more sustained.

SID 5 (Rev. 3/06) Page 12 of 39

The effect of the plant activator cis-jasmone on both the biosynthetic and activating genes was measured in different tissues using real time PCR in a selection of varieties. 12 day old seedlings were chosen because at this stage constitutive levels are low and no longer decreasing significantly. In the variety Welford a transient increase in gene expression of the Bx genes in the coleoptile was detected six hours after cis-jasmone treatment. By 24 hours after treatment levels had returned to normal (Figure 16 A). No effect was observed on the expression of these genes in the root or leaf tissue but a similar increase in the glucosidases, of a comparable magnitude was detected in the root tissue (Figure 16B). Other varieties were also tested for the effects of cis-jasmone on Bx gene expression and although this aspect of the work is still in progress, preliminary data suggests that the varieties that contain higher levels of constitutive expression of the Bx genes show lower levels of induction.

The investigations will now be expanded by including additional varieties of wheat for testing. These will include Durum wheat varieties, Triticum durum, from Syria that demonstrate varying degrees of resistance to insects, and diploid wheat lines, Triticum monocccum, including take all resistant lines available from the WGIN program. This work is being taken forward in a BBSRC industrial partnership Crop Science Initiative project in collaboration with the British Wheat Breeders and work here, and new work in this direction, will be needed to bring into use via LINK.

Assessment of DIMBOAPreliminary work was carried out in previous Defra project PS2101 (see final report PS2101, 2006) where liquid phase extraction (LPE) and vapour phase extraction (VPE) were used to evaluate the impact of the plant activator, cis-jasmone, on the secondary metabolism of wheat, Triticum aestivum, var. Solstice. The LPE demonstrated that levels of hydroxamic acids (HAs), particularly the most generally active, 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), were significantly higher in leaves and roots of T. aestivum treated with cis-jasmone when compared with untreated plants (Blassioli Moraes et. al., in press). This suggested that DIMBOA may play a role in the induced resistance, since DIMBOA, and a number of related compounds in its biosynthetic pathway, have been shown to be active against a range of insect pests, including aphids. To determine whether levels of inducability are variable among wheat varieties, a more rapid quantification of the HAs using HPLC was also established and the effects of cis-jasmone treatment on 14 winter and one spring UK wheat variety (Axona) was investigated. Ten seedlings of each winter variety plus ten Axona seedlings as a reference, grown separately in small pots in vermiculite, were sprayed with cis-jasmone, formulated with the wetter Ethylan BV, with the wetter alone or untreated, and left for 48h for induction to occur. After 48h, the plants were carefully removed from the vermiculite, the roots, leaves and stems were separated and the pooled plant parts were weighed and stored at -20°C to disrupt the plant cells. While still frozen, the plant parts were cut into small pieces and homogenised in 20ml distilled water after which they were left to hydrolyse for 1hr. The samples were then centrifuged to remove the solid phase and a 50µl aliquot of each of the liquid samples was subjected directly to HPLC analysis. Analysis has to be done soon after extraction since DIMBOA eventually breaks down under aqueous conditions. Unfortunately, the levels of DIMBOA found in these samples were too variable, both between and within varieties extracted at different times, to draw any firm conclusions. An alternative extraction procedure that stabilises the DIMBOA has now been devised and is available for future studies.

Laboratory bioassay of aphid development on cis-jasmone treated wheat varietiesTo determine if wheat varieties differ in levels of induced defence after treatment with cis-jasmone, the development of the grain aphid, Sitobion avenae, was investigated in laboratory bioassays on four winter wheat varieties. These had been investigated for volatile production and/or field tested for susceptibility to cereal aphids in PS2105 (see final report PS2105, 2006). Since most of the preceding laboratory investigation with cis-jasmone and cereal aphids had been done with the spring wheat, Axona, this variety was used as a standard in each trial. Seedlings, at the two leaf stage, were sprayed with a water and wetter based formulation of cis-jasmone at a rate of 50g a.i./ha, or with the wetter formulation alone, and the two sets of plants were kept in separate compartments of a glass house for

SID 5 (Rev. 3/06) Page 13 of 39

48h until tested. Neonate aphid nymphs were weighed and caged on the plants for 7 days after which they were re-weighed and the mean relative growth rate (MRGR) was calculated. The nymphs were returned to plants of the same treatment and the time of production of their own first nymph was recorded (D). The number of nymphs produced was recorded for an equivalent time (FD). Nymphs were removed every other day to prevent overcrowding effects. At the end of the trial the intrinsic rate of population increase (rm), a measure of fecundity, was calculated using the equation (FD/D) x C, where C = a constant calculated by Wyatt and White, (1977). Aphid performance was compared in each trial using an unpaired t test (Figure 17 shows the results for measurement of rm). Development varied on the different varieties indicating that there may be differences in the natural constitutive levels of defence between varieties. There were also differences in the induced effects between varieties. However, when induced with cis-jasmone, none of the winter varieties tested reduced the rm of S. avenae as significantly as Axona. The rm was reduced on all the varieties, significantly so on Solstice and Welford.

In addition, the development of the other cereal aphids, the rose-grain aphid, Metopolophium dirhodum (Figure 18), and the bird cherry-oat aphid, Rhopalosiphum padi, was determined on a limited number of wheat varieties treated with cis-jasmone. Neither of these species showed a significant effect of cis-jasmone treatment on development rates, either for weight-gain of for fecundity. These results confirm the findings of studies in PS2105 (see final report PS2115, 2006) where the specialist aphids tested so far were shown to be more sensitive to cis-jasmone treatment to their host plants than the more polyphagous aphid species feeding on the same plants. Here, cis-jasmone treatment has a positive effect on S. avenae, while it may be neutral for the other species. When considering the use of this plant activator in pest management strategies, the effects of cis-jasmone treatment on crop varieties and on aphid species complexes should be studied in detail to determine the most efficacious crop plant targets. In addition, a fuller understanding of the mode of action of cis-jasmone activation of defence related pathways within the target plant is required and will be a target for future research.

