Ozone‐induced foliar damage and release of stress volatiles is highly dependent on stomatal openness and priming by low‐level ozone exposure in Phaseolus vulgaris

Acute ozone exposure triggers major emissions of volatile organic compounds (VOCs), but quantitatively, it is unclear how different ozone doses alter the start and the total amount of these emissions, and the induction rate of different stress volatiles. It is also unclear whether priming (i.e. pre‐exposure to lower O₃ concentrations) can modify the magnitude and kinetics of volatile emissions. We investigated photosynthetic characteristics and VOC emissions in Phaseolus vulgaris following acute ozone exposure (600 nmol mol^?1 for 30 min) under illumination and in darkness and after priming with 200 nmol mol^?1 O₃ for 30 min. Methanol and lipoxygenase (LOX) pathway product emissions were induced rapidly, followed by moderate emissions of methyl salicylate (MeSA). Stomatal conductance prior to acute exposure was lower in darkness and after low O₃ priming than in light and without priming. After low O₃ priming, no MeSA and lower LOX emissions were detected under acute exposure. Overall, maximum emission rates and the total amount of emitted LOX products and methanol were quantitatively correlated with total stomatal ozone uptake. These results indicate that different stress volatiles scale differently with ozone dose and highlight the key role of stomatal conductance in controlling ozone uptake, leaf injury and volatile release.     

The piercing-sucking herbivores Lygus hesperus and Nezara viridula induce volatile emissions in plants

Plant volatiles induced by herbivory are often used as olfactory cues by foraging herbivores and their natural enemies, and thus have potential for control of agricultural pests. Compared to chewing insects and mites, little is known about plant volatile production following herbivory by insects with piercing-sucking mouthparts. Here, we studied factors (insect life stage, gender, the role of salivary glands, and type of bioassay used for volatile induction) that influence the induction of plant volatiles by two agriculturally important hemipterans, Lygus hesperus and Nezara viridula. Feeding on intact cotton by virgin females of L. hesperus induced 2.6-fold greater volatile response compared to that induced by mated females, possibly due to increased feeding activity by virgin females. This plant volatile response was associated with elicitors present in the insect's salivary glands as well as to the degree of mechanical injury. Feeding injury by N. viridula females also increased volatile emissions in intact maize by approximately 2-fold compared to control plants. Maize seedlings injured by N. viridula emitted higher amounts of the monoterpene linalool, the sesquiterpenes (E)-beta-caryophyllene, alpha-trans-bergamotene, and (E,E)-beta-farnesene, and the homoterpene (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene, but not amounts of green leaf volatiles, compared to uninjured plants. Emissions from intact maize injured by adult males were lower than those emitted by adult females of the same age and did not differ from those emitted by uninjured plants. Similarly, feeding by virgin female N. viridula followed by excision led to 64% higher quantities of volatiles compared to untreated plants. Volatile emission in excised plants, however, was considerably greater than in intact plants, suggesting that careful consideration must be given to bioassay design in studies of herbivore-induced plant volatiles. Salivary gland extracts of N. viridula led to sesquiterpene emissions approximately 2.5-fold higher than for controls, although no significant differences were observed for green leaf volatiles, monoterpenes, and homoterpenes. These results indicate that L. hesperus and female N. viridula feeding induce volatile production in plants, and that volatile production is affected by gender and life stage of the bug. Although oviposition and mechanical injury by stylets may increase release of volatiles, elicitors from salivary glands of L. hesperus and N. viridula also seem to play a role in the emission of plant volatiles.     

Herbivore-induced Blueberry Volatiles and Intra-plant Signaling

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack^1,2. These HIPVs are mainly regulated by the defensive plant hormone jasmonic acid (JA) and its volatile derivative methyl jasmonate (MeJA)^3,4,5. Over the past 3 decades researchers have documented that HIPVs can repel or attract herbivores, attract the natural enemies of herbivores, and in some cases they can induce or prime plant defenses prior to herbivore attack. In a recent paper⁶, I reported that feeding by gypsy moth caterpillars, exogenous MeJA application, and mechanical damage induce the emissions of volatiles from blueberry plants, albeit differently. In addition, blueberry branches respond to HIPVs emitted from neighboring branches of the same plant by increasing the levels of JA and resistance to herbivores (i.e., direct plant defenses), and by priming volatile emissions (i.e., indirect plant defenses). Similar findings have been reported recently for sagebrush⁷, poplar⁸, and lima beans⁹..Here, I describe a push-pull method for collecting blueberry volatiles induced by herbivore (gypsy moth) feeding, exogenous MeJA application, and mechanical damage. The volatile collection unit consists of a 4 L volatile collection chamber, a 2-piece guillotine, an air delivery system that purifies incoming air, and a vacuum system connected to a trap filled with Super-Q adsorbent to collect volatiles^5,6,10. Volatiles collected in Super-Q traps are eluted with dichloromethane and then separated and quantified using Gas Chromatography (GC). This volatile collection method was used n my study⁶ to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants. These methods are described here. Briefly, undamaged blueberry branches are exposed to HIPVs from neighboring branches within the same plant. Using the same techniques described above, volatiles emitted from branches after exposure to HIPVs are collected and analyzed.     

