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SID 5 Research Project Final Report

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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.

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Project identification

1. Defra Project code PS2101

2. Project title

Identification and provision of potential semiochemical tools for use in integrated crop management.

3. Contractororganisation(s)

Rothamsted ResearchHarpendenHerts. AL5 2JQUK                         

54. Total Defra project costs £ 1383750

5. Project: start date................ 01 April 2003

end date................. 31 March 2006

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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 In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain.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 project was to identify and provide semiochemicals, i.e. 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 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.

Objectives1. Identify new plant stress signals that can act as plant activators and determine effects up to the third trophic level.Healthy barley plants, Hordeum vulgare, exposed to volatile emissions from barley infected with the pathogen leaf blotch (Rhynchosporium secalis) were more resistant to subsequent infection with the pathogen. Volatiles produced by the infected plant, were identified as limonene, (E)-ocimene and trace amounts of 6-methyl-5-hepten-2-one. When healthy barley plants were exposed to synthetic (E)-ocimene they showed induced resistance, comparable with the plant-plant experiment. In addition, we have shown that when barley plants are exposed to the volatiles from thistle plants, Cirsium spp., they become less attractive to cereal aphids compared to unexposed plants. Volatiles from Cirsium spp., identified by GC/MS, implicated (E)-ocimene as the potential plant activator. (E)-ocimene, shown above for the first time to induce defence in barley against pathogen attack, is also a key compound that increases in the volatile profile of many crop plants when they are exposed to the plant activator cis-jasmone. In addition, it is a key component mediating host location by the aphid parasitoid, Aphidius ervi. Thus, it would appear that (E)-ocimene may have a dual role as a plant stress indicator, mediating tritrophic interactions and as a plant activator.When analysed by GC/MS, the volatiles from an aphid infested potato was found to contain the stress related compounds, 4,8-dimethyl-(E)-nona-1,3,7-triene, 4,8,12-trimethyl-(E,E)-trideca-1,3,7,11-tetraene and cis-jasmone, which were not produced by untreated plants. The potato aphid, Macrosiphum euphorbiae, was attracted to volatiles from the uninfested potato but not to the aphid infested plant while the peach-potato aphid, Myzus persicae, was repelled by the infested plant. Healthy, untreated potato plants were exposed to the volatiles from an aphid infested potato and the volatiles produced by the receiving plants were found to contain significant levels of 4,8-dimethyl-(E)-nona-1,3,7-triene, 4,8,12-trimethyl-(E,E)-trideca-1,3,7,11-tetraene, 6-methyl-5-hepten-2-one, (β)-caryophyllene and cis-jasmone, which were not produced by unexposed plants. This is the first time that production of the plant activator cis-jasmone by an induced crop plant has been demonstrated.

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In olfactometer studies with synthetic samples, M. persicae, was significantly repelled by the 4,8,12-trimethyl-(E,E)-trideca-1,3,7,11-tetraene but was unaffected by the 4,8-dimethyl-(E)-nona-1,3,7-triene. In contrast M. euphorbiae was not significantly affected by 4,8,12-trimethyl-(E,E)-trideca-1,3,7,11-tetraene. At the higher trophic level, the aphid parasitoid, Aphidius ervi, was not affected by 4,8-dimethyl-(E)-nona-1,3,7-triene, a ubiquitous herbivore stress compound known to attract other parasitoid species, but was significantly attracted to 4,8,12-trimethyl-(E,E)-trideca-1,3,7,11-tetraene, demonstrating, for the first time, that the newly identified stress related volatile from the potato plant/plant interaction is involved in host location by both aphids and aphid parasitoids.

2. Define host plant location and avoidance of unsuitable potential hosts so as to identify new semiochemicals. In addition to pests, this will also include parasitoids and predators.Female orange wheat blossom midges, Sitodiplosis mosellana, were attracted to a 5 component synthetic blend of volatiles, identified from the susceptible growth stage of wheat, when they were presented in the natural ratio and concentration. The blend was as attractive as the natural air entrainment sample of the ears. In subsequent assays, the blend was reduced by one component, the 6-methyl-5-hepten-2-one was omitted, and yet the females were still significantly attracted to the 4 component blend. However, when the 5 component blend was presented in an unnatural ratio, (15ng instead of 5ng of 6-methyl-5-hepten-2-one) the blend was not attractive. Thus, for this insect these data provide clear evidence that host recognition is conferred by specific ratios of ubiquitous compounds. 6-Methyl-5-hepten-2-one acts as a spacing cue for the aphid Rhopalosiphum padi and is also attractive to aphid parasitoids. However, here 6-methyl-5-hepten-2-one would seem to be acting as a key indicator of a stressed i.e. a non-appropriate host plant for the midge.Although the aphid alarm pheromone (E)-β-farnesene has considerable potential for the development of novel aphid control strategies all renewable sources investigated contain additional compounds that inhibit the alarm response. Thus, an alternative approach to exploitation of this pheromone was taken. Arabidopsis thaliana plants were transformed, with an (E)-β-farnesene synthase gene cloned from Mentha × piperita, and to emit high levels of pure (E)-β-farnesene, which elicited potent effects on behaviour of the aphid Myzus persicae, both in terms of alarm and repellent responses. In addition, air entrainment samples of the plants showed a strong GC-EAG response from aphid parasitoid Diaeretiella rapae and in laboratory behavioural assays these plants elicited a strong arrestant response from the parasitoid. This is the first time that a plant has been transformed to produce an insect pheromone and demonstrate that the resulting emission affects behavioural responses at two trophic levels. These studies show production of an insect pheromone in a transgenic plant and clearly indicate the potential value for aphid control of plants expressing an (E)-β-farnesene synthase gene.

3. Determine the potential value of exploiting rhizosphere allelopathy, in controlling soil-pest/plant interactions, and suppressing competitive weeds.In the course of this work we found a dramatic induction by the plant activator cis-jasmone of allelophathic agents in wheat i.e. hydroxamic acids (HAs) or benzoxazinones/benzoxazolin-2-(3H)-ones, with the generally most active component being 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA). The overwhelming evidence that the HAs play a significant role in pest resistance scientifically justifies exploring the possibility that the HA pathway can be exploited to achieve environmentally benign pest resistance. Liquid phase extraction (LPE) using N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSFTA) as a derivatising reagent, and vapour phase extraction (VPE) in combination with trimethylsilyl diazomethane (TMSCHN2) as a derivatising reagent were used to evaluate the impact of cis-jasmone on the secondary metabolism of wheat. Liquid phase extraction allowed the measurement of hydroxamic acids DIMBOA, HMBOA and MBOA, and phenolic acids such as trans-p-coumaric acid, syringic acid, p-hydroxybenzoic acid, vanilic acid and cis and trans-ferulic acid. Using LPE, levels of DIMBOA were significantly higher in leaves and roots of wheat treated with cis-jasmone when compared with untreated plants.Using RTPCR and real time relative q RTPCR analysis to measure the expression of the major genes (the Bx genes) in the HAs pathway, we found that, consistent with the observation that HA levels decrease as the plant matures beyond the seedling stage, expression of all of the Bx genes is highest in young plants. Accumulation of HAs, measured by HPLC, is highest in the leaf tissue but the expression of all the genes is higher in the stem than in leaf tissue. This suggests that some degree of transport of the compound from the stem to the leaves is taking place. Preliminary studies clearly demonstrate that when applied to leaves cis-jasmone induces Bx gene expression in certain selected varieties. This offers the possibility of “switching on” production of HAs to protect plants when they are under threat from pest colonisation, rather than the more metabolically costly constitutive expression where HAs are produced constantly.

4. Identify new strategies for exploiting insect predators and parasitoids via semiochemical tools.Female aphid parasitoids with varying host ranges (Aphidius eadyi, A. ervi and Praon volucre) significantly avoided leaves previously visited by intraguild predatory ladybirds, Coccinella septempunctata and Adalia bipunctata. The avoidance responses shown by the two Aphidius species were stronger to trails of C. septempunctata than to those of A. bipunctata. However, P. volucre avoided trails of both ladybird species to a similar degree. Bioassays with these three parasitoid species and the hydrocarbons n-tricosane, n-pentacosane and n-heptacosane, which are components of the trails of both C. septempunctata and A. bipunctata, indicated that A. eadyi showed more sensitive avoidance responses to n-tricosane than the other two species, all three species showed similar responses to n-pentacosane, and only P. volucre showed avoidance responses to n-heptacosane. Quantitative analyses of each hydrocarbon showed

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that n-pentacosane and n-heptacosane occur in significantly greater amounts in C. septempunctata trails than in those of A. bipunctata. The trails of the two species also differ qualitatively in the other hydrocarbons present. Such compounds may play a role in mediating intraspecific ladybird interactions, specifically the oviposition deterrent response of adult A. bipunctata following detection of intraspecific larval footprints. Other work on avoidance of C. septempuncata footprints by A. ervi revealed virtually identical hydrocarbon profiles for adults and larvae. Thus, it can be expected that these compounds do indeed play a role in C. septempunctata oviposition deterrent behaviour, and that a similar outcome can be expected for A. bipunctata. Further studies are required to confirm the role of these compounds.

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).