Field experiment with point source formulation of cis -jasmone against different wheat varieties A randomised block trial, to investigate the effect of cis-jasmone on the colonisation by and development of cereal aphid populations on different wheat varieties, was conducted in the field. Replicated plots of four different winter wheat varieties, three chosen from the laboratory study described above (Solstice, Hereward and Welford) and Consort, the standard variety used in all previous field studies with cis-jasmone (Bruce et. al., 2003a), were either treated with point sources of cis-jasmone released from pvc rope, or left untreated as controls. Visual assessments of aphid populations on 100 tillers per plot were made approximately weekly, weather permitting, throughout the trial. An assessment of orange wheat blossom midge, Sitodiplosis mosellana, infestation was made on 25 ears per plot in late June and yields were taken at the end of the experiment. Aphid numbers were low and patchily distributed and no significant differences were obtained when data were subjected to ANOVA. Numbers of wheat blossom midge were very low and no treatment effects were observed. Since pest pressure was low there were no effects on yield. However, the total aphid numbers on the different plots did show a convincing trend for most varieties (Figures 19-26). Overall, S. avenae numbers were lower in most treated plots compared to control plots except for Welford where the colonisation in the cis-jasmone plots was less clear. In addition, despite being apparently unaffected by the induced defences of wheat under laboratory conditions (see above), numbers of M. dirhodum, the predominant aphid in this season, were comparatively lower in the treated plots except for Hereward where no apparent treatment effect was seen (Figure 24). This could be accounted for by the semiochemical effect of cis-jasmone, acting purely as a repellent for this aphid, whilst attracting parasitoids and predators to reduce aphid numbers. The possibility of exploiting differential inducability between wheat varieties offers great potential for targeted pest control. Future research to understand the mechanism underpinning induced response in wheat, particularly in relation to the BBSRC funded project identifying the genes upregulated by cis-jasmone in Arabidopsis, will be pursued. In the opinion of the researcher, the efficacy of different formulations as repellents or induction agents also requires further investigation.

SID 5 (Rev. 3/06) Page 14 of 39

Milestone 7. Investigate the effects of root exudates on seed germinationRoot exudates were collected from plants grown hydroponically in aerated nutrient solution for approximately one month. Exudates collected included wheat (cvs Axona and Solstice), which were expected to contain hydroxamic acids e.g. DIMBOA (Wu et. al. 2000), and a number of other associated plant species, including the common weed black grass, Alopecurus myosuroides, and white clover, Trifolium repens, which is often sown under cereals in organic farming. In addition, to provide an authentic standard, DIMBOA was extracted from maize seedlings, using a large scale method based on Larsen and Christensen, (2000) and chemically authenticated using HPLC, NMR and GC/MS techniques. The exudates and a solution of DIMBOA were tested for potential herbicidal effects such as the suppression or stimulation of seed germination between species.

Isolation of DIMBOA from Maize SeedlingsMaize seeds (Zea mays, cv Hudson) were grown in the dark and, 7 days after germination, the shoots were harvested and stored at -18°C overnight, to disrupt the cells. Frozen shoots were crushed in a mortar with distilled water and then left for approximately 1 hour at room temperature, to allow the glucoside to be hydrolysed to DIMBOA. The solution was then filtered to remove the plant material and Amberlite XD7 resin was added to the extract and the mixture was stirred for 1 hour, to ensure the Amberlite ‘bonded’ to all the DIMBOA present. The solution was then filtered off and the DIMBOA was washed off the Amberlite with acetone. The acetone was then evaporated leaving a brown aqueous solution, which was extracted with diethyl ether and dried with magnesium sulphate. The ether was then evaporated leaving brown oil from which DIMBOA formed as reddish crystals. The identity of the DIMBOA was confirmed using HPLC, NMR and GC/MS techniques, the latter with derivatised material (Figure 27).

Germination testsThree replicate Petri dishes (9cm diameter) each with one filter paper, 2ml of exudate (or water as a control, since the level of nutrient left in the root exudates sample was unknown, but probably fairly depleted) and 10 or 20 test seeds, depending on the seed size, were set up for each root exudate and seed type tested. Seeds were surface sterilised in 1% sodium hypochlorite before being tested. Once the seeds had been added, each dish was covered with cling film to help reduce evaporation. The dishes were kept at 22°C and inspected regularly to record seed germination. Root exudates of Axona and Solstice wheat, were tested against seeds of the weed black grass and spring oilseed rape (Brassica napus cv Topic), which is often controlled with herbicide when it occurs as volunteer plants in following cereal crops. In addition, exudates of black grass were tested against Solstice wheat and spring oilseed rape, and white clover exudate was tested against seeds of spring oilseed rape, black grass and Solstice wheat.

There were no significant effects of any of the root exudates on the germination of the seeds tested (paired t tests). However, there were some interesting trends for the germination of black grass shown in Figures 28-30. The exudates of both wheat varieties, and to a greater extent the white clover exudate, slowed the germination of black grass although the data were not significant due to variation across the replicates. In addition, a 1mg/ml solution of DIMBOA in 10% aqueous acetone, and 10% aqueous acetone and water as controls, were tested against Solstice wheat, and a number of weed species including black grass, charlock (Sinapsis arvensis), spring oilseed rape (as an example of a volunteer plant), common poppy (Papaver rhoeas), wild oat (Avena fatua) and mayweed (Matricaria perforata). These trials recorded germination and development of the seeds. The charlock and mayweed had poor germination and were discarded from the trials. The results for the germination of the other target species are shown in Figures 31-35. In all the tests, the acetone treatment had some effect on germination, but, except for the wheat, the DIMBOA showed a greater suppression of germination and most of the weed seeds that did germinate died or had stunted development. These results suggest that wheat varieties with high inducible or constitutive levels of hydroxamic acids would have a dramatic effect on competitive weeds and volunteer plants.

These preliminary trials are very promising, but will be repeated to confirm the results and the chemical composition of the root exudates, particularly the clover, need to be investigated further, since this

SID 5 (Rev. 3/06) Page 15 of 39

offers an alternative approach to the management of weeds, many of which, e.g. black grass and wild oat, have developed resistance to a range of herbicides. Other work, developing DIMBOA and other hydroxamic acids against insects, could be exploited and developed to provide an additional alternative weed control system. Objective 4. This objective has focused on providing the semiochemical tools for the manipulation of polyphagous predators such as ladybirds and lacewings, particularly in relation to understanding interactions between different members of natural enemy guilds since they are frequently in competition with each other for the same food resource. In addition, such semiochemicals can be used to manipulate these predators in push-pull strategies e.g. by moving them from field margins into the fields in the spring and early summer when they can exert a major impact on pest populations. We have already identified the semiochemicals derived from ladybird foraging on plants that are used by aphid parasitoids to avoid oviposition in aphid colonies that are already attacked by the predators (Nakashima, et. al., 2004 & 2006). A formulation of these semiochemicals has been provided for field testing in the RELU project ‘Re-bugging the System: Promoting Adoption of Alternative Pest Management Strategies in Field Crop Systems’ in 2006 and 2007. However, chemical identification and behavioural studies have shown that there are additional semiochemicals in these predator “chemical footprints” that mediate both inter- and intra-specific interactions. These interactions relate specifically to the oviposition deterrent responses of adults following detection of intraspecific larval footprints.

Milestone 8. Isolate and identify footprint compounds mediating inter- and intra-specific interactions for ladybird and lacewing speciesRecent studies have shown that for ladybirds, footprint components comprise a mixture of “generic” straight-chain hydrocarbon compounds, specifically n-tricosane, n-pentacosane and n-heptacosane, and species-specific branched hydrocarbons. For the seven spot ladybird, Coccinella septempunctata, major specific components include methyl, di- and trimethylalkanes, and for the two spot ladybird, Adalia bipunctata, these include 7-methyltricosane and 9-methyltricosane.Whilst the n-alkanes have been shown to elicit the oviposition avoidance response by aphid parasitoids the branched alkanes have been implicated in mediating the intraspecific interactions. To confirm this latter aspect, samples of ‘footprints’ were obtained by solvent extraction of surfaces on which larval ladybird species had walked.

Composition of larval tracks of A. bipunctataTwo-spot ladybirds, A. bipunctata, came from a stock culture maintained in the laboratory at 18± 1°C and a photoperiod of 16 h light and 8 h darkness. They were reared in 5-liter plastic boxes containing a piece of corrugated filter paper on which females laid eggs. Three time a week the ladybirds were fed an excess of pea aphids, Acyrthosiphon pisum. Two stems of broad bean, Vicia faba L., were added to each box to improve the survival of the aphids.

Larvae were taken from the stock culture, isolated in 5 cm diameter Petri dishes and starved 16 hours. At the end of that period, each female was then carefully placed in a 12 x 75mm glass tube where it remained without any food for 24 hours. Each tube was sealed with a cotton plug. Beetles treated in this way did not produce faeces. They did not contaminate the tube by reflex bleeding as it is the case when larvae interact with one another or are roughly handled. As the ladybirds were hungry, they spent most of the time walking. After 24 h the larvae were removed and returned to the stock culture. This was repeated 30 times and all the tubes were kept under the same conditions as the stock culture. In total, 50 batches were prepared, leading to a total of 1500 larval track extracts.

For each batch, hexane (1 ml) was poured into the first tube, which was agitated by a circular movement for about 5 seconds. The solvent was then taken up with a microsyringe and transferred to the second tube and so on until the 30th tubes. All the tubes were similarly washed a second time with another ml of hexane. The resulting 2 ml of hexane extract were mixed in a small vial and completely evaporated off under a gentle stream of nitrogen. The dry residue was dissolved in hexane and 1 l of this solution

SID 5 (Rev. 3/06) Page 16 of 39

analysed using a VG Autospec GC-mass spectrometer (GC-MS) coupled to a non-polar DB-1 column (30oC/5 min, then 5oC/min to 280oC). The operating conditions were: cool on-column injector mode; carrier gas helium. The mass spectra were recorded in the electron impact mode at 70 eV, source temperature 250oC. The peaks detected on the chromatograms were identified on the basis of retention data and fragmentation patterns. The identifications were confirmed by comparison of spectra with those recorded in the NIST library.GC-MS analysis suggested that there were 39 alkanes in the tracks of larvae. There were also fatty acid methyl esters that were not identified. The extract included two putative oviposition deterrent pheromone (ODP) compounds, 11, 19 diMeC31 and 13, 17 diMeC31.

Synthesis of the 13,17-diMeC31 compound was initiated starting from the homochiral (S)-(-)-β-citronellol. Tosylation of the primary alcohol proceeded in a smooth manner (93% yield), followed by cuprate-mediated addition of decylmagnesium bromide to incorporate the 13,17-dimethyl structure. Allylic oxidation using selenium dioxide proceeded in moderate yield (28%), with allylic alcohol being converted to the corresponding iodide in one step (69% yield). The iodide was converted to the phenyl sulphonate in high yield (83%). Julia-type addition of the sulphonate to tridecyl magnesium bromide, followed by removal of the sulphonate group and catalytic hydrogenation will generate 13,17-diMeC31 for subsequent behavioural tests.

A new botanical resource for the ladybird footprint compounds n-tricosane, n-pentacosane and n-heptacosane has been investigated, using the wax-type material which is generated from extraction of wheat straw, Triticum aestivum, using liquid/supercritical carbon dioxide. A whole straw extract and a hydrocarbon fraction of the extract, collected by liquid chromatography, have been shown by GC analysis to contain these footprint compounds, and were tested in an aphid parasitoid foraging bioassay. In replicated trials, single female Aphidius ervi were introduced onto a single treated or control wheat seedling and the parasitoids behaviour was observed and recorded until she left the plant. Data were analysed by a paired Wilcoxon test (Figures 36 & 37) Here, the data showed unequivocally that foraging A. ervi spent significantly less time on wheat seedlings sprayed with the wax extracts compared to control plants (whole extract p<0.03; hydrocarbon fraction p<0.05). Botanical resources for the putative A. bipunctata compounds are still being investigated.

Concluding Comments

This report outlines the progress made in four separate, but related, objectives all aimed at the common goal of using semiochemicals and semiochemical approaches in novel and environmentally friendly pest control strategies. As such, each has made progress towards being part of IPM with some nearer to field implementation than others. Specifically:

Objective 1. (E)-ocimene has been shown to have potential for inducing defence in barley against pathogen and aphid attack. Importantly, it can be delivered in a stabilized form as an essential oil. Likewise, formulations suitable for field deployment have been developed for another plant activator, cis-jasmone. In the short-term, these compounds will be field-tested (PS2113, RELU, LINK) and could be available for practical use in IPM in the short to medium term.

Objective 2. It has been shown that modifying ratios of semiochemicals produced by plants can interfere with host location by insects. Ratios have differing effects on species using the same compounds. This approach clearly has promise for IPM, but will need to be delivered by selective breeding programmes to produce varieties with different volatile profiles, and thus should be seen as a medium to long-term approach.

Objective 3. Rhizosphere allelopathy has been investigated by molecular analysis of hydroxamic acid (HA) biosynthesis. This has been a fundamental study, but it has brought us to a position where we can show that there is potential to alter the production of HAs and hence affect plant/pest interactions. This work will continue through BBSRC CSI funding. The implementation of this approach is again best

SID 5 (Rev. 3/06) Page 17 of 39

achieved via selective breeding programmes and hence has a medium to long-term application to IPM. It has also been shown that the HA, DIMBOA, affects germination and growth of neighbouring weeds. This is preliminary work and would require further in depth study and thus, offers promise of a practical use in IPM in the long-term

Objective 4. The exploitation of predators and parasitoids to control pests has been studied previously, mainly using semiochemicals and cultivation practices (e.g. in the LINK project “3D Farming”) to enhance populations and deliver them to the field simultaneously with pest arrival. More recently we have identified new semiochemicals derived from ladybird “footprints” that can be used to manipulate aphid parasitoids, either by driving them out of areas such as crop margins and into the neighbouring crop to control colonizing aphids, or to keep them away from areas of crop that have been treated with insecticide. Promising results have been obtained in preliminary trials in the RELU project and, in addition, natural sources of the active semiochemicals have been identified from wheat straw waxes, and this approach has potential in many crop/pest situations in the medium term. In our current work we have also investigated intra-guild interactions, specifically interactions between ladybirds. This is in the early stages of study, although has great potential in the long term to elucidate these behavioural interactions, particularly with respect to the threat to indigenous ladybird populations from the invading harlequin ladybird, Harmonia axyridis.

Intellectual Property arising from this reportA patent has been filed on cis-jasmone as a plant stress related signal that can be used to effect defence against insect pests (e.g. aphids) in crops and also cause the plants to attract organisms antagonistic to the pests (e.g. aphid parasitoids).

Technology TransferThe original filings related to non-Defra funded work and were promulgated through BTS. Now since the practical and molecular genetic opportunities are more clearly defined the patents have moved to the plant bioscience company PBL.

Options for new workUnder Research Council funding, the molecular mechanisms underpinning the high activity and persistent effects of cis-jasmone are underway in collaborative work with the CPI Division of Rothamsted Research. Promoter sequences for genes upregulated by cis-jasmone have been linked to marker genes, which were subsequently upregulated selectively with cis-jasmone. This will contribute to greater understanding and could be exploited by linking the promoter sequences to other valuable genes. Some such promoter sequences are already naturally present in elite cereal varieties.Varietal differences in response to cis-jasmone may offer new opportunities for further understanding on field exploitation in delivering semiochemically based effects.At the end of each Milestone report above there are specific options for new work including i) investigation of the molecular mechanisms mediating the effects of (E)-ocimene and exploitation of its generic effects as a plant activator and ii) the exploitation of the allelopathic effects, and underlying chemistry, of root exudates on weed germination and development for new weed control methods.

Knowledge TransferPresentations on research results

S.M. Cook, L.E. Smart, M.P. Skellern, N.P. Watts, I.H. Williams (2006) Development of a push-pull strategy for control of oilseed rape pests. Proceedings of the International Symposium on Integrated Pest Management in Oilseed Rape 3-5 April, 2006, Gottingen, Germany

Bruce, T.J.A., Hooper, A.M., Jones, O.T., Martin, J.L., Oakley, J., Smart, L.E. & Wadhams, L.J. (2006) Development of monitoring traps for the orange wheat blossom midge, Sitodiplosis mosellana, in the UK. Abstracts International Society of Chemical Ecology meeting Barcelona 15-19 July 2006.

SID 5 (Rev. 3/06) Page 18 of 39

L E Smart, M A Birkett, T J A Bruce, K Chamberlain, L M Field, J L Martin, J A Pickett, L J Wadhams and C M Woodcock, M H Beale, A K Huttly, R Parker, A L Phillips, I M Prosser, P R Shewry and Y Zhang (2006) Influencing aphid and parasitoid behaviour with transgenic plants releasing the aphid alarm pheromone Abstracts Royal Entomological Society meeting “ENTO 06” University of Bath, 20-22 September, 2006.

