Self-defence for plants

chembytes e-zine 1998 - Self-defence for plants Page 1 of 8 Self-defence for plants Used alongside conventional pesticides and other farming practices, Nature's chemical messengers will play an important role in future crop protection, say Mark Luszniak and John Pickett The delicate codling moth may look innocuous, but these little orchard pests can wreak havoc on valuable apple crops. Pesticides work, but to be effective farmers have to spray their crops during a very short period of time in order to kill the pest larvae before the insects burrow into the fruit. Further, the broad spectrum pesticides used against codling also kill the many other insect pest enemies, making way for new pests. Now though, farmers have another weapon with which to fight back using one of the moth's own sex messenger chemicals to disrupt mating. By placing between 600 and 700 slow release dispensers per hectare (10 4 m 2 ) of orchard to release this chemical a synthetic version of the female moth sex attractant or pheromone farmers confuse male moths trying to locate females releasing the same natural message. Prolonged use of the technique over several seasons in the US has markedly reduced codling populations, and increased levels of the natural enemies of other pests such as aphids and leafminers. However, pesticides may still be used if pheromonal control breaks down, for example because of unusual climate conditions. Methods exploiting natural chemical messengers, collectively known as semiochemicals, are becoming increasingly familiar. Semiochemicals are more clearly defined as compounds that act as signals modifying animal or plant behaviour, or development, for example by regulating the organism's hormone balance. 1 When moth sex pheromones were discovered 40 years ago, they seemed to offer the solution to the public's generally ill-founded fears about pesticides. 2 So far, however, they have had limited impact on worldwide sales of agrochemicals. Between 1990 and 1996, annual worldwide sales of insecticides averaged 5000m, of which 80 per cent was generated by organophosphates, carbamates, pyrethroids, organochlorines and benzoyl ureas. The remaining 20 per cent included only a small contribution from semiochemicals. In 1990, a survey showed that semiochemical-based insect monitoring and control systems were recorded for 257 pest species - comprising 89 pests for field crops, 79 for vegetables, 63 for orchards and 55 for forests. 3 New ideas

chembytes e-zine 1998 - Self-defence for plants Page 2 of 8 New ideas Two major developments are now set to revolutionise the use of semiochemicals. First is the realisation that semiochemicals cannot be used alone, but should be combined with population-reducing agents such as highly selective pesticides or biological control agents. Secondly, the use of biotechnology is essential, either for producing bulk semiochemicals that are already generated by higher plants as secondary metabolites, or for producing insect pheromones and their precursors by closely related metabolic pathways. Modifying higher plant genetics to produce semiochemicals within the crop plants themselves (for their own defence, or the defence of other crops and animals) is now in some cases a reality. Until it becomes feasible to transfer packets of 'metabolite-yielding' genes from one plant to another, however, we must resort to other approaches, such as increasing the expression of key genes controlling metabolism. Alternatively, we might transfer a single alien gene, preferably from another plant, so that a substrate already generated by the wild type plant is diverted into producing a metabolite that is useful in crop protection. Rüdiger Hain's group at Bayer AG in Leverkusen, Germany, has demonstrated this approach 4 by transferring the gene for a stilbene synthase enzyme from groundnut to tobacco. The synthase enzyme introduced into the tobacco plant converts the precursors p-coumaryl-scoa (1) and malonyl-scoa (2) naturally produced by the plant to resveratrol (3), (Scheme 1). Resveratrol is a type of phytoalexin - compounds produced by a plant in response to damage caused by a pathogenic process. Scheme 1. Production of resveratrol via the enzyme stilbene synthase Another example illustrating the power of biotechnology approaches is provided by the cyclic hydroxamic acid Dimboa, a secondary metabolite produced in maize. Dimboa confers resistance to the European corn borer pest by interfering with feeding behaviour, and to northern corn leaf blight and stalk rot. Researchers have recently identified 5 and demonstrated the role of each of five genes responsible for biosynthesising Diboa the immediate precursor of Dimboa in maize. It should therefore now be possible to transfer these genes, and subsequently confer the advantages of Dimboa synthesis, into other plant species. Aphid attack However, the main insect pests of northern Europe are aphids, which not only damage crops by feeding, but can also further weaken plants by transferring plant viruses. Most pest aphids belong to the sub-family

chembytes e-zine 1998 - Self-defence for plants Page 3 of 8 Aphidinae. These aphids have evolved to exploit herbaceous flowering plants, which grow rapidly in the summer and are colonised by the asexually reproducing summer form of the aphids. To pass through the sexual stages essential for overwintering, many of these insects must return to their primary host, usually a tree. This migration occurs by asexually reproducing females moving to the primary host, where true sexual females are produced. These then release the sex pheromone which helps the males not only locate females ready for reproduction, but also to locate the primary host itself. For example in autumn the black bean aphid, a major pest on legumes and sugar beet in the summer, has to return to its primary host, the spindle tree, to mate. Our research group at IACR-Rothamsted in Harpenden has successfully identified the sex pheromones (4-7, Fig 1) for many pest species in the Aphidinae by work on aphid electrophysiology coupled with high resolution gas chromatography (see Box). We have been able to synthesise all of these cyclopentanoid compounds in the laboratory, although with considerable difficulty for diastereoisomers (6) and (7). The activity of these pheromones can be synergised by volatile components released by the primary host, but together or alone, they can act as potent attractants for male aphids. In field trials involving collaborators around the world, thousands of male aphids have been caught in simple water traps releasing the cyclopentanoids in far greater numbers than were caught in control traps. Fig 1. Attracting interest - a selection of potentially useful aphid sex pheremones Perhaps the most exciting development in this area was the discovery that aphid parasitoids are also attracted to aphid sex pheromone components. Aphid parasitoids are small wasps that lay their eggs in aphid bodies, ultimately leading to the aphid's death, and the release of a young wasp. They almost certainly use aphid pheromones as specific semiochemicals, known as kairomones, to locate their hosts in the autumn. Electrophysiological studies on the antennae of parasitoids show that they retain the ability to detect the aphid sex pheromone not only during aphid migration, but throughout the summer. In field trials at Rothamsted, we have successfully used these synthetic pheromones to increase parasitism of aphids on cereals and beans. Fungus traps However, catching male aphids or attracting parasitoids are by no means effective methods for total aphid population control. Together with various collaborators, we are therefore currently investigating the potential for introducing fungal pathogens, such as Verticillium lecanii, into male aphid

chembytes e-zine 1998 - Self-defence for plants Page 4 of 8 introducing fungal pathogens, such as Verticillium lecanii, into male aphid traps. Males attracted to the trap via the sex pheromones will pick up the fungal pathogen and unwittingly transfer the spores to the mating population on the primary host. Because the pathogen will ultimately kill the infected aphids, this method further reduces aphid populations. The success of initial field trials has created a considerable demand for the aphid cyclopentanoids. Although we have devised synthetic routes that are amenable to producing cyclopentanoids on a medium scale, ultimately the aim is to employ biotechnology approaches. Together with various industrial partners, at IACR-Rothamsted we have recently begun work to extract aphid sex pheromone components and precursors from catmint plants (Nepeta species). This approach will provide us with large quantities of enantiomerically pure nepatalactones and nepetalactols for commercial use as future crop protection agents with non-toxic modes of action. Fig 2. Making it hard to swallow - a few antifeed compounds One example from our research at Rothamsted is the dialdehydic isoprenoid polygodial (8), which is effective in preventing the transmission of yellow dwarf virus in barley via the bird-cherry-oat aphid. Originally, we obtained polygodial via liquefied carbon dioxide extraction of Polygonum hydropiper, but are again exploring the possibility of exploiting new industrial crops to produce these antifeedants efficiently. Drimenol (9) is a precursor to polygodial and other dialdehydic drimane antifeedants, and is produced in higher plants by the action of a cyclase enzyme. Such a cyclase enzyme is present not only in higher plants, but also in Basidiomycetes, which like the related mushrooms, may be more amenable to exploitation by molecular genetics. A u g o se is th a (F 2 co th d th co o p b in w th ta

chembytes e-zine 1998 - Self-defence for plants Page 5 of 8 Signalling change Ajugarin I (10) is an antifeedant obtained naturally from the bugle plant Ajuga remota. It can be used in very low concentrations to deter feeding by Coleoptera such as the mustard beetle and also the major world pest, the Colorado potato beetle. 6 Spraying the top parts of the plants with electrostatically charged droplets of a solution of ajugarin I deters the insects from feeding and forces them to the lower regions where their population is reduced with selective, insect growth regulating pesticides. Compared with conventional sprays, an electrostatic assisted spray is generally more efficient, 7 because the droplets adhere to the plant, including the undersides of leaves, and less material is wasted needlessly spraying the soil. 14- Hydro-15-hydroxyajugapitin (11) is potentially even more useful 8 as an antifeedant than ajugarin I. However, it is available in only low yield from, for example, the groundpine plant A. chamaepitys. It is hoped that plant breeding or genetic engineering techniques will provide a new industrial crop source of this antifeedant in the UK or an A. remota crop for use in Africa. Yet another useful compound implicated with plant defence is methyl salicylate, which was correctly predicted by our collaborator at the Swedish University of Agricultural Sciences in Uppsala, Jan Pettersson, to act as a dispersal agent for the spring migrants heading for their secondary host. In the field, we have used methyl salicylate in slow release formulations over three seasons to cause aphids to disperse, consistently reducing the aphid population in a cereal crop by up to 50 per cent. Although this level of control is highly reproducible, increasing the methyl salicylate dose does not increase the dispersal effect. This lack of dose response, once the chemical takes effect, is typical of semiochemicals and emphasises the need to use them as one component of an overall pest management strategy. However, incomplete control using one particular semiochemical has the advantage of impeding the development of resistant pest species. Apart from aphids, many other insects also show a highly specific response to methyl salicylate. 9 We therefore propose that methyl salicylate is a volatile and thereby external signal related to the internal signalling compound salicylic acid. Salicylic acid is produced in plants via the phenylalanine ammonia lyase pathway, a pathway known to produce many secondary metabolites, some of which are used for plant defence. Clearly, there is a role for transgenic cereal crops that could release methyl salicylate as an inherent semiochemical defence in the leaves. Another metabolite resulting from the phenylalanine ammonia lyase pathway is 1,2-dimethoxybenzene, which has been shown by electrophysiology to be a highly active compound pivotal in the interaction between another sucking insect, the brown planthopper, and its host, rice. Genetic manipulation of rice plants could allow disruption of the process by which plants produce 1,2-dimethoxybenzene, by preventing the biosynthesis of its precursor compound, catechol. Jasmonic acid and its corresponding ester, methyl jasmonate, also have important roles as signals in plant defence. For example, methyl jasmonate

chembytes e-zine 1998 - Self-defence for plants Page 6 of 8 important roles as signals in plant defence. For example, methyl jasmonate induces the production of proteinase inhibitors in certain plants. 10 This has the effect of preventing insects from digesting their food, by inhibiting the necessary proteinase enzymes. When methyl jasmonate is allowed to permeate into the air above oilseed rape plants, for example, this induces the production of indolylglucosinolates, secondary metabolites involved in the defence of certain vegetables. This could have the net effect of deterring any unadapted herbivores from feeding on the plants and preventing any further progression of disease. Pushing and pulling Nevertheless, it is rare for a single semiochemical to be very effective when used alone. Instead, the usual approach is a 'push-pull' strategy which involves 'pushing' the insects away from the harvestable, economic crops, and 'pulling' them onto a trap crop where their population is reduced by a biological control agent (such as a fungal pathogen) or highly specific but slow-acting pesticide. Thus, antifeedants, non-host volatiles, parasitoid attractants and compounds associated with plant defence can be used to achieve the 'push', while the sex pheromone and host volatiles can be used to 'pull' the insects onto the trap crop. Together with our Kenyan collaborators from the International Centre for Insect Physiology and Ecology in Nairobi, we have recently demonstrated a successful example of such a 'push-pull' strategy for maize and sorghum. 11 Damage by moth caterpillars often dramatically reduces the yields of these important cereal crops, vital staples in the African diet. By intercropping these crops with Melinis minutiflora (molasses grass) we have successfully 'pushed' these stem-borer insects away from the main crop, significantly reducing the level of infestation. In addition, volatile compounds produced by the molasses grass increased larval parasitism of stem-borers by the wasp Cotesia sesamiae. The 'pull' for our strategy is provided by planting yet another plant around the maize - the forageable crop Napier grass. Napier grass is much more attractive to the stem-borers than the cereal host, but interferes with the normal development of the stem-borer larvae. Following the success of the individual 'push-pull' components, we are bringing both aspects together to develop a simple protocol for effectively controlling stem-borer populations. The benefits of a 'push-pull' strategy include a lower requirement for broad spectrum pesticides, saving these valuable materials for a 'fire fighting' role. In addition, there is less risk of producing populations of resistant insects. Because the components of a 'push-pull' strategy are not individually greatly effective, they do not select for resistance as strongly as conventional toxicant pesticides. Further, genetically modified plants that produce the key semiochemicals could offer an environmentally cleaner solution for manufacture than conventional synthesis. Tuning in to semiochemicals When an insect detects a volatile semiochemical, usually via its antenna, the molecular

chembytes e-zine 1998 - Self-defence for plants Page 7 of 8 When an insect detects a volatile semiochemical, usually via its antenna, the molecular recognition process occurring within the olfactory receptors causes cells to 'fire', leading to minute electrical pulses. Electrophysiology allows us to measure these pulses and investigate the effects of different chemical signals. We currently use two techniques to identify semiochemicals. 12 Electroantennography (EAG) involves measuring changes in electrical potential across the whole antenna, and has the advantage of exposing volatile components within a mixture of chemicals to all the receptors on the antenna. Using EAG coupled to gas chromatography (GC), we can monitor the eluant from the GC and simultaneously locate the active components. Alternatively, with greater difficulty, we can use tungsten microelectrodes to record from individual olfactory cells; this technique is known as single-cell recording (SCR) and it allows us to determine which receptors respond to which electrophysiologically active components. By using coupled GC-EAG or GC-SCR, it is possible to pinpoint exactly which components within a complex mixture, often comprising hundreds of volatile chemicals, are eliciting a response from the insect. The amplifiers and software used in our electrophysiological studies were provided by Syntech in The Netherlands. Acknowledgements: This work was in part supported by the UK Ministry of Agriculture Fisheries and Food. The Institute of Arable Crops Research (IACR) receives grant-aided support from the UK Biotechnology and Biological Sciences Research Council. Mark Luszniak is a postdoctoral researcher in the chemical ecology group and John Pickett FRS is head of the biological and ecological chemistry department, at IACR-Rothamsted, Harpenden, Herts AL5 2JQ. References 1. J. A. Pickett et al in Proceedings of phytochemical diversity: a source of new industrial products, p 220. Cambridge: RSC, 1997. 2. lnsect pheromone research - new directions, R. T. Cardé and A. K. Minks (eds). London: Chapman and Hall, 1996. 3. O. T. Jones in Proceedings of the British crop protection conference

chembytes e-zine 1998 - Self-defence for plants Page 8 of 8 3. O. T. Jones in Proceedings of the British crop protection conference on pests and diseases, p 1213. Brighton: British Crop Protection Council, 1994. 4. R. Hain et al, Plant Mol. Biol., 1990, 15, 325; R. Hain in Proceedings of the British Crop protection conference on pests and diseases, p 757. Brighton: British Crop Protection Council, 1992. 5. M. Frey et al, Science, 1997, 277, 696. 6. J. A. Pickett et al in Proceedings of the British Crop protection conference on pests and diseases, p 1041. Brighton: British Crop Protection Council, 1988. 7. A. J. Arnold and B. J. Pye in ibid, 1979, p 109; A. J. Arnold and B. J. Pye, ibid, 1981, p 661. 8. F. Camps, J. Coll and O. Dargallo, Phytochemistry, 1984, 23, 2577. 9. J. A. Pickett et al, J. Chem. Ecol., 1994, 20, 2565. 10. E. E. Farmer and C. A. Ryan, Proc. Natl. Acad. Sci. USA, 1990, 87, 7713. 11. J. A. Pickett et al, Nature (London), 1997, 388, 631. 12. L. J. Wadhams, Chromatography and isolation of insect hormones and pheromones, p 289. New York: Plenum, 1990. Go to e-zine 1998 back issues Go to e-zine current issue networks & societies conferences & events chemsoc careers learning resources chembytes infozone web links 109 visual elements periodic table Terms and Conditions This web site is copyright of Royal Society of Chemistry, 2000