BackgroundThe purpose of this Project was to identify and provide the semiochemical tools for benign means of crop protection that will ultimately replace or reduce dependence on conventional pesticides. The Project also demonstrated a major research effort into developing alternatives to use of synthetic chemical toxicants in pest control particularly insecticides, which are the most likely group of pesticides to impinge upon human health and the environment The work includes studies of plant stress compounds acting as activators to switch on defence effects in plants and of semiochemicals that control key stages of plant colonisation and population development of UK pests and extends to preliminary work identifying selected agents having “allelopathic” activity and to semiochemicals for the exploitation of natural enemies.Previous work in PI0339 and 0341 had identified cis-jasmone, functioning as a signal in activating plant defence and that, by signalling plant damage, not only repels herbivorous insects, but also attracts predators and parasitoids, resulting in reduced colonisation by pests. This work provided a lead to the identification of more signals within this project.All work on the delivery and development of semiochemicals for alternative plant protection technologies for the future exploit some form of “push-pull” or stimulo-deterrent diversionary strategy. For example, pheromones can be used to direct pests away from the crop and attract them to areas or “trap crops” where they can be controlled. However, work in the former PI03 Programme indicated that pheromones seldom act alone and often require, and are synergised by, other semiochemicals, particularly those from the host plant. Leads from this previous work were studied to improve understanding and utilisation of “push-pull” strategies.

Objectives1. Identify new plant stress signals that can act as plant activators and determine effects up to the third trophic level.2. Define host plant location and avoidance of unsuitable potential hosts so as to identify new semiochemicals. In addition to pests, this will also include parasitoids and predators.3. Determine the potential value of exploiting rhizosphere allelopathy, in controlling soil-pest/plant interactions, and suppressing competitive weeds.

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

Priority for investigation was given to semiochemicals functioning as plant signals in activating plant defence. However, semiochemicals acting directly on pests and beneficial organisms were also identified and provided for use here and in the associated projects. Semiochemicals were secured, wherever possible, from botanical sources. Chemical synthesis provided an integral part of the authentication process and quality assurance for semiochemical structure determination and a back-up where there was delay or problems encountered in providing immediate botanical sources of semiochemicals.

The outcome of work towards each Objective is reported separately, but where there is significant overlap in the results for individual milestones, these have been combined and reported together for clarity. 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. Identify new plant stress signals that can act as plant activators and determine effects up to the third trophic levelThis objective builds on the plant activator studies that lead to the identification of cis-jasmone but was extended to incorporate other signalling phenomena and to determine the effects of plant activator treated crops/plants on natural enemies.

01/01 Identification of new plant stress signals. In chimney cage experiments, healthy barley plants, Hordeum vulgare, exposed to volatile emissions from barley infected with the pathogen leaf blotch (Rhynchosporium secalis) were more resistant to subsequent infection with the pathogen and in replicated experiments the response (pathology of resistance) was totally reproducible. In each trial, volatiles produced by the infected plant, “the emitter”, were collected by air entrainment and compared to those of an uninfected plant. The volatile profiles were variable, but the consistent compounds emitted by infected plants were identified as limonene, (E)-ocimene and trace amounts of 6-methyl-5-hepten-2-one. To establish if any of these compounds were contributing to the induction of defence against the pathogen, healthy plants were exposed to the individual compounds (E)-ocimene (synthesised in this project in S01/02) and 6-methyl-5-hepten-2-one in the ppm concentration range and the plants were then challenged with the pathogen. Healthy plants were also exposed to methyl jasmonate, methyl salicylate, cis-jasmone, ethylene and 4,8-dimethyl-(E)-nona-1,3,7-triene (synthesised in this project in 02/03), all compounds known either to induce plant defence or that are associated with the volatile profile of herbivore induced plants. (E)-Ocimene and methyl jasmonate induced resistance, comparable with the plant-plant experiment, across the range of concentrations tested. 6-Methyl-5-hepten-2-one and methyl salicylate showed low induction of resistance only at the highest concentration. Healthy plants exposed to infected plants, but not subsequently challenged with pathogen, released similar volatiles to the infected emitter. GC analysis of the volatiles collected from plants, following infection, showed that elevated production of these volatiles, in exposed and pathogen infected plants, occurs over a four to five day period. In the case of pathogen infection, elevated production of these key volatiles is initiated prior to visible signs of infection (lesions). Laboratory behavioural studies have shown that 6-methyl-5-hepten-2-one is not only an epideitic (spacing) pheromone for the bird cherry oat aphid, Rhopalosiphum padi, (Quiroz et. al., 1997) but is also a key attractant mediating host location by insects antagonistic to pests, particularly aphid parasitoids (Du et. al., 1998; see below).

Laboratory studies have shown that in chimney cage experiments where barley plants are exposed to the volatiles from intact and uninfested thistle plants, Cirsium spp., they become less attractive to cereal aphids compared to unexposed plants (Glinwood et. al., 2004). In olfactometer tests, Cirsium-exposed barley was significantly less attractive than unexposed plants to R. padi, indicating that there was a change in the volatile profile of exposed barley plants. In other

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behavioural assays, settlement of both R. padi and the grain aphid, Sitobion avenae, was significantly reduced on Cirsium-exposed plants. In addition, 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. Since no major differences were observed in the volatile profiles obtained from Cirsium plants during the scotophase and photophase, including (E)-ocimene (see below), it is likely that the diel periodicity observed in the induction of barley relates to a differential sensitivity of the receiving plant to the phytopheromone. However, confirmation of this must await full characterisation of the phytopheromone. Nevertheless, these observations support those described above in that plant to plant interactions can lead to effects at higher trophic levels. Preliminary air entrainment studies of Cirsium spp. and subsequent GC/MS investigations have identified and implicated the monoterpene hydrocarbon (E)-ocimene as a potential plant activator. (E)-ocimene has already been shown to induce defence in barley against pathogen attack (see above), but is also a key compound that increases in the volatile profile of many crop plants e.g. beans (Birkett, et. al., 2000), wheat, potatoes and peppers when they are exposed to the known plant activator cis-jasmone (Figs 1 & 7 and see below). In addition, (E)-ocimene is a key component mediating host location by the aphid parasitoid, Aphidius ervi (Du et. al., 1998). Thus, it would appear that (E)-ocimene may have a dual role as a plant stress indicator, mediating tritrophic interactions and as a plant activator.In further laboratory studies, barley seedlings were exposed for 24h to low (ppb) levels of synthetic (E)-ocimene (provided in S01/02 below) in hexane in sealed 30 litre chambers. Control plants were exposed to the solvent alone. Individual plants were fully contained in chimney cages after treatment. On day 3 after treatment, alate R. padi were introduced into the chimney cages and their settlement was recorded after 2, 5 and 24h. After 24h the alatae were removed and the number of nymphs they had produced was recorded. These nymphs were then allowed to develop for a further seven days after which the total aphid population per plant was recorded (Fig 2). Although there were no significant differences between treatments, the numbers of alatae settled and nymphs produced at 24h and the total number of aphids recorded on the plants at the end of the trial, were all lower on the (E)-ocimene treated plants as compared to the untreated controls. Although exposure of barley plants to (E)-ocimene at the ppm level induced significant pathogen resistance in exposed barley plants (see above), these studies to investigate induced effects on aphid development were conducted with more physiologically relevant stimulus concentration levels i.e. ppb. Whilst it is recognised that two very different induced effects are investigated here, it is likely that the high chemical instability of (E)-ocimene contributed to the lack of significant effects against aphid development when presented at low stimulus concentrations. To overcome this, and based on our experience with other unstable semiochemicals, e.g. (E)-β-farnesene, we will seek a natural plant essential oil source of (E)-ocimene that will provide greater stability for the compound.

S01/01 Define new biological systems in which plant/plant interactions can be detected at the third trophic level.Air entrainments were collected from aphid infested potato, variety Desiree, and broad bean plants, variety Sutton, (100 aphids each) and from uninfested control plants. These samples were tested against the potato aphid, Macrosiphum euphorbiae, the peach-potato aphid, Myzus persicae, and the black bean aphid, Aphis fabae, in 4-arm olfactometer bioassays. A. fabae, although not attracted to the uninfested bean volatiles, was repelled by volatiles from aphid infested beans, although not significantly. M. euphorbiae was significantly attracted to uninfested potato odours (mean time spent: in treated arm 4.13 min, in control arm 2.70 min, t test P=0.007) but not to aphid infested potato, while M. persicae was repelled by the volatiles from the aphid infested potato. When analysed by GC/MS, the entrainment sample from the aphid infested potato was found to contain significant levels of the stress related compounds, 4,8-dimethyl-(E)-nona-1,3,7-triene, 4,8,12-trimethyl-(E,E)-trideca-1,3,7,11-tetraene and cis-jasmone, which were not present in volatile profiles of untreated plants. We have already demonstrated that cis-jasmone acts as a plant activator and further experiments were conducted to determine whether infested potatoes

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could induce changes in uninfested plants.Healthy untreated potato plants were exposed individually for 24 hours in a chimney cage to the volatiles from a potato infested with 100 aphids. The volatiles produced by the receiving plants were collected by air entrainment and when analysed by GC/MS were found to contain significant levels of the stress related compounds, 4,8-dimethyl-(E)-nona-1,3,7-triene, 4,8,12-trimethyl-(E,E)-trideca-1,3,7,11-tetraene, 6-methyl-5-hepten-2-one, (β)-caryophyllene and cis-jasmone, which were not present in volatile profiles of unexposed plants. This is the first time that we have demonstrated production of the plant activator cis-jasmone in an induced crop plant. In olfactometer studies with the synthetic samples of 4,8,12-trimethyl-(E,E)-trideca-1,3,7,11-tetraene and 4,8-dimethyl-(E)-nona-1,3,7-triene (synthesised in this project in 02/03), the peach-potato aphid, M. persicae, was significantly repelled by the 4,8,12-trimethyl-(E,E)-trideca-1,3,7,11-tetraene (Fig 3), but was unaffected by the 4,8-dimethyl-(E)-nona-1,3,7-triene. In contrast, the potato aphid, M. euphorbiae was not significantly affected by 4,8,12-trimethyl-(E,E)-trideca-1,3,7,11-tetraene. Laboratory olfactometer assays were conducted to investigate the effects of these compounds at higher trophic levels. The generalist aphid parasitoid, A. ervi, was presented with 4,8-dimethyl-(E)-nona-1,3,7-triene, (β)-caryophyllene and 4,8,12-trimethyl-(E,E)-trideca-1,3,7,11-tetraene. Surprisingly, the parasitoid was not affected by 4,8-dimethyl-(E)-nona-1,3,7-triene, a ubiquitous herbivore stress compound known from other work by us to attract other parasitoid species (Khan et. al, 1997). There was also a weak but non-significant attraction to (β)-caryophyllene. However, A. ervi was significantly attracted to 4,8,12-trimethyl-(E,E)-trideca-1,3,7,11-tetraene, (Fig 4), demonstrating, for the first time, that the newly identified stress related volatile from the potato plant/plant interaction is involved in host location by both aphids and aphid parasitoids (also see 01/03 and 02/02).

01/02 Determine effects of plant activators on aphidophagous and polyphagous predators.In previous studies we have shown that attraction of the major pest insect predator the 7 spot ladybird Coccinella septempunctata, to cis-jasmone (Birkett et. al., 2000). In the current programme work was done to investigate the effects of cis-jasmone treated plants on the behaviour of this ladybird. Using defined behaviours such as time spent walking, still, cleaning and foraging on extra floral nectaries, detailed foraging behavioural assays using Observer software (Noldus©) were conducted with both 2 and 7 spot ladybirds (Adalia bipunctata and C. septempunctata) on untreated bean plants and plants that had been pre-treated with the plant activator cis-jasmone 24, 48, and 72h previously. A. bipunctata were very inactive in these trials and no differences in behaviour were observed for this ladybird. However, C. septempunctata spent significantly longer on plants 48h after cis-jasmone treatment than on untreated control plants (Fig 5). The main behaviour that was significantly increased on cis-jasmone treated plants was the time spent still. This effect has also been noted for other beneficial species including aphid parasitoids and lacewing larvae (see later). Unfortunately, the C. septempunctata failed to respond to the volatiles collected from cis-jasmone treated beans in an olfactometer bioassay, although this will not prevent field utilization/investigation of this effect. In similar behavioural assays, larvae of the aphid predatory lacewing Chrysoperla carnea were observed and compared on cis-jasmone treated and untreated plants. As seen with the ladybirds, time spent on the cis-jasmone treated plants was significantly increased and again time spent still was the main increase (Fig 6)

S01/02 Provision of newly identified plant activators.(E)-Ocimene (>95% purity by GC) was synthesised by 1,4 addition of sulphur dioxide to isoprene, followed by anionic, regioselective dimethylallylation and subsequent removal of the cyclic sulfone protecting group. The importance in generating highly pure material, on a sufficiently large scale, while the molecule is inherently unstable provided some difficulties, but our experience of handling labile semiochemicals, including use of an inert atmosphere, overcame this.

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01/03 Select crop plant cultivars with maximal emission of plant activators.(S01/03 Identify crop plant cultivars, or near relatives that respond maximally to plant activators.)

As seen with the study of (E)-ocimene in 01/01, we have identified multifunctional roles for some potential plant activators and stress induced volatiles. Thus, for example cis-jasmone itself shows significant activity against a range of pest and insects antagonistic to these pests in addition to its effects as a plant activator. In order to exploit this multifunctional role we have developed new longer lasting field formulations (see report for PS2105) and investigated the plant activator effects of cis-jasmone on a range of crop plants to identify elite varieties that respond maximally to treatment with this plant activator.In initial laboratory studies, a number of different crop plants were exposed to cis-jasmone and the changes in their volatile profiles, compared to untreated plants were monitored by air entrainment and GC analysis (Figs 1 & 7). As described above, one of the main compounds found to be increased in treated plants was (E)-ocimene, a key semiochemical mediating host location by aphid parasitoids (Du et. al., 1998), and for which we now have preliminary evidence for its role as a plant activator (see above). Although induction of (E)-ocimene by cis-jasmone treatment varied between replicates for many of the crop plants investigated there were clear differences between elite varieties. For example, there were no significant differences from cis-jasmone treated and untreated pepper variety Ferrari, but there was a significant increase in production of both (E)-ocimene and 6-methyl-5-hepten-2-one, already described by us to be highly valuable defence semiochemicals, by cis-jasmone treated pepper variety Bell Boy (Fig 1). cis-Jasmone treated potato plants, variety Desiree, showed elevated levels of (E)-ocimene, (β)-caryophyllene 4,8-dimethyl-(E)-nona-1,3,7-triene, 4,8,12-trimethyl-(E,E)-trideca-1,3,7,11-tetraene and traces of germacrene D (Fig 7) Some of these compounds (4,8-dimethyl-(E)-nona-1,3,7-triene, 4,8,12-trimethyl-(E,E)-trideca-1,3,7,11-tetraene, and (β)-caryophyllene) were also identified from a potato plant, which was exposed to an aphid infested potato (see S01/01). Further studies are required to determine whether this effect is variety dependant. A more detailed study to identify elite varieties with maximal response to plant activators was conducted with winter wheat. Although wheat seedlings produce very low levels of volatiles even after treatment with cis-jasmone, we have demonstrated a significant reduction in cereal aphid settlement and development and an increase in time spent by aphid parasitoids on treated plants (see report for PS2105) (Bruce et. al., 2003a,b,c). In addition, there is considerable up regulation of genes in the pathway to the production of the hydroxamic acids (HAs), which have been shown to be active against a range of insect pests, including aphids, and work on which is described in Objective 3 below. Since wheat ears are attacked by aphids, particularly the grain aphid, S. avenae, and the newly emerging major pest, the orange wheat blossom midge, Sitodiplosis mosellana, the effects of cis-jasmone on a range of elite varieties were investigated at this susceptible growth stage. As found in other studies (see Objective 02/01), a number of key compounds, particularly β-caryophyllene, (E)-ocimene and 6-methyl-5-hepten-2-one were shown to increase on cis-jasmone treatment. There was a significant variation in levels of these compounds produced by the different elite lines following cis-jasmone treatment. Some varieties e.g. Napier, Consort and ECO22 had trace levels of key volatiles and did not show any increase in production of 6-methyl-5-hepten-2-one after treatment with cis-jasmone Other varieties e.g. Lynx and most particularly Hussar showed an increase in all three compounds after treatment (Fig 8). Interestingly, when Hussar was infested with aphids, air entrainment samples showed that all three compounds were elevated, but less so than in the cis-jasmone treatment. A similar picture was observed with Lynx except that levels of 6-methyl-5-hepten-2-one were much higher following aphid infestation. The most elevated compound in Lynx and Hussar was β-caryophyllene and this compound alone, a synthetic blend of the three compounds and the natural sample from Hussar were tested against S. avenae in an olfactometer bioassay. Neither the β-caryophyllene alone or the synthetic blend significantly reduced the amount of time spent in the treated arm of the olfactometer, but the natural air entrainment sample of cis-jasmone treated Hussar was significantly repellent to this aphid (Fig 9). This indicates the presence of other compounds in the natural sample that

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contribute to the repellent effect. After more sensitive GC/MS analysis, traces of 4,8,12-trimethyl-(E,E)-trideca-1,3,7,11-tetraene were found in the Hussar entrainment sample. This is one of the key aphid repellent compounds already identified from induced potato plants (see S01/01). In replicated olfactometer studies, S. avenae virginoparae were significantly repelled by a synthetic sample of 4,8,12-trimethyl-(E,E)-trideca-1,3,7,11-tetraene (Fig 10), indicating that this is the key repellent compound for this aphid in this instance. Work on the aphid parasitoids is reported in Objective 2 - 02/02.

Objective 2 Define host plant location and avoidance of unsuitable potential hosts so as to identify new semiochemicals. In addition to pests, this will also include parasitoids and predators. This objective addresses the question as to whether host specificity is conferred by distinctive key components from the host plant, or by specific blends of ubiquitous components. In the former case, we used insects specifically adapted to toxic hosts to attempt to identify non-toxic volatile semiochemicals that are characteristic of those plant taxa in the hope of identifying new repellent non-host cues or the semiochemicals that can interfere with host plant colonisation. In addition to pest insects, this work also investigated effects on natural enemies as it is important to determine whether or not host plant volatiles that disrupt colonisation by pest herbivores also interfere with the foraging behaviour of predators and parasitoids.