Cereals 2006/ HGCA: Orange wheat blossom midge pheromone trap development. Nocton Estate, Lincolnshire 14-15 June 2006Rothamsted Research Association/ HGCA Joint Workshop on Orange Wheat Blossom Midge control, Crowmarsh Battle Farm, Preston Crowmarsh, Wallingford, 4th May 2006Presentations by Professor Pickett for 2006/7

VOCBAS-ESF workshop on Improving the interpretation and application of plant volatile analysis, Wageningen, “New frontiers for the identification of biologically active VOCs from highly sensitive biological systems, induced gene upregulation and gene over-expression versus knockouts”, 16.3.06

Molecular Ecotoxicology students at University of Nottingham, “Plants and plant products in pest control”, 23.3.06

NACS Meeting, Atlanta, Georgia, Symposium on Plant Response to Biotic Insults, “Production of semiochemical and allelobiotic agents as a consequence of aphid feeding”; 29.3.06

Andersonian Chemical Society Centenary Lecture, University of Strathclyde, Glasgow, “Semiochemistry: chemical signalling between ourselves, our food and our pests”, 5.4.06

Molecular Ecotoxicology students at University of Nottingham, “Semiochemicals and pest control”, 27.4.06

Molecular Ecotoxicology students at University of Nottingham, “Integrated pest management”, 4.5.06

RELU meeting, Hampshire, “Manipulating insect pests with semiochemicals”; 5.5.06 LINK/NNFCC Seminar at Rothamsted Research, presentation on SEMIOCHEM projects,

"Development of semiochemical production from catmints and some unexpected uses"; 9.5.06 School of Chemistry, University of Cardiff, “Semiochemistry: chemical signalling between

ourselves, our food and our pests”, 25.5.06 Meeting on Structure and Function of Insect Odorant Binding Proteins, Max Planck Institute for

Biophysical Chemistry, Goettingen, Germany, “Insect olfaction: chemical aspects”, 29.5.06 Swedish University of Agricultural Sciences Biocenter, Uppsala, “Production of semiochemical

and allelobiotic agents as a consequence of aphid feeding”, 1.6.06 Lord De Ramsey and staff at Rothamsted Research, “The challenge of pests to the food chain”,

27.6.06 DFID/BBSRC Joint Funding Scheme: Sustainable Agriculture, Rothamsted Manor,

“Opportunities and challenges of biological research for development”, 6.9.06 The First International Conference on Glucosinolates, Max-Planck Institute for Chemical

Ecology, Jena, 10-14 September, 2006, “Exploiting the “push-pull” crop protection system using transgenic oilseed rape”, 12.9.06

5th Annual London Area Molecular Plant Sciences (LAMPS) meeting at Rothamsted Research, plenary lecture, “Defence signals from plants under stress of herbivory”, 19.9.06

ESAC1 committee meeting, Newark Liberty Marriott Hotel, USA, ESAC Sponsor’s Summary, 28-29 September 2006

School of Biological Sciences, University of Bristol, “Stress signalling in plants and animals”, 23.10.06

Manchester Literary and Philosophical Society, University of Manchester, “Learning from how insects perceive odours”, 26.10.06

SCI meeting, Plant-derived Natural Products: A Resource for Bioactive Compounds, at Syngenta, Jealott’s Hill, “Development of natural plant activators for pest control and disease monitoring”, 28.11.06

Department of Stress and Development Biology, Leibniz Institute of Plant Biochemistry, Halle BRD, “Potential for exploiting biotic stress signalling in plants”, 21.12.06

SID 5 (Rev. 3/06) Page 19 of 39

Vegetable Agronomists Association Members’ Day, PGRO, Peterborough, “Farming and the Future”, 11.1.07

EU ENDURE meeting, 20-23 February, INRA Sophia Antipolis, France, “Semiochemicals in pest control”, 22.2.07

University of Nottingham, School of Biosciences, Sutton Bonington, “Current Issues in Crop Science” teaching modules (D236A3 and D2DA09). Two lectures: 1) “Prospects for biological control”; 2) “How to deliver semiochemicals?”; 26.2.07

Leicester Literary & Philosophical Society, New Walk Museum, Leicester,“Pheromones and other scents for the alleviation, worldwide, of many pest problems in plant, animal and human health”, 26.2.07

BSc Hons Biology 3rd year undergraduate students on module C13687 Managing Pests and Pollution, University of Nottingham, “Plants and plant products in pest control”, 9.3.06

State Key Laboratory of Integrated Management of Insects and Rodent Pests in Agriculture, Shanghai, China, “Pheromones and other semiochemicals for the alleviation of pest problems in plant, animal and human health”, 25.3.07

Nanyang Normal University, Nanyang, China, “Pheromones and other semiochemicals for the alleviation of pest problems in plant, animal and human health”, 27.3.07

Zhejiang Academy of Agricultural Sciences, Zhejiang, China, “Potential for exploiting biotic stress signalling in plants”, 30.3.07

Institute of Applied Entomology, Zhejiang University, Zhejiang, China, “Pheromones and other semiochemicals for the alleviation of pest problems in plant, animal and human health”, 31.3.07

SID 5 (Rev. 3/06) Page 20 of 39

Annex 1. Figures

SID 5 (Rev. 3/06) Page 21 of 39

Settling of R. padi on barley exposed to (E)-ocimene fraction of efore for small scale production) ngs using a methos based on ape repespectively, on.Nepeta cataria oil for 48h

0

1

2

3

4

5

6

7

8

Control ocimene

P= 0.004

Settling of R. padi on barley exposed to (E)-ocimene fraction of Nepeta cataria oil for 24h

0

1

2

3

4

5

6

7

Control ocimene

P= 0.01

Figure 1.

Figure 2.

Figure 3. GC traces of entrainment samples of volatiles produced by Cirsium vulgare under light and dark conditions. 1 = (Z)-3-hexenylacetate, 2 = (Z)-ocimene, 3 = (E)-ocimene, 4 = (E,E)-α-farnesene.

SID 5 (Rev. 3/06) Page 22 of 39

1

3

2

1

3

2

4

C. vulgare dark

C. vulgare light

Development of Lipahis erysimi on turnip rape (Agena) and oilseed rape (Topic) treated with (E )-ocimene

0

0.1

0.2

0.3

0.4

0.5

0.6

8 day w t MRGR rm

TRs P=0.007 for 8 day wt, NS for MRGR, P=0.005 for rm ; OSRs P<0.001 for 8 day wt and MRGR and P=0.0014 for rm ;

TR con

TR ocimene

OSR con

OSR ocimene

Figure 4.

Emission of cis -jasmone from rape plant treated with different formulations

0

50

100

150

200

0 10 20 30 40 50 60 70

time after treatment (h)

rele

ase

rate

(ng/

h)

Formulation 3Formulation 2Formulation 1

Figure 5.

SID 5 (Rev. 3/06) Page 23 of 39

Sitobion avenae alate settling no choice test on wheat seedlings treated with formulated cis-jasmone

0

1

2

3

4

5

6

7

8

9

alates settled nymphs produced after 24h

P=0.01 for settlement

mea

n no

.

blank formulation cis-jasmone formulation

Figure 6.

Figure 7. Gas Chromatographs of entrained samples from untreated and salicylic acid treated turnip rape plants showing differences in volatiles.

SID 5 (Rev. 3/06) Page 24 of 39

Figure 8. Effect of SA treatment of turnip rape on infestation by pollen beetles in a polytunnel arena trial.

Figure 9. Effect of MeJa treatment of oilseed rape on infestation by pollen beetles in a polytunnel arena trial.

SID 5 (Rev. 3/06) Page 25 of 39

Figure 10.

Figure 11.

SID 5 (Rev. 3/06) Page 26 of 39

Aphid performance on oilseed rape plants treated with methyl jasmonate or a control

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

BrevicoryneSprayed

Brevicorynecontrol

Myzus sprayed Myzus control

wei

ght (

mg)

mass after 6 days

Birth weight

Aphid performance on turnip rape plants treated with salicylic acid or a control

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

BrevicoryneSprayed

Brevicorynecontrol

Myzus sprayed Myzus control

wei

ght (

mg)

mass after 6 days

Birth weight

Figure 12. Occurrence of 3 key semiochemicals for orange wheat blossom midge in the volatile profiles of wheat varieties.

Figure 13. Coupled GC-EAG trace of the antennal response of the black bean aphid, Aphis fabae to volatiles collected from bean plants, Vicia faba. The upper trace is the GC and the lower trace the EAG.

SID 5 (Rev. 3/06) Page 27 of 39

Figure 14. The hydroxamic acid biosynthetic pathway in wheat.

SID 5 (Rev. 3/06) Page 28 of 39

SID 5 (Rev. 3/06) Page 29 of 39

6 7 8 9 10 12 14

6 7 8 9 10 12 14Days post germination

Days post germination

A

B

C

D

Figure 15. Changes in Bx 2 (A, B and C) and Bx 3 (D) gene expression during early development in A and D, leaf, B, stem and C, root tissue from the variety Welford. Expression is relative to the amount at 10 days which is set at a value of one.

Figure 16. Changes in Bx 2 (A) and glucosidase (B) gene expression in the coleoptile (A) and in the root (B) in response to treatment with cis-jasmone. Expression was measured by real time PCR and values are relative to time 0, which was set as a value of one.

A

B

Figure 17

Figure 18

SID 5 (Rev. 3/06) Page 30 of 39

Intrinsic rate of population increase (rm ) for Sitobion avenae on wheat varieties treated with cis -jasmone

0.2

0.22

0.24

0.26

0.28

0.3

0.32

Solstice P=0.028; Herew ard NS (P=0.38); Welford P=0.047; Option NS (P=0.061); Axona P<0.001

Solstice con

Solstice c-j

Herew ard con

Herew ard c-j

Welford con

Welford c-j

Option con

Option c-j

Axona con

Axona c-j

Intrinsic rate of population increase of Metopolophium dirhodum on wheat varieties treated with cis -jasmone

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

rm

No significant differences

axona conaxona c-j

welford con

welford c-j

Sitobion avenae on winter wheat Consort +/- cis-jasmone; total aphids on 400 tillers

0

10

20

30

40

50

60

70

01-Jun 7 12 20 29 06-Jul

control

cis-jasmone

Figure 19

Sitobion avenae on winter wheat Hereward +/- cis-jasmone; total aphids on 400 tillers

0

10

20

30

40

50

60

70

80

01-Jun 7 12 20 29 06-Jul

control

cis-jasmone

Figure 20

Sitobion avenae on winter wheat Solstice +/- cis-jasmone; total aphids on 400 tillers

0102030405060708090

100

01-Jun 7 12 20 29 06-Jul

control

cis-jasmone

Figure 21

SID 5 (Rev. 3/06) Page 31 of 39

Sitobion avenae on winter wheat Welford +/- cis-jasmone; total aphids on 400 tillers

0

10

20

30

40

50

60

01-Jun 7 12 20 29 06-Jul

control

cis-jasmone

Figure 22

Metopolophium dirhodum on winter wheat Consort +/- cis-jasmone; total aphids on 400 tillers

0

20

40

60

80

100

120

140

160

01-Jun 7 12 20 29 06-Jul

control

cis-jasmone

Figure 23

Metopolophium dirhodum on winter wheat Hereward +/- cis-jasmone; total aphids on 400 tillers

0

20

40

60

80

100

120

01-Jun 7 12 20 29 06-Jul

control

cis-jasmone

Figure 24

SID 5 (Rev. 3/06) Page 32 of 39

Metopolophium dirhodum on winter wheat Solstice +/- cis-jasmone; total aphids on 400 tillers

020

4060

80100

120140

160180

200

01-Jun 7 12 20 29 06-Jul

control

cis-jasmone

Figure 25

Metopolophium dirhodum on winter wheat Welford +/- cis-jasmone; total aphids on 400 tillers

0

20

40

60

80

100

120

140

160

01-Jun 7 12 20 29 06-Jul

control

cis-jasmone

Figure 26

Figure 27. Gas Chromatograph of extract of maize seedlings showing main constituent to be the derivatized hydroxamic acid, DIMBOA.

SID 5 (Rev. 3/06) Page 33 of 39

min0 5 10 15 20 25

pA

0

200

400

600

800

FID2 B, (I:\DEPTS\BCH\LESLEY\050407F.D)

16.

519

19.

465

21.

270

Germination of Black grass seeds in root exudate from wheat cv Axona

0102030405060708090

day 4 day 5 day 6 day 8

% g

erm

inat

ion

water

Axona exudate

Figure 28

Germination of Black grass seeds in root exudate from wheat cv Solstice

0

10

20

30

40

50

60

70

day 4 day 5 day 6 day 8

% g

erm

inat

ion

water

Solstice exudate

Figure 29

Germination of black grass seeds in root exudate from white clover

0102030405060708090

day 4 day 5 day 6 day 8

% g

erm

inat

ion

water White clover exudate

Figure 30

SID 5 (Rev. 3/06) Page 34 of 39

Germination of Black grass seeds in presence of DIMBOA

0

5

10

15

20

25

30

35

40

45

50

Day 4 Day 7 Day 10

60 seeds per treatm ent; DIMBOA - 0.063m g/cm 2

% g

erm

inat

ion

w ater

10% acetone

DIMBOA

Figure 31

Germination of wild oat seeds in presence of DIMBOA

0

10

20

30

40

50

60

70

Day 7 Day 10

30 seeds per treatm ent; DIMBOA - 0.063m g/cm 2

% g

erm

inat

ion

w ater

10% acetone

DIMBOA

Figure 32

Germination of OSR seed var. Topic in presence of DIM BOA

0

20

40

60

80

100

120

Day 3 Day 4 Day 7

60 seeds/treatment; DIMBOA - 0.063mg/cm2; seeds in DIMBOA dying as root emerges

% g

erm

inat

ion

water

10% acetone

DIMBOA

Figure 33

SID 5 (Rev. 3/06) Page 35 of 39

Poppy seed germination in DIMBOA

0102030405060708090

100

day 4 day 5 day 6 day 8

% g

erm

inat

ion

water 10% acetone DIMBOA

Figure 34

Germination of wheat cv Solstice on DIMBOA

0102030405060708090

100

day 4 day 5 day 6 day 8

DIMBOA 1mg/ml

% g

erm

inat

ed

water

10% acetone

DIMBOA

Figure 35

SID 5 (Rev. 3/06) Page 36 of 39

Foraging by Aphidius ervi on wheat seedlings treated with wheat straw extract

0

200

400

600

800

1000

1200

1400

control w ax

n=7; P=0.03

Tim

e sp

ent (

sec)

Figure 36

Foraging by Aphidius ervi on wheat seedlings treated with wheat straw wax alkanes

0

50

100

150

200

250

300

350

400

450

500

control treated

n=10; P=0.049

Tim

e sp

ent (

sec)

Figure 37

References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.

SID 5 (Rev. 3/06) Page 37 of 39

Agelopoulos, N.G., Hooper, A.M., Maniar, S.P., Pickett, J.A. and Wadhams, L.J. (1999) A novel approach for isolation of volatile chemicals released by individual leaves of a plant in situ. Journal of Chemical Ecology. 25, 1411-1425.Beale, M.H. Birkett, M.A. Bruce, T.J. Chamberlain, K. Field, L.M. Huttly, A.K. Martin, J.L. Parker, R. Phillips, A.L. Pickett, J.A. Prosser, I.M. Shewry, P.R. Smart, L.E. Wadhams, L.J. Woodcock, C.M. and Zhang, Y. (2006) Aphid alarm pheromone produced by transgenic plants affects aphid and parasitoid behaviour. Proceedings of the National Academy of Sciences USA 103, 10509-10513.Birkett, M.A., Bruce, T.J., Martin, J.L., Smart, L.E., Oakley, J.N. and Wadhams, L.J. (2004) Responses of female orange wheat blossom midge, Sitodiplosis mosellana, to wheat panicle volatiles. Journal of Chemical Ecology. 30 (7) 1319-1328.Birkett, M., Campbell, C.A.M., Chamberlain, K., Guerrieri, E., Hick, A.J., Martin, J.L., Matthes, M. Napier, J., Pettersson, J., Pickett, J.A., Poppy, G., Pow, E.M., Pye, B.J., Smart, L.E., Wadhams, G., Wadhams, L.J. and Woodcock, C.M. (2000) New roles for cis-jasmone as an insect semiochemical and in plant defense. Proceedings of the National Academy of Sciences of the USA 97: 9329-9334.Birkett, M.A., Chamberlain, K., Khan, Z.R., Pickett, J.A., Toshova, T., Wadhams, L.J. and Woodcock, C.M. (2006) Electrophysiological responses of the lepidopterous stemborers Chilo partellus and Busseola fusca to volatiles from wild and cultivated host plants. Journal of Chemical Ecology 32, 2475-2487.Blassioli Moraes, M.C., Birkett, M.A., Bromilow, R.H., Gordon-Weeks, R., Martin, J.L., Pye, B.J., Smart, L.E. and Pickett, J.A. cis-Jasmone induces accumulation of defence compounds in wheat, Triticum aestivum. Phytochemistry (in press).Bruce, T.J. Martin, J.L., Pickett, J.A., Pye, B.J., Smart, L.E. and Wadhams, L.J. (2003a) cis-Jasmone treatment induces resistance in wheat plants against the grain aphid, Sitobion avenae (Fabricius) (Homoptera: Aphididae). Pest Management Science, 59, 1031-1036.Bruce, T.J. A., Pickett, J.A. and Smart, L.E. (2003b) cis-Jasmone switches on plant defence against insects. Pesticide Outlook 14: 96 – 98.Bruce, TJA, LJ Wadhams & CM Woodcock. (2005). Insect host location: a volatile situation. Trends in Plant Science 10:269-274.Chamberlain, K. Khan, Z.R. Pickett, J.A. Toshova T.and Wadhams L.J. (2006) Diel periodicity in the production of green leaf volatiles by wild and cultivated host plants of stemborer moths, Chilo partellus and Busseola fusca. Journal of Chemical Ecology 32, 565-577.Cook, S.M. Khan Z.R. and Pickett J.A. (2007) The use of push-pull strategies in integrated pest management. Annual Review of Entomology 52, 375-400.Cook, S. M. Rasmussen, H. B. Birkett, M.A. Woodcock, C. M. Murray, D. A. Pye, B. J. Watts, N. P. Williams, I. H. (2007). Behavioural and chemical ecology of the success of turnip rape (Brassica rapa) trap crops in protecting oilseed rape (Brassica napus) from the pest Meligethes aeneus. Arthropod Plant Interactions 1: 57-67. Cook, S.M. Smart, L.E. Martin, J.L. Murray, D.A. Watts N.P. and Williams I.H. (2006) Exploitation of host plant preferences in crop protection strategies for oilseed rape (Brassica napus). Entomologia experimentalis et applicata 119, 221-229.Couty, A. van Emden, H. Perry, J.N. Hardie, J. Pickett, J.A. and Wadhams, L.J. (2006) The roles of olfaction and vision in host-plant finding by the diamondback moth, Plutella xylostella. Physiological Entomology 31, 134-145.Glinwood, R., Ninkovic, V., Pettersson, J. and Ahmed, E. (2004) Barley exposed to aerial allelopathy from thistles (Cirsium spp.) becomes less acceptable to aphids. Ecological Entomology 29, 188-195.Hooper, A.M. Farcet, J-B. Mulholland N.P. and Pickett J.A. (2006) Synthesis of 9-methylgermacrene B, racemate of the sex pheromone of Lutzomyia longipalpis (Lapinha), from the renewable resource, Geranium macrorrhizum essential oil. Green Chemistry 8, 513-515.Khan, Z.R. Hassanali A. and Pickett J.A. (2006) Managing polycropping to enhance soil system productivity: a case study from Africa. In Biological Approaches to Sustainable Soil Systems, ed. N. Uphoff, CRC Press, pp. 575-586.Khan, Z.R. Midega, C.A.O. Hassanali, A. Pickett, J.A. Wadhams L.J. and Wanjoya A. (2006) Management of witchweed, Striga hermonthica, and stemborers in sorghum, Sorghum bicolor, through intercropping with greenleaf desmodium, Desmodium intortum. International Journal of Pest Management 52, 297-302.Khan, Z.R. Pickett, J.A. Wadhams, L.J. Hassanali A. and Midega C.A.O. (2006) Combined control of Striga hermonthica and stemborers by maize-Desmodium spp. intercrops. Crop Protection 25, 989-995.Larsen, E. and Christensen, L.P. (2000) Simple method for large scale isolation of the cyclic arylhydroxamic acid DIMBOA from maize (Zea mays L.) J. Agric. Food Chem. 48, 2556-2558.Nakashima, Y., Birkett, M.A., Pye, B.J., Pickett, J.A. and Powell. W. (2004) The role of semiochemicals in the avoidance of the seven-spot ladybird Coccinella septempunctata (Coleoptera: Coccinelidae) by the aphid parasitoid, Aphidius ervi (Hymenoptera: Braconidae). J. Chem. Ecol. 30; 1103-1115.Nakashima, Y., Birkett, M.A., Pye, B.J., Powell, W. (2006) Chemically mediated intraguild predator avoidance by aphid parasitoids: Interspecific variability in sensitivity to semiochemical trails of ladybird predators J. Chem. Ecol., 32: 1989-1998. Pettersson, J. Ninkovic, V. Glinwood, R. Al Abassi, S. Birkett, M. Pickett J. and Wadhams L. (2005) Foraging behaviour of Coccinella septempunctata (L.): volatiles and allelobiosis. Proceedings International Symposium on Biological Control of Aphids and Coccids, Tsuruoka, Japan, September 25-29, 2005, 187-192.Pickett, J.A. Bruce, T.J.A. Chamberlain, K. Hassanali, A. Khan, Z.R. Matthes, M.C. Napier, J.A. Smart, L.E. Wadhams L.J. and Woodcock, C.M. (2006) Plant volatiles yielding new ways to exploit plant defence. In: Chemical ecology: from gene to ecosystem, pp. 161-173. Editors M. Dicke and W. Takken. (Springer, Netherlands).Pickett, J.A. Birkett, M.A. Blassioli Moraes, M.C. Bruce, T.J.A. Chamberlain, K. Gordon-Weeks, R. Matthes, M.C. Napier, J.A. Smart, L.E. Wadhams, L.J. & Woodcock, C.M. (2007) cis-Jasmone as an allelopathic agent in inducing plant defence. Allelopathy Journal 19: 109-118Schönrogge, K. Gardner, M.G. Elmes, G.W. Napper, E.K.V. Simcox, D.J. Wardlaw, J.C. Breen, J. Barr, B. Knapp, J.J. Pickett J.A. and Thomas J.A. (2006) Host propagation permits extreme local adaptation in a social parasite of ants. Ecology Letters 9, 1032-1040.Van Emden, H.F. (1969) Plant resistance to Myzus persicae induced by a plant regulator and measured by aphid relative growth rate. Entomol. Exp. Appl. 12: 125-131.Wu, H.W., Haig, T. Pratley J. et al. (2000). Distribution and exudation of allelochemicals in wheat, Triticum aestivum. Journal of Chemical Ecology 26, 2141-2154.Wyatt, I.J. and White, P.F. (1977) Simple estimation of intrinsic increase rates for aphids and tetranychid mites. J. Applied Ecology 14: 757-766.

SID 5 (Rev. 3/06) Page 38 of 39

SID 5 (Rev. 3/06) Page 39 of 39