Changes in composition of essential oils and volatile emissions of Minthostachys mollis, induced by leaf punctures of Liriomyza huidobrensis

Plant defensive mechanisms against herbivores include chemical changes following damage. Effects of feeding punctures produced by Liriomyza huidobrensis (pea leafminers) adult females on the plant's dominant monoterpenes, pulegone and menthone were assessed by monitoring essential oil composition at 24, 48, and 120 h; emission of volatiles was also measured 24 and 48 h after wounding. We studied such changes in Minthostachys mollis, a Lamiaceae species native to Central Argentina with medicinal and aromatic uses. Leaf puncturing resulted in reduced menthone throughout the experiment and increased pulegone concentration in M. mollis essential oil during the first 48 h. The adjacent undamaged leaves showed similar changes, suggesting a systemic response. Composition of volatiles released from damaged leaves was also altered, most noticeably by increasing pulegone and diminishing menthone emissions. Such herbivore-induced chemical changes in aromatic plants are economically relevant, since the quality of essential oils and volatile emissions are altered.     

Detection of plant volatiles after leaf wounding and darkening by proton transfer reaction "time-of-flight" mass spectrometry (PTR-TOF)

Proton transfer reaction-time of flight (PTR-TOF) mass spectrometry was used to improve detection of biogenic volatiles organic compounds (BVOCs) induced by leaf wounding and darkening. PTR-TOF measurements unambiguously captured the kinetic of the large emissions of green leaf volatiles (GLVs) and acetaldehyde after wounding and darkening. GLVs emission correlated with the extent of wounding, thus confirming to be an excellent indicator of mechanical damage. Transient emissions of methanol, C5 compounds and isoprene from plant species that do not emit isoprene constitutively were also detected after wounding. In the strong isoprene-emitter Populus alba, light-dependent isoprene emission was sustained and even enhanced for hours after photosynthesis inhibition due to leaf cutting. Thus isoprene emission can uncouple from photosynthesis and may occur even after cutting leaves or branches, e.g., by agricultural practices or because of abiotic and biotic stresses. This observation may have important implications for assessments of isoprene sources and budget in the atmosphere, and consequences for tropospheric chemistry.     

Herbivore-induced plant volatiles mediate behavioral interactions between a leaf-chewing and a phloem-feeding herbivore

Plants respond adaptively to herbivore stress in order to maintain fitness. Upon herbivore attack, plants emit blends of volatile organic compounds (VOCs) that differ from those that are constitutively emitted. These defense responses are typically specific to the identity of the attacking herbivore and often linked to the herbivore's feeding guild (e.g. chewing, phloem-feeding). Herbivores use plant volatiles to locate suitable host plants and changes in volatile emissions can affect host-plant location. Therefore, herbivores from separate feeding guilds can interact indirectly through the modulation of plant responses. In this study we tested how damage by an herbivore from one feeding guild affected the host-plant choice of an herbivore from a separate feeding guild, and vice versa. A chewing herbivore, the Colorado potato beetle (Leptinotarsa decemlineata), and a phloem feeding herbivore, the green peach aphid (Myzus persicae), were assayed in olfactometers to assess behavioral responses to odors emitted by potato plants (Solanum tuberosum) that were damaged by herbivores from the other feeding guild. Leptinotarsa decemlineata oriented more frequently towards undamaged plants compared to M. persicae damaged plants. Surprisingly, M. persicae preferred plants that were damaged by L. decemlineata, although previous studies had shown that they perform worse on these plants. Distinct differences were detected in the volatile profiles of herbivore-damaged and undamaged plants. Leptinotarsa decemlineata induced stronger volatile emissions compared to undamaged control plants, while M. persicae tended to suppress volatile emissions. These herbivores demonstrate contrasting induction of plant volatiles and behavioral responses. Exploring the nature of co-occurring herbivores and how they perceive potential hosts can play a significant role in understanding the ecological functions and community dynamics of plant plasticity and interactions with a variety of herbivores.     

Changes in photosynthetic rate and stress volatile emissions through desiccation‐rehydration cycles in desiccation‐tolerant epiphytic filmy ferns (Hymenophyllaceae)

Exposure to recurrent desiccation cycles carries a risk of accumulation of reactive oxygen species that can impair leaf physiological activity upon rehydration, but changes in filmy fern stress status through desiccation and rewatering cycles have been poorly studied. We studied foliage photosynthetic rate and volatile marker compounds characterizing cell wall modifications (methanol) and stress development (lipoxygenase [LOX] pathway volatiles and methanol) through desiccation–rewatering cycles in lower‐canopy species Hymenoglossum cruentum and Hymenophyllum caudiculatum, lower‐ to upper‐canopy species Hymenophyllum plicatum and upper‐canopy species Hