Plants can defend themselves against herbivores by attracting natural enemies of the herbivores. The cues for attraction are often complex mixtures of herbivore-induced plant volatiles, making it difficult to demonstrate the role of specific compounds. After herbivory by lepidopteran larvae, maize releases a mixture of volatiles that is highly attractive to females of various parasitic wasp species. We identified the terpene synthase TPS10 that forms (E)-␤-farnesene, (E)-␣-bergamotene, and other herbivory-induced sesquiterpene hydrocarbons from the substrate farnesyl diphosphate. The corresponding gene is expressed in response to herbivore attack and is regulated at the transcript level. Overexpression of tps10 in Arabidopsis thaliana resulted in plants emitting high quantities of TPS10 sesquiterpene products identical to those released by maize. Using these transgenic Arabidopsis plants as odor sources in olfactometer assays showed that females of the parasitoid Cotesia marginiventris learn to exploit the TPS10 sesquiterpenes to locate their lepidopteran hosts after prior exposure to these volatiles in association with hosts. This dissection of the herbivore-induced volatile blend demonstrates that a single gene such as tps10 can be sufficient to mediate the indirect defense of maize against herbivore attack.parasitoid attraction ͉ plant defense signal ͉ terpene biosynthesis ͉ volatile terpenes I n the last two decades, researchers have frequently observed that herbivore damage to certain plants induces the emission of volatile organic compounds that attract natural enemies of the herbivores. Termed ''indirect defense,'' this phenomenon has been reported in Ͼ15 different plant species to date (1-4). For example, after damage by lepidopteran larvae to maize foliage, the plant releases a complex mixture of volatiles containing a large number of mono-and sesquiterpenes, lipoxygenase pathway products, and indole (5), with the mixture varying among cultivars and between maize and related species of the Poaceae (6, 7). The high number of compounds that make up this and other herbivore-induced volatile mixtures has so far impeded the identification of the compound(s) actually responsible for signaling herbivore enemies. Attempts to dissect the volatile signal emitted by herbivore-damaged leaves of lima bean (1) and maize (8), for instance, could not identify a specific compound responsible for enemy attraction, suggesting that mixtures constitute the active signal. However, the application of individual plant volatiles, such as methyl salicylate and the sesquiterpene alcohol nerolidol, in behavioral assays have been occasionally reported to attract parasitoids (9) and predatory mites (10-12).The volatile signal emitted by maize seedlings after attack by lepidopteran larvae attracts females of the parasitic braconid wasp Cotesia marginiventris (Hymenoptera), which oviposit in the larvae (5). To determine the role of specific volatiles in attracting C. marginiventris, we sought to learn more about the regulation of vola...

SummaryPlants counteract attack by herbivorous insects using a variety of inducible defence mechanisms. The production of toxic proteins and metabolites that instantly affect the herbivore's development are examples of direct induced defence. In addition, plants may release mixtures of volatile organic compounds (VOCs) that indirectly protect the plant by attracting natural enemies of the herbivore. Recent studies suggest that these VOCs can also prime nearby plants for enhanced induction of defence upon future insect attack. However, evidence that this defence priming causes reduced vulnerability to insects is sparse. Here we present molecular, chemical and behavioural evidence that VOC-induced priming leads to improved direct and indirect resistance in maize. A differential hybridization screen for inducible genes upon attack by Spodoptera littoralis caterpillars identified 10 defence-related genes that are responsive to wounding, jasmonic acid (JA), or caterpillar regurgitant. Exposure to VOCs from caterpillar-infested plants did not activate these genes directly, but primed a subset of them for earlier and/or stronger induction upon subsequent defence elicitation. This priming for defence-related gene expression correlated with reduced caterpillar feeding and development. Furthermore, exposure to caterpillar-induced VOCs primed for enhanced emissions of aromatic and terpenoid compounds. At the peak of this VOC emission, primed plants were significantly more attractive to parasitic Cotesia marginiventris wasps. This study shows that VOC-induced priming targets a specific subset of JAinducible genes, and links these responses at the molecular level to enhanced levels of direct and indirect resistance against insect attack.

The sesquiterpene (E)-b-caryophyllene is emitted by maize (Zea mays) leaves in response to attack by lepidopteran larvae like Spodoptera littoralis and released from roots after damage by larvae of the coleopteran Diabrotica virgifera virgifera. We identified a maize terpene synthase, Terpene Synthase 23 (TPS23), that produces (E)-b-caryophyllene from farnesyl diphosphate. The expression of TPS23 is controlled at the transcript level and induced independently by D. v. virgifera damage in roots and S. littoralis damage in leaves. We demonstrate that (E)-b-caryophyllene can attract natural enemies of both herbivores: entomopathogenic nematodes below ground and parasitic wasps, after an initial learning experience, above ground. The biochemical properties of TPS23 are similar to those of (E)-b-caryophyllene synthases from dicotyledons but are the result of repeated evolution. The sequence of TPS23 is maintained by positive selection in maize and its closest wild relatives, teosinte (Zea sp) species. The gene encoding TPS23 is active in teosinte species and European maize lines, but decreased transcription in most North American lines resulted in the loss of (E)-b-caryophyllene production. We argue that the (E)-b-caryophyllene defense signal was lost during breeding of the North American lines and that its restoration might help to increase the resistance of these lines against agronomically important pests.

Summary• Root colonization by arbuscular mycorrhizal fungi (AMF) was investigated in industrially polluted grassland characterized by exceptionally high phosphorus levels (up to 120 g kg − 1 soil).• Along a pollution-induced nitrogen gradient, soil and tissue element concentrations of Artemisia vulgaris plants and their mycorrhizal status were determined. Additionally, we compared mycorrhization rates and above-ground biomass of A. vulgaris at N-fertilized and control plots in the N-poor area.• Despite high soil and tissue P concentrations, plants from N-deficient plots, which were characterized by low tissue N concentrations and N : P ratios, were strongly colonized by AMF, whereas at a plot with comparable P levels, but higher soil and plant N concentrations and N : P ratios, mycorrhization rates were significantly lower. Correlation analyses revealed a negative relationship between percentage root colonization of A. vulgaris by AMF and both tissue N concentration and N : P ratio. Accordingly, in the fertilization experiment, control plants had higher mycorrhization rates than N-fertilized plants, whereas the species attained higher biomass at Nfertilized plots.• The results suggest that N deficiency stimulates root colonization by AMF in this extraordinarily P-rich field site.

The current discussion on the safety of transgenic crops includes their effects on beneficial insects, such as parasitoids and predators of pest insects. One important plant trait to consider in this context is the emission of volatiles in response to herbivory. Natural enemies use the odours that result from these emissions as cues to locate their herbivorous prey and any significant change in these plant-provided signals may disrupt their search efficiency. There is a need for practical and reliable methods to evaluate transgenic crops for this and other important plant traits. Moreover, it is imperative that such evaluations are done in the context of variability for these traits among conventional genotypes of a crop. For maize and the induction of volatile emissions by caterpillar feeding this variability is known and realistic comparisons can therefore be made. Here we used a six-arm olfactometer that permits the simultaneous collection of volatiles emitted by multiple plants and testing of their attractiveness to insects. With this apparatus we measured the induced odour emissions of Bt maize (Bt11, N4640Bt) and its near-isogenic line (N4640) and the attractiveness of these odours to Cotesia marginiventris and Microplitis rufiventris, two important larval parasitoids of common lepidopteran pests. Both parasitoid species were strongly attracted to induced maize odour and neither wasp distinguished between the odours of the transgenic and the isogenic line. Also wasps that had previously experienced one of the odours during a successful oviposition divided their choices equally between the two odours. However, chemical analyses of collected odours revealed significant quantitative differences. The same 11 compounds dominated the blends of both genotypes, but the isogenic line released a larger amount of most of these. These differences may be due to altered resource allocation in the transgenic line, but it had no measurable effect on the wasps' behaviour. All compounds identified here had been previously reported for maize and the differential quantities in which they were released fall well within the range of variability observed for other maize genotypes.

After herbivore attack, plants release a plethora of different volatile organic compounds (VOCs), which results in odor blends that are attractive to predators and parasitoids of these herbivores. VOCs in the odor blends emitted by maize plants (Zea mays) infested by lepidopteran larvae are well characterized. They are derived from at least three different biochemical pathways, but the relative importance of each pathway for the production of VOCs that attract parasitic wasps is unknown. Here, we studied the importance of shikimic acid derived VOCs for the attraction of females of the parasitoids Cotesia marginiventris and Microplitis rufiventris. By incubating caterpillar-infested maize plants in glyphosate, an inhibitor of the 5-enolpyruvylshikimate-3-phospate (EPSP) synthase, we obtained induced odor blends with only minute amounts of shikimic acid derived VOCs. In olfactometer bioassays, the inhibited plants were as attractive to naive C. marginiventris females as control plants that released normal amounts of shikimic acid derived VOCs, whereas naive M. rufiventris females preferred inhibited plants to control plants. By adding back synthetic indole, the quantitatively most important shikimic acid derived VOC in induced maize odors, to inhibited plants, we showed that indole had no effect on the attraction of C. marginiventris and that M. rufiventris preferred blends without synthetic indole. Exposing C. marginiventris females either to odor blends of inhibited or control plants during oviposition experiences shifted their preference in subsequent olfactometer tests in favor of the experienced odor. Further learning experiments with synthetic indole showed that C. marginiventris can learn to respond to this compound, but that this does not affect its choices between natural induced blends with or without indole. We hypothesize that for naïve wasps the attractiveness of an herbivore-induced odor blend is reduced due to masking by nonattractive compounds, and that during oviposition experiences in the presence of complex odor blends, parasitoids strongly associate some compounds, whereas others are largely ignored.

Plant volatile compounds induced by herbivore attack have been demonstrated to provide a signal to herbivore enemies such as parasitic wasps that use these volatiles to locate their hosts. However, in addition to herbivore-induced volatiles, plants often release volatiles constitutively. We assessed the interaction between herbivore-induced and constitutively released volatiles of maize in the attraction of the wasp Cotesia marginiventris that parasitizes herbivorous lepidopteran larvae feeding on maize. Experiments were carried out with olfactometers in which the sources of volatiles were transgenic Arabidopsis thaliana plants overexpressing maize sesquiterpene synthases that produce blends of herbivore-induced or constitutive compounds. We found that the constitutive volatiles of maize terpene synthase 8 (TPS8) were attractive to C. marginiventris, just like the herbivore-induced volatiles of TPS10 studied earlier. A mixture of both the TPS8 and TPS10 volatile blends, however, was more effective in parasitoid attraction, indicating that constitutively released sesquiterpenes enhance the attraction of those induced by herbivores. While C. marginiventris did not distinguish among the volatiles of TPS8, TPS10, nor those of another maize sesquiterpene synthase (TPS5), when these blends were combined, their attractiveness to the wasp appeared to increase with the complexity of the blend.

Many parasitic wasps that exploit herbivores as their hosts make use of herbivoreinduced plant odours to locate their victims and these wasps often exhibit an ability to learn to associate specific plant-produced odours with the presence of hosts. This associative learning is expected to allow generalist parasitoids to focus on cues that are most reliably associated with current host presence, but evidence supporting this hypothesis is ambiguous. Using a sixarm olfactometer we compared the responses of three generalist larval endoparasitoids, Cotesia marginiventris (Hymenoptera: Braconidae), Microplitis rufiventris (Hymenoptera: Braconidae) and Campoletis sonorensis (Hymenoptera: Ichneumonidae), to the induced odours of three plant species: maize (Zea mays), cowpea (Vigna unguiculata), and cotton (Gossypium hirsutum). We tested the responses of naïve females as well as of females that were first conditioned by parasitising host larvae feeding on one of the plant species. Despite similarities in biology and host range the three wasp species responded entirely differently. Naïve C. marginiventris and C. sonorensis chose equally among the induced odours of the three plants, whereas naïve M. rufiventris, which may have a somewhat more restricted host range, tended to prefer the odour of maize. After conditioning, most C. marginiventris females chose the odour of the plant species that they had experienced, but conditioned M. rufiventris showed an even stronger preference for maize odours, independently of the plant they had experienced. Cotesia sonorensis did not show any change in its preference after conditioning. We speculate that its extremely broad host range allows C. sonorensis females to use fixed responses to cues commonly associated with plants damaged by Lepidoptera. These results imply that different generalist parasitoids may employ different foraging strategies and that associative learning is not necessarily part of it.