02/01 Identify key semiochemicals mediating recognition of appropriate/non-appropriate host plants based on ratios of ubiquitous plant volatiles. The winter wheat variety ECO22 is particularly susceptible to the orange wheat blossom midge, S. mosellana, and GC-MS analysis of an entrained sample of ears of this variety at early anthesis identified 5 compounds: α-pinene, 6-methyl-5-hepten-2-one, 3-carene, acetophenone and 2-dodecanone, which were electrophysiologically active against the midge in GC coupled EAG studies (Birkett et al., 2004). When presented as a 5 component synthetic blend, in the natural ratio and concentration, the blend was as attractive to female midges in an olfactometer assay as the natural air entrainment sample of the ears. In subsequent assays, the blend was reduced by one component, the 6-methyl-5-hepten-2-one was omitted, and yet the females were still significantly attracted to the 4 component blend. However, when the 5 component blend was presented in an unnatural ratio, (15ng instead of 5ng of 6-methyl-5-hepten-2-one) the blend was not attractive (Fig 11). Thus, for this insect these data provide clear evidence that host recognition is conferred by specific ratios of ubiquitous compounds. As described above, 6-methyl-5-hepten-2-one acts as a spacing cue for R. padi and is also attractive to aphid parasitoids. However, here 6-methyl-5-hepten-2-one would seem to be acting as a key indicator of a stressed i.e. a non-appropriate host plant for the midge. Surprisingly no trace of phenylacetaldehyde, which we have shown to be a potent attractant for the orange wheat blossom midge parasitoid, Macroglenes penetrans, was found in the volatiles from any wheat variety either untreated or cis-jasmone treated. When the volatiles from another susceptible variety Lynx were collected by air entrainment a number electrophysiologically active components, in addition to those identified from ECO22, were identified. These were 3-carene, acetophenone, (Z)-3-hexenyl acetate, 2-ethyl hexanol, 1-octen-3-ol, 2-tridecanone and nonanal. Levels of 1-octen-3-ol were too low to allow determination of the full stereochemistry. A synthetic blend in the natural ratio of 6 of the 7 components (comprising the compounds detailed above but excluding 1-octen-3-ol since the chirality had not been determined) were presented to female midges in the olfactometer and they were as attractive as the natural air entrainment sample. Surprisingly, when the synthetic blend was reduced to the three most electrophysiologically active components 3-carene, acetophenone and (Z)-3-hexenyl acetate, it was as attractive to female midges in the olfactometer as the full blend (Fig 12). Using this simplified blend, slow release dispensers were designed to release the three components in the correct ratio at high and low rates and these were tested in field traps in PS2105. However, although stable under controlled temperature and wind speed conditions in the laboratory the formulations were not field stable and ratio integrity was lost for 3-carene in particular (Fig 13). The key aspect of future work will be to develop stable formulations maintaining release integrity

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in the field.

S02/01 Identify non-toxic semiochemicals mediating host location relating specifically to toxic host plants To address further the question as to whether host recognition is conferred by distinctive key components from the host plant and to attempt to identify additional host location cues relating to toxic host plants, coupled GC-EAG and GC-MS studies were conducted with the cinnabar moth, Tyria jacobaeae, and volatiles from its host plant ragwort, Senecio jacobea. The electrophysiologically active compounds in the air entrainment samples from S. jacobea were identified as the ubiquitous plant volatiles (Z)-3-hexenal, (E)-2-hexen-1-ol, benzaldehyde, myrcene, benzyl alcohol and phenylacetaldehyde. Thus, even for this specialist insect, feeding on a toxic plant, host recognition appears to be based on a collection of common plant volatiles rather than taxonomically specific semiochemicals.In extension of this, a number of toxic plants including ragwort, foxglove, Digitalis purpurea, deadly nightshade, Atropa belladonna and garlic chives, Allium tuberosum, were tested in an olfactometer against the grain aphid, S. avenae. However, with the exception of ragwort no repellent effects were observed. Even with ragwort repellent effects against S. avenae were only observed when the plants were at an early growth stage (mean time spent: in treated arm 0.93 min; in control arm 2.93 min, t test P=0.002). The volatiles from ragwort plants, (flowers and foliage) were entrained and analysed and the major physiologically active components were phenylacetaldehyde (44% of total volatiles), 2-phenylethanol (9.3%), limonene (4.4%) and benzaldehyde (4.4%) with several other minor components. However, it is possible that nitrogen containing compounds may be lost during the isolation protocols used here. Further studies using more benign isolation techniques, particularly vacuum distillation, will be required to address this issue.

02/02 Establish roles of newly identified semiochemicals at higher trophic levels.As described in S01/01 above, untreated potato plants, exposed individually for 24 hours, to the volatiles from an aphid infested potato in a chimney cage produced significant levels of the stress related compounds, 4,8-dimethyl-(E)-nona-1,3,7-triene, 4,8,12-trimethyl-(E,E)-trideca-1,3,7,11-tetraene, 6-methyl-5-hepten-2-one, (β)-caryophyllene and cis-jasmone, which were not present in volatile profiles of untreated plants (S01/01). In addition, trace but significant levels of 4,8,12-trimethyl-(E,E)-trideca-1,3,7,11-tetraene were detected in cis-jasmone treated Hussar wheat variety. As detailed in 01/03 air entrainment samples from the cis-jasmone treated Hussar were repellent to the grain aphid S. avenae as was synthetic 4,8,12-trimethyl-(E,E)-trideca-1,3,7,11-tetraene.In previous wind tunnel studies we have shown that the aphid parasitoid, A. ervi, is attracted to 6-methyl-5-hepten-2-one and (E)-ocimene production of which is induced by cis-jasmone treatment (Birkett et. al., 2000). In the present Project we have demonstrated attraction of this parasitoid to cis-jasmone treated wheat and determined the key semiochemicals mediating this. We have shown that in addition to 6-methyl-5-hepten-2-one and (E)-ocimene, this parasitoid is also significantly attracted to 4,8,12-trimethyl-(E,E)-trideca-1,3,7,11-tetraene in an olfactometer bioassay (Fig 14) (see S01/01 above). Further intensive observational assays, using specialist software (Noldus©), were performed to determine fully the role of this compound on parasitoid behaviour and host location. The behaviour of adult mated female A. ervi, collected from a laboratory culture (i.e. they had experienced contact with aphids) was observed on small wheat seedlings. As reported previously (PS2105) the parasitoids spent longer foraging on cis-jasmone treated wheat than on the untreated control. In extension of this work, the behaviour of A. ervi mated females was observed on wheat seedlings baited with a dispenser releasing the 4,8,12-trimethyl-(E,E)-trideca-1,3,7,11-tetraene, and compared to behaviour on untreated plants. The parasitoids spent significantly longer on the treated plants compared to the untreated ones (Fig 15). Thus 4,8,12-trimethyl-(E,E)-trideca-1,3,7,11-tetraene appears to have a dual role in host location for this parasitoid, functioning both as an attractant and an arrestant. Surprisingly, the closely related 4,8-dimethyl-(E)-nona-1,3,7-triene was not active in these bioassays.Volatiles collected from wheat and shown to be active in GC/EAG and in laboratory studies in

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02/01 for female wheat blossom midge, S. mosellana, were field tested using yellow sticky traps or water traps, baited with either individual compounds e.g. 6-methyl-5-hepten-2-one at a range of release rates or mixtures of the plant volatiles, in PS2105. There was no significant attraction of female midges or the midge parasitoid, M. penetrans, to any of the treatments. In the case of the mixtures, this was probably due to the difficulty of releasing these volatiles in the correct ratios under field conditions (see 02/01). However, as observed in previous studies in PI0341 there was very strong attraction of the parasitoid to phenylacetaldehyde (see S04/02). This compound, when released at relatively high levels (10mg/lure/day) also attracted adult lacewings, Chrysoperla carnea, and caught 36 individuals in a baited yellow water trap over a two month period compared to none in an untreated trap. When the release rate was reduced to 1.7mg/day very few C. carnea were captured.

S02/02 Extension of host recognition to other pest/plant taxa.Since host recognition for insects feeding on toxic plants was found to be based on a collection of common plant volatiles rather than taxonomically specific semiochemicals (see S01/01) this line of investigation was switched to the study of aphid response to non-host plants. Laboratory behavioural studies showed that the grain aphid, S. avenae, was repelled by the volatiles from potato leaves and by air entrainment samples of potato and basil plants, Ocimum basilicum. The major physiologically active compound conferring the repellent effect of basil was germacrene D, which was identified previously in the essential oil of Hemizygia petiolata and shown to be highly active against S. avenae (Bruce et. al., 2005a) and shown to be induced in potato plants treated with the plant activator, cis-jasmone (see Objective 1). In addition, a number of non-host plant essential oils were investigated. Of these, the essential oil of cumin, Cumina cyminum, was repellent to S. avenae and the key compound shown to be α-pinene.

02/03 Make available the most promising host recognition semiochemicals.The synthesis of 4,8,12-trimethyl-(E,E)-trideca-1,3,7,11-tetraene (see S01/01, 02/02) was achieved starting from the commercially available (E,E)-farnesol (Fig 16). This latter was catalytically oxidized using a perruthenate derivative to give the corresponding aldehyde. The Wittig reaction, reaction between a phosphonium ylide and an aldehyde, allowed the formation of the desired product with high yield and more importantly high chemical and stereochemical purity (>98% purity by GC). In the same way, the synthesis of 4,8-dimethyl-(E)-nona-1,3,7-triene (>99% purity by GC) was achieved starting from the commercially available geraniol.

Many of the compounds identified in S02/02 are unstable. However, our previous experience with such compounds has shown high compound stability in essential oils. Thus we have searched for essential oils rich in the key compounds. Ylang ylang contains significant quantities of (-)-germacrene D and was shown to be significantly effective in laboratory behavioural assays against S. avenae (mean time spent: in treated arm 1.63 min, in control arm 3.31min, P=0.032). Slow release formulations were developed and provided for evaluation in the field in PS2105. In an attempt to exploit the aphid alarm pheromone (details of studies with Hemizygia petiolata essential oil are presented in PS2105) a search was conducted for essential oils enriched in (E)-β-farnesene. One such oil, German chamomile, Matricaria recutita L, was found to contain 38% (E)-β-farnesene. However, despite this no alarm response was observed for the peach-potato aphid, Myzus persicae, in laboratory bioassays and further studies identified low levels of bicyclogermacrene, shown previously to inhibit the alarm pheromone response for this aphid.

Since all renewable sources of (E)-β-farnesene investigated, contain additional compounds that inhibit the alarm pheromone response an alternative approach to exploitation of this pheromone was taken. In a collaborative BBSRC project “Putting insects off the scent: modifying plant semiochemistry to disrupt plant-insect interactions” we used high levels of expression, in Arabidopsis thaliana plants, of an (E)-β-farnesene synthase gene cloned from Mentha × piperita, to cause emission of pure (E)-β-farnesene as determined by air entrainment and analysis of the volatile samples by gas chromatography. These plants elicited potent effects on behaviour of the

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aphid M. persicae, both in terms of alarm (Fig 17) and repellent responses (Fig 18). In addition, the air entrainment sample showed a strong GC-EAG response from aphid parasitoid Diaeretiella rapae (Fig 19) and in laboratory behavioural assays these plants elicited a strong arrestant response from the parasitoid (see also report for PS2105). This is the first time that a plant has been transformed to produce an insect pheromone and demonstrate that the resulting emission affects behavioural responses at two trophic levels (Beale et. al. submitted). These studies show production of an insect pheromone in a transgenic plant and clearly indicate the potential value for aphid control of plants expressing an (E)-β-farnesene synthase gene. This would seem to be particularly appropriate for improving the level of aphid control achieved by aphid parasitoids. The applied value of the current work will be determined by experiments with transgenic crop plants such as oilseed rape, Brassica napus. These will be produced also by using promoter sequences that facilitate the specific induction of (E)-β-farnesene production following exposure to natural plant activators, at times when aphids are expected to attack. This work could form the first generation of new genetically modified crops that produce natural plant products active by non-toxic mechanisms and which would be totally different to plants expressing non-plant genes for toxic products, such as Bacillus thuringiensis endotoxins.

Many of the electrophysiologically active compounds, particularly sesquiterepene hydrocarbons, identified in this Project are not commercially available or require complex and expensive synthesis by conventional chemical routes. However, essential oils provide a rich source of these compounds. A micro-scale preparative GC technique has been developed that allows isolation of these compounds from essential oils or other botanical sources e.g. β-bourbonene, δ-elemene and germacrene D from gum haggar, in sufficient quantities for structure confirmation by micro-probe NMR (Fig 20). This authenticated material is then available for confirmation of tentative MS identifications by GC co-injection and laboratory behavioural studies in other Objectives, particularly for the isolation and identification of lacewing semiochemicals, (Objective 4) and in associated Projects.

S02/03 Determine the means of modifying key ratios of semiochemicals in a crop environment.Slow release dispensers of (Z)-3-hexanol and (Z)-3-hexenyl acetate were developed for use in field trials in PS2105 specifically to interfere with host location by augmentation of key components.

Objective 3. Determine the potential value of exploiting rhizosphere allelopathy, in controlling soil-pest/plant interactions, and suppressing competitive weeds.This Objective initiated studies on allelopathy and the identification of selected allelopathic agents. Allelopathy is defined as ‘any direct or indirect harmful or beneficial effect by one plant (including microorganisms) on another through production of chemical compounds that escape into the environment’. Allelopathic compounds enter the region around plants by a number of routes including volatilisation from the leaves, exudation from the roots, leaching from leaves or from plant litter by rain, and by decomposition of plant residues. Such compounds can have direct biocidal and signalling effects on plants and soil organisms. Recent work by us has demonstrated more subtle allelopathic interactions including signalling between plants via the rhizosphere showing that aphid-infested broad bean plants can induce uninfested plants to release parasitoid-attracting volatile semiochemicals by means of a chemical activator conveyed from plant to plant via the root system. Another research project involving ourselves on the control of the parasitic witchweed, Striga hermonthica, by intercropping with legumes of Desmodium spp. has highlighted the potential of allelopathic compounds in controlling pernicious weeds and studies will be extended to investigate legumes used in UK cropping systems.

03/01 Develop methods for collection, isolation and bioassay of root and leaf exudates that affect neighbouring plants.As a means of investigating the transmission of plant signals through the rhizosphere, a simple

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but very versatile experimental system has been developed. Using peristaltic pumps, nutrient solution is circulated through vessels in which the emitter and receiver plants are growing hydroponically. Aphids or other herbivores are easily confined on either plant, and volatile compounds can be collected, using standard entrainment equipment. The identification of possible root signals requires the nutrient solution from the emitter to be pumped through suitable SPE cartridges to collect such compounds, which are then eluted with solvents for analysis by HPLC or GC and mass spectrometry. This system allows for the first time the simultaneous collection of both aerial and rhizosphere signals from the same emitter plant and aerial signals from the receiving plant.

In previous studies we have shown that rhizosphere signals i.e. exudates from the roots of beans, Vicia faba, infested with pea aphid, Acyrthosiphon pisum, elicit modification of the secondary metabolism of an intact and uninfested receiving bean plant so as to make it more attractive to the aphid parasitoid A. ervi (Chamberlain et. al, 2001). In order to confirm that the plants were not stressed under the hydroponic culture conditions, volatiles were collected from uninfested and infested beans. The volatile profiles of beans grown hydroponically were identical to those obtained from beans grown conventionally in compost. Collection of the volatiles from hydroponically-grown aphid infested beans were analysed by gas chromatography (GC) and GC-MS and showed that the levels of five compounds were significantly higher than in uninfested beans. One compound, the aphid alarm pheromone, (E)-β-farnesene, was only present in the infested bean entrainment sample. The other 5 compounds which were present at higher levels in the aphid infested plants comprised (Z)-3-hexen-1-ol, (Z)-3-hexen-1-yl acetate, (E)-ocimene, (E)-caryophyllene and 6-methyl-5-hepten-2-one. This is the same profile of compounds identified from A. pisum infested beans grown conventionally. In particular, we have shown in previous studies that 6-methyl-5-hepten-2-one is specifically associated with A. pisum herbivory on beans and is not produced by other aphids feeding on beans e.g. Megoura viciae or Aphis fabae. As detailed previously, 6-methyl-5-hepten-2-one and (E)-ocimene are key components mediating host location by the aphid parasitoid, A. ervi. Thus hydroponically grown beans are behaving in exactly the same way as plants grown in compost (also see rhizosphere signalling studies in PS2105) and thereby justify this practical approach to exploitation of rhizosphere signalling and allelopathy.In order to identify the rhizosphere signal derived from A. pisum infested plants the nutrient solution from aphid infested and uninfested plants was extracted onto solid phase cartridges (700mg C2/ENV+) and the adsorbed organic material eluted with redistilled diethyl ether. Subsequent GC analysis of these samples showed no major difference between infested and uninfested samples suggesting that either the signal was not produced, was involatile or that the solid phase extraction was ineffective. However, the finding that there was no change in the volatile profile of plants exposed to the root exudates from aphid infested plants as compared to control plants, exposed only to root exudates from uninfested plants, suggests that, in these experiments, there was either no production of the rhizopshere signal or that the receiver plants were not responding. Further studies would be needed to investigate this. However, all would require replicated wind tunnel assays of challenged receiver plants with the parasitoid, A. ervi (see PS2105). Since aphid parasitoids are highly susceptible to fluctuations in barometric pressure resulting in large day to day variability in their responsiveness this approach was not deemed feasible. Attempts to follow changes in the receiver plants by coupled GC-EAG with the parasitoid have been precluded by the low levels of volatiles produced.

Studies have been conducted to investigate the allelopathic effect of couch grass, Elytrigea repens, root exudate on the germination and subsequent seedling growth of wheat. The nutrient solution from hydroponically-grown couch grass was circulated over wheat seeds (cv. Axona) and the results compared with a control comprising nutrient solution only with no couch grass root exudate. Germination of the seeds was not affected but there was an almost significant (P=0.058) increase in shoot length after 5 days. The mean shoot lengths (4 experiments, 33

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seedlings in all) were:- control 2.519 ± 0.242; treated 3.173 ± 0.328. There was no significant effect on root weight although the appearance of the roots differed between control and treatment. The roots of treated plants were numerous, but shorter and thicker than those of the untreated control plants. Similar studies to investigate potential allelopathic effects of couch grass exudates on black grass, Alopecurus myosuroides, have been confounded by the low germination of the field collected seed.

S03/01 Identify biological systems in which plant – plant interactions via the rhizosphere can be detected in changes in plant physiology. Allelopathic effects have been studied by bioassay with insect herbivores or their parasitoids. Methods to correlate bioassay results with changes in the expression of marker genes for resistance are being developed. This will allow rapid in vitro tests on receiver plants to be carried out and will facilitate the identification of allelopathically active signalling compounds in the medium. To identify suitable markers that are strongly up-regulated in the receiver plant after contact with nutrient solution from the emitter, changes in gene expression in these plants are being assessed using PCR. Currently expression of genes encoding members of the pathogenesis related protein families, including those that have been demonstrated to be up regulated in cereals by aphid infestation, is being investigated.

The nutrient solution in which a bean plant infested with aphids was growing was circulated through another vessel in which an uninfested bean plant was growing for several days. The shoots of both plants were contained in glass chambers and filtered air pumped in to them. The volatiles produced by the uninfested plant were collected for 24h periods on Porapak Q and subsequently eluted by gas chromatography. The differences from a blank sample (untreated plant connected to another untreated plant) were very small. However, one compound that was induced in several different collections was 3-octanone and will be investigated further (see also report for PS2105). Nutrient solution from these experiments was saved for further analysis. In addition, nutrient solutions from hydroponically grown couch grass, E. repens, black grass, A. myosuroides and white clover, Trifolium repens, have also been collected and stored at 15°C for bioassays and biochemical analysis

03/02 Identify newly isolated rhizosphere signalling compounds.An extraction protocol for the isolation, by solid-phase extraction (SPE), of rhizosphere semiochemicals, particularly root exudates, has been developed, and extracts have been analysed by high-pressure liquid chromatography (HPLC). Root exudate has been collected from couch grass, E. repens, and further compounds, in addition to the carboline already identified, were isolated and identified as tryptophan, 5-hydroxytryptophan and 5-hydroxyindoleacetic acid and are being tested for effects on barley plants and cereal aphid behaviour.

In laboratory studies with the carboline and barley variety Siberia, grown in hydroponic solution in 30ml blacked out glass vials, it was found that the solvent acetone, used to formulate the carboline, had a significantly detrimental effect on root growth on treated plants and this effect was investigated prior to starting new studies with other plant species. A number of solvent formulations were tried, but all had similar effects on root development. Although only weakly soluble, we were able to obtain a significant reduction (in PS2105) in alate R. padi settlement on barley plants treated with carboline in water alone after 24h in a no choice test. However, the carboline treatment resulted in significant necrosis of the roots of treated plants.

Liquid phase extraction (LPE) using N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSFTA) as a derivatising reagent, and vapour phase extraction (VPE) in combination with trimethylsilyl diazomethane (TMSCHN2) as a derivatising reagent were used to evaluate the impact of the plant activator, cis-jasmone, on the secondary metabolism of wheat, Triticum aestivum, var. Solstice. Liquid phase extraction allowed the measurement of hydroxamic acids DIMBOA, HMBOA and

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MBOA, and phenolic acids such as trans-p-coumaric acid, syringic acid, p-hydroxybenzoic acid, vanilic acid and cis and trans-ferulic acid. Using LPE, levels of DIMBOA were significantly higher in leaves and roots of T. aestivum treated with cis-jasmone when compared with untreated plants. The amount of DIMBOA in leaves treated with cis-jasmone was 108.1 ± 15.2 mg/Kg of fresh weight, and in untreated leaves was 42.9 ± 12.2 mg/Kg. In roots, the concentrations recorded were 729.2 ± 156.1 for treated roots and 162.3 ± 37.3 mg/Kg for untreated roots. Similar results were obtained for some phenolic acids, such as trans-ferulic acid and vanillic acid in roots. Using vapour phase extraction, it was possible to measure HBOA, benzoxazoline-3-one, ferulic acid, syringic acid and coumaric acid. Quantitative analyses of HBOA in leaves ranged from 12.6 ± 2.6 mg/Kg in cis-jasmone treated plants and 0.6 ± 0.2 mg/Kg in untreated plants, while in roots the concentrations were 34.2 ± 10.1 mg/Kg for cis-jasmone treated plants and 0.8 ± 0.1 mg/Kg for untreated plants.

03/03 Establish modes of action, in the receiving plant, of possible herbicidal or phytotoxic agents Although compounds are not yet available for establishing the mode of action on the receiving plant of herbicidal or phytotoxic agents, we have found, against all expectation, a dramatic induction by cis-jasmone of allelophathic agents i.e. hydroxamic acids (HAs) or benzoxazinones/benzoxazolin-2-(3H)-ones, with the generally most active component being 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and have therefore concentrated on this aspect. Highly significant (ten fold) differences in constitutive levels of these compounds have been measured in young wheat seedlings, including UK varieties, and those with high concentrations of HAs display greater resistance to aphids, e.g. effects on settling, growth rate and fecundity, than those with low concentrations (Sicker, et al., 2000). Artificial feeding experiments confirmed that the levels detected in plant tissues are sufficient to deter insects. Wide ranging HA concentrations both within the roots and in root exudates have been measured in wheat varieties that correlate with allelopathic activity and this could be important for the development of weed control. However, significantly, there are no reports of measurable concentrations of these compounds being detected in the grain, indicating that varieties that produce high levels of HAs would not represent a risk to human health. The biosynthetic pathway and associated genes for the HAs were first elucidated in maize (Sicker, et al. 2000). The molecular characterisation and chromosomal localisation of the HA biosynthetic genes involved in hexaploid wheat have also been completed (Nomura et al. 2003). The genes in the pathway, consistent with the situation that is emerging for a number of defence secondary metabolites, are clustered together in the genome of the producer plants. The overwhelming evidence that the HAs play a significant role in pest resistance scientifically justifies exploring the possibility that the HA pathway can be exploited to achieve environmentally benign pest resistance. Evidence that the HA pathway can be induced is provided by the observation that HA concentrations increase in response to aphid feeding (Sicker et al. 2000). Taking the advantages of cis-jasmone into account, we plan to investigate if it induces HA accumulation and if this effect contributes to the cis-jasmone induced aphid resistance in wheat that we have observed.For this work we have developed end point RTPCR and real time relative q RTPCR analysis to measure the expression of the major genes (the Bx genes) in the pathway. We have found that, consistent with the observation that HA levels decrease as the plant matures beyond the seedling stage, expression of all of the Bx genes is highest in young plants, progressively decreasing over 14 days post germination. Accumulation of HAs, measured by HPLC, is highest in the leaf tissue but the expression of all the genes is higher in the stem than in leaf tissue with Bx5 being the most highly expressed. This suggests that some degree of transport of the compound from the stem to the leaves is taking place. Preliminary studies clearly demonstrate that cis-jasmone when applied to leaves as an aqueous emulsion using the non-ionic food grade surfactant Ethylan BV (EBV), induces Bx gene expression in certain selected varieties. Good indication has been obtained for increased expression of all Bx genes in roots after cis-jasmone treatment in the variety Solstice using

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endpoint RTPCR, with the maximum increase appearing between 20 and 30 hours after the leaves were sprayed with the reagent. Induction was also observed in stem, but not leaf tissue in this variety (Fig 21).

S03/03 Provision of newly identified allelopathic agents. 2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) was isolated from maize, Zea mays, plant material. Plants were frozen at -20°C, with the disruption of the cell wall enabling efficient extraction of the glycoside, which is cleaved enzymatically during thawing by -glucosidase. The plant material was homogenized after thawing in the presence of ethyl acetate as the extraction solvent. Subsequently, DIMBOA was extracted with a saturated NaHCO3

solution, reprotonated with HCl, and re-extracted in ethyl acetate. After evaporation the substance was purified by washing with diethyl ether.

Objective 4. Identify new strategies for exploiting insect predators and parasitoids via semiochemical tools.This objective provided the semiochemical tools to investigate manipulation of a range of aphidophagous and polyphagous predators, such as lacewings, ladybirds and carabids. In addition, it provided tools to manipulate interactions between different natural enemies. Potentially, different natural enemies are in competition with each other for the same aphid resources. For example, parasitoids need to avoid laying eggs in aphid colonies that are already being attacked by predators such as ladybirds. Work in a related project, AR0305 (Utilising populations of natural enemies for control of aphids) has shown that they do this by detecting semiochemicals left on the plant surface by a foraging ladybird, which cause them to leave and move on to another plant. Such semiochemicals could be useful for manipulating the distribution of parasitoids, for example to remove them from areas at risk from sprays or from horticultural crops about to be harvested on which they would be regarded as contaminants. Such potential needs to be assessed.

04/01 Identify semiochemical cues, other than the aphid sex pheromone, mediating predator/prey interactions. Coupled GC-EAG recordings of the antenna of the hoverfly, Episyrphus balteatus, located regions of activity from oilseed rape flower volatiles. 7 active compounds have been identified by GC-MS (benzaldehyde, myrcene, phenylacetaldehyde, limonene, acetophenone, methyl benzoate and methyl salicylate). Active compounds e.g. (E)-ocimene, linalool and 2-phenyl ethanol were also identified from bean flower volatiles.

S04/02 Identify floral volatiles and determine roles in foraging behaviour of adult hoverflies and parasitoids. Of the floral volatiles identified in 04/01, 2-phenylethanol was significantly attractive to the predaceous hoverfly E. balteatus in field traps. Protocols for the study of the flight behaviour of E. balteatus in response to volatiles have been established. In field trapping trials, the flower volatile phenylacetaldehyde attracted large numbers of the wheat midge parasitoid Macroglenes penetrans. Interestingly, this compound is not present in the volatile profile of wheat either in ear or in flower, the pertinent growth stages for the parasitoid. A live trap is being developed for field collection of M. penetrans for GC-EAG studies.

S04/01 Identify semiochemicals mediating host location by insects that impact negatively on beneficial insects. Field traps, containing a slow release formulation of the ladybird kairomone precoccinelline, identified from the 7 spot lady bird, Coccinella septempunctata, were designed and deployed to attract the ladybird parasitoid Dinocampus coccinellae, however, none were caught. The harlequin ladybird, Harmonia axyridis, has been described as the most invasive ladybird in the world. Originating in Asia, it has been mass produced as an effective biocontrol agent, but after release in the USA it has become the predominant species, out-competing and replacing

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other ladybird species. A similar pattern has been developing since its release in continental Europe. In 2004, it arrived in SE England and has spread rapidly across the UK. As it is a much more efficient predator and is capable of producing several generations each year, there is concern that through intraguild predation and depletion of food resources it will marginalise or even replace UK native species. To provide semiochemical tools for reducing such negative impacts, the chemical ecology of H. axyridis has been studied, in particular the aggregation behaviour of adults prior to overwintering, and interaction with its’ natural enemy, D. coccinellae. To study the chemically-mediated interactions of H. axyridis, a large scale extract (~500 adults) was prepared using chloroform as the solvent (24h, ambient temperature). The crude extract was then subjected to vacuum distillation (24h) to provide a wholly volatile fraction, which was concentrated to ca. 100 l under a gentle stream of nitrogen. The extract was subjected to coupled GC-organoleptic evaluation and coupled GC-MS analysis, leading to the identification of 2-isopropyl-3-methoxypyrazine and 2-isobutyl-3-methoxypyrazine. The former compound has already been identified as an aggregation pheromone component for the seven-spot ladybird, C. septempunctata, and the presence of an additional component for H. axyridis suggests that they play specific, similar roles in the aggregation behaviour of H. axyridis. In laboratory behavioural assays using a 2-arm olfactometer, adult H. axyridis were attracted to both compounds (Fig 22), although due to behavioural variation, the response was not always statistically significant. The wholly volatile extract of adult H. axyridis prepared as described above was used in coupled GC-EAG studies of D. coccinellae with an extract of H. axyridis demonstrated that this ladybird does not release the alkaloids that the parasitoid uses to find C. septempunctata. As part of a programme to minimise the effects of this ladybird on native species we therefore need to look further for the semiochemical involved in location of this pest by parasitoids. 04/02 Identify semiochemicals mediating interspecific interactions within natural enemy guilds.The role of semiochemicals in mediating intraguild interactions between an aphid predator and an aphid parasitoid were investigated, using the ladybird, C. septempunctata, and the aphid parasitoid, Aphidius ervi. The effect of C. septempunctata adult and larval trails on A. ervi behaviour and parasitism rates was investigated, using a dual-choice leaf square bioassay. Female parasitoids significantly avoided leaves previously visited by C. septempunctata adults and larvae. The behaviourally active period was lost after 24 hr. Ethanol extracts of C. septempunctata adults and larvae, when applied to leaf squares, also induced avoidance responses by A. ervi. Vacuum distillation of the adult ethanol extract generated a distillate containing volatile components, and a residue containing components with little or no volatility. Leaf square bioassays with the distillate and the residue showed that the latter was responsible for the biological activity. Liquid chromatography of the residue, and testing of the fractions, showed that the active compounds were non-polar hydrocarbons. Two of the hydrocarbons identified by gas chromatography (GC) and coupled GC-mass spectrometry (GC-MS), n-tricosane (C23H48) and n-pentacosane (C25H52), when tested individually at levels found in the adult extract, significantly induced avoidance by A. ervi. Further investigation of the larvae extract, and footprint chemicals deposited by adults in glass Petri dishes, confirmed the presence of the hydrocarbons. When the chemicals were tested together, an additive effect was observed. Parasitism rates of the pea aphid, A. pisum, on broad bean plants, V. faba, which had been sprayed with a mixture of the chemicals, were significantly lower than those on control plants. The effect, however, was no longer evident after 24 hr. Field stable formulations of these hydrocarbons have been developed and will be tested in the RELU project: Re-Bugging the System: Promoting Adoption of Alternative Pest Management Strategies in Field Crop Systems

The avoidance responses of aphid parasitoids with varying host ranges (Aphidius eadyi, A. ervi and Praon volucre) to chemical trails deposited by intraguild predatory ladybirds, C. septempunctata and Adalia bipunctata, were investigated. Females of all three parasitoid species significantly avoided leaves previously visited by C. septempunctata or A. bipunctata adults. The

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avoidance responses shown by the two Aphidius species were stronger to trails of C. septempunctata than to those of A. bipunctata. However, P. volucre avoided trails of both ladybird species to a similar degree. Dose-responses of these three parasitoid species to the hydrocarbons n-tricosane (C23H48), n-pentacosane (C25H52) and n-heptacosane (C27H56), which are components of the trails of both C. septempunctata and A. bipunctata, were evaluated. Dual-choice bioassays indicated that 1) A. eadyi showed more sensitive avoidance responses to n-tricosane than did the other two parasitoid species, 2) all three species showed similar responses to n-pentacosane across a range of doses, and 3) only P. volucre showed avoidance responses to n-heptacosane. Quantitative analyses of each hydrocarbon in the trails of the two ladybird species showed that n-pentacosane and n-heptacosane occur in significantly greater amounts in C. septempunctata trails than in those of A. bipunctata. The trails of the two species also differ qualitatively in the other hydrocarbons present. Compounds identified specifically for C. septempunctata included 13-, 9- and 7-methylheptacosane, 9,13-, 7,11-dimethylheptacosane and 7,11,15- trimethylheptacosane. Compounds identified specifically for A. bipunctata were n-heneicosane, 9- and 7-methyltricosane. Studies elsewhere have implied that such compounds may play a role in mediating intraspecific ladybird interactions (Hemptinne et al., 2001), specifically the oviposition deterrent response of adult A. bipunctata following detection of intraspecific larval footprints. Previous work on avoidance of C. septempuncata footprints by A. ervi (Nakashima et al., 2004) revealed virtually identical hydrocarbon profiles for adults and larvae. Thus, it can be expected that these compounds do indeed play a role in C. septempunctata oviposition deterrent behaviour, and that a similar outcome can be expected for A. bipunctata. Further studies are required to confirm the role of these compounds.

04/03 Identify pheromones of lacewings and parasitoids.The structures of components of the volatile secretions of three lacewing species were elucidated by a combination of GC-MS, GC-EAG, microreactions, chemical synthesis and NMR spectroscopy. The cuticular washings of male and female Chrysoperla carnea and air entrainment of male Peyerimhoffina gracilis contained (4Z,7Z)-tridecadiene as a minor component and (Z)-4-tridecene as the major component. Cuticular washings of male and female C. cognata also produced these two components and in addition produced undecane, (Z)-4-undecene and skatole. (Z)-4-tridecene was shown to be electrophysiologically active to P. gracilis and in field experiments, (Z)-4-tridecene significantly reduced the attractiveness of the field attractant neomatatabiol to male P. gracilis (Fig 23). In Korea, (Z)-4-tridecene also significantly reduced the attractiveness of the field attractant nepetalactol to C. cognata. We propose (Z)-4-tridecene is a semiochemical for P. gracilis and C. cognata. As similar activity has been reported in C. carnea and it may prove to be a general feature within the Chrysopidae.

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.

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Varietal differences in response to cis-jasmone may offer new opportunities for further understanding on field exploitation in delivering semiochemically based effects.

Knowledge TransferPresentations on research results

Gordon-Weeks, R., Smart, L., Chamberlain, K., Wadhams, L. and Pickett, J. The role of lipid transfer proteins in defence against aphids and in response to allelopathic chemicals in barley cultivars. 13th International Workshop on Plant Membrane Biology 6-10 July 2004 Montpellier.

Wadhams, L.J. Temporal and spatial scale effects, and potential complementarity between CBC and semiochemical technologies. Imperial College, Wye Campus,17.10.04.

Wadhams, L.J. New chemical signals in plant protection against herbivores and weeds. Rothamsted Research Association 25.11.04

Presentations by Prof. J.A. Pickett

- Department of Chemical Ecology, Universidad de La Frontera, Temuco, Chile, 20.1.04, 1) “Chemical ecology: general principles and approaches adopted by Rothamsted Research and its collaborators”, 2) “Methods in chemical ecology: from electrophysiological analysis to sustainable semiochemical production”, 3) “Exploiting chemical ecology in IPM: successful applications, including the East African experience”

- Defra, Chief Scientist’s Group (Howard Dalton) 3.2.04, “Bugging the bugs: pheromones and other semiochemicals in crops, animal health and food safety”,

- University of Lund, Sweden 9.2.04, Infochemicals in pest control and conservation biology, 1) “Semiochemicals and pest control” and 2), “Integrated pest management: the value of a trap crop, extending to the push-pull system”

- University of Nottingham, Molecular Ecotoxicology 18.3.04, “Plants and plant products in pest control” - Workshop on Semiochemicals and Microbial Antagonists: Their Role in Integrated Pest Management in Latin America, March 22-26 2004, CATIE, Turrialba, Costa Rica, “Developing new semiochemical-based strategies for pest management: solving the problems of sustainable production, registration and delivery”

- Defra review workshop, Potential to reduce insecticide use against BYDV vectors through genetic improvement to crops, HGCA, London (with Ruth Gordon-Weeks) 23.4.04, “DIMBOA and induced aphid resistance in wheat” - AAB Centenary Meeting, London 26.5.04, “The promise of innovative research in plant stress signalling for crop protection in world agriculture” - Danish Agricultural and Veterinary Research Council, Copenhagen, Summer Meeting at Rothamsted Research 15.6.04, “Novel strategies from plant stress signalling for the control of insect pests”, - Imperial College GSLSM Summer Symposium as Guest Lecturer 15.7.04, “Confusing stories of sex and other pheromones”,

- XXII International Congress of Entomology, Brisbane, Australia, 15-21 August 2004: 1)

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“Herbivore-induced plant volatiles identified from interactions at higher trophic levels: persistent effects on plants in the laboratory and field”, 2) “The plant signal cis-jasmone - genetics to applied ecology”, 3) “Habitat management for stem borers and striga control in Africa: underpinning mechanisms and long term biotechnological opportunities” - German Botanical Society, Braunschweig, Plant-Herbivore Interactions Symposium 8.9.04, “Exploiting chemical signals in plant protection against herbivores”,

- John Innes Centre, Norwich, colloquium on Plant-Insect Interactions 21.9.04, “Plant activators from plant-insect interactions”,

- Annual Meeting European PhD course “Insect Biotechnology”, Maratea, Italy 20.10.04, "Confusing stories of sex and other pheromones: intraspecific and interspecific insect-insect interactions"

- University of Newcastle, plenary lecture to celebrate opening of new Institute for Research on Environment and Sustainability 12.11.04, "New ways to reduce the potential environmental impact of pesticides: natural plant activators in protection against herbivores and weeds" - BBSRC-CREST Joint International Workshop (UK/Japan Partnership Awards) – A post-genomic approach to understanding and exploiting multitrophic interactions / Linking genes to ecology – multitrophic interactions in the 21st century. Kyoto, 17-19 November. “Plant activators from plant/insect interactions”

- AAB Centenary Meeting, St. Catherine’s College, Oxford, Advances in applied biology: providing new opportunities for consumers and producers in the 21st century, St. Catherine’s College, Oxford 15.12.04, “Exploiting rhizosphere interactions in pest and weed management”

- Inter-Institute Brassica workshop, JIC Norwich , “Stimulo-deterrent diversionary (push-pull) strategy for transgenic oilseed rape modified for pest colonisation signals, i.e. kairomone or host plant attractant, 5.1.05

- “The challenge of pests to the food chain, the essential need to control pests, and the need for the food chain and science to collaborate”, Pesticides and the Food Chain seminar, Rothamsted Research, Keynote Address, 10.5.05

- “cis-Jasmone as an allelopathic agent by plant defence induction”, Fourth International Allelopathy Congress, Wagga Wagga, Australia, 24.8.05

- “The demise of pesticides – will the food chain collapse?”, Guest speaker at the BCPC President’s Dinner, Glasgow (Challenge of Pests to the Food Chain) 1.11.05

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AppendixFigures

Fig 1. GC trace of the volatiles produced by sweet pepper, variety Bell Boy, 48h after treatment with cis-jasmone.

Settlement and nymph production by Rhopalosiphum padi on (E )-ocimene treated barley

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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.

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Beale, M.H, Birkett, M. A., Bruce, T. J. A., Chamberlain, K., Field, LM, 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., Woodock, C.M and Zhang, Y. Aphid alarm pheromone produced by transgenic plants affects aphid and parasitoid behavior. (submitted)Birkett, M. A., Agelopoulos, N., Jensen, K-M. V., Jespersen, J. B., Pickett, J. A., Prijs, H. J., Thomas, G., Trapman, J. J., Wadhams, L. J. & Woodcock, C. M. (2004). The role of volatile semiochemicals in mediating host location and selection by nuisance and disease-transmitting cattle flies. Medical and Veterinary Entomology 18, 313-322.Birkett, M. A., Bruce, T. J. A., Martin, J. L., Smart, L. E., Oakley, J. & Wadhams, L. J. (2004). Responses of female orange wheat blossom midge, Sitodiplosis mosellana, to wheat panicle volatiles. Journal of Chemical Ecology 30, 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., Dodds, C. J., Henderson, I. F., Leake, L. D., Pickett, J. A., Selby, M. J. & Watson, P. (2004). Antifeedant compounds from three species of Apiaceae active against the field slug, Deroceras reticulatum (Muller). Journal of Chemical Ecology 30, 563-576.Blande, J. D., Pickett, J. A. & Poppy, G. M. (2004). Attack rate and success of the parasitoid Diaeretiella rapae on specialist and generalist feeding aphids. Journal of Chemical Ecology 30, 1781-1795.Bruce, T.J.A., Birkett, M.A., Blande, J., Hooper, A.M., Khambay, B., Martin, J.L., Prosser, I., Smart, L.E. and Wadhams, L.J. (2005) Response of economically important aphids to components of Hemizygia petiolata essential oil. Pest Management Science 61 (11) 1115-1121.Bruce, T.J. A., 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 - 1036Bruce, T.J. A., Pickett, J.A. and Smart, L.E. (2003b) Cis-jasmone switches on plant defence against insects. Pesticide Outlook 14: 96 – 98Bruce, T.J. A., Pickett, J.A. and Smart, L.E. (2003c) Developing plant activators for the field. Rothamsted Research Annual Report 2002 – 2003Chamberlain, K., Guerrieri, E., Pennacchio, F., Pettersson, J., Pickett, J.A., Poppy, G.M., Powell, W., Wadhams L.J. and Woodcock C.M. (2001) Can aphid-induced plant signals be transmitted aerially and through the rhizosphere? Biochemical Systematics and Ecology 29, 1063-1074.Cook, S. M., Smart, L. E., Rasmussen, H. B., Bartlet, E., Martin, J. L., Murray, D. A., Watts, N. P., Williams, I. H. (2003) Push-Pull strategies to reduce insecticide input to oilseed rape (Brassica napus): Potential of low alkenyl glucosinolate oilseed rape varieties (push!), and turnip rape (Brassica rapa) trap crops (pull!). Proceedings of the 11th International Rapeseed Congress: Toward enhanced value of cruciferous oilseed crops by optimal production and use of high quality seed components. Copenhagen, Denmark, July 2003, Vol. 3, 1015-1017.Du, Y., Poppy, G.M., Powell, W., Pickett, J.A., Wadhams, L.J. and Woodcock, C.M. (1998) Identification of semiochemicals released during aphid feeding that attract parasitoid Aphidius ervi. Journal of Chemical Ecology 24, 1355-1367.Falk, K. L., Vogel, C., Textor, S., Bartram, S., Hick, A. J., Pickett, J. A. & Gershenzon, J. (2004). Glucosinolate biosynthesis: demonstration and characterization of the condensing enzyme of the chain elongation cycle in Eruca sativa. Phytochemistry 65, 1073-1084.Glinwood, R., Pettersson, J., Ahmed, E., Ninkovic, V., Birkett, M.A. and Pickett, J.A. (2003) Change in acceptability of barley plants to aphids after exposure to allelochemicals from couch-grass (Elytrigia repens). Journal of Chemical Ecology 29, 261-274.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.Goldansaz, S. H., Dewhirst, S., Birkett, M. A., Hooper, A. M., Smiley, D. W. M., Pickett, J. A., Wadhams, L. J. & McNeil, J. N. (2004). Identification of two sex pheromone components of the potato aphid, Macrosiphum euphorbiae (Thomas). Journal of Chemical Ecology 30, 819-834.Hemptinne, J-l., Lognay, G., Doumbia, M. and Dixon, A.F.G. (2001) Chemical nature and persistence of the oviposition deterring pheromone in the tracks of the larva of the two spot ladybird, Adalia bipunctata (Coleoptera: Coccinellidae). Chemoecology 11, 43-47.Hooper, A. M., Napper, E. K. V. & Pickett, J. A. (2004). Insect alarm pheromones. In Encyclopedia of entomology J. L. Capinera, ed,. Springer Verlag, Heidelberg.Hooper, A. M. & Pickett, J. A. (2004). Semiochemistry. In Encyclopedia of supramolecular chemistry J. L. Atwood, ed, 1270-1277. Marcel Dekker, New York.Khan, Z.R., Ampong-Nyarko, K., Chiliswa, P., Hassanali, A., Kimani, S., Lwande, W., Overholt, W.A., Pickett, J.A., Smart, L.E., Wadhams, L.J. and Woodcock, C.M. (1997) Parasitism in African cereal pests increased by intercropping. Nature 388: 631-632.Nakashima, Y., Birkett, M. A., Pye, B. J., Pickett, J. A. & Powell, W. (2004). The role of semiochemicals in the avoidance of the seven-spot ladybird, Coccinella septempunctata, by the aphid parasitoid, Aphidius ervi. Journal of Chemical Ecology 30, 1103-1116.Ngumbi, E.N., Ngi-Song, A.J., Njagi, E.N.M., Torto, R., Wadhams, L.J., Birkett, M.A., Pickett, J.A., Overholt, W.A. & Torto, B. (2005). Responses of the stem borer larval endoparasitioid Cotesia flavipes (Hymenoptera: Braconidae) to plant derived synomones: Laboratory and field cage experiments. Biocontrol Science and Technology, 15 (3): 271-279 Nomura, T. et al. (2003). Planta, 217, 776-782.Pettersson, J, Ninkovic, V, Glinwood, R, Birkett, M.A, and Pickett, J.A (2005) Foraging in a complex environment – semiochemicals support searching behaviour of the seven spot ladybird. European Journal of Entomology 102, 365-370.Pope, T.W., Campbell, C.A.M., Hardie, J., Wadhams, L.J. (2004). Electroantennogram responses of the three migratory forms of the damson-hop aphid, Phorodon humuli, to aphid pheromones and plant volatiles. Journal of Insect Physiology 50 (2004) 1083-1092.Quiroz, A., Pettersson, J., Pickett, J.A., Wadhams, L.J. and Niemeyer, H.M. (1997) Semiochemicals mediating spacing behaviour of bird-cherry-oat aphid, Rhopalosiphum padi, feeding on cereals. Journal of Chemical Ecology 23, 2599-2607.Sicker, D. et al. (2000) Int. Rev. Cytol. 198, 319-346.Tsanuo, M. K., Hassanali, A., Hooper, A. M., Khan, Z., Kaberia, F., Pickett, J. A. & Wadhams, L. J. (2003). Isoflavanones from the allelopathic aqueous root exudate of Desmodium uncinatum. Phytochemistry 64, 265-273.Wadhams, L.J. (2003) Host location mechanisms in insects. SEB Abstracts/ Comparative Biochemistry and Physiology Part A 134, S63 A8.13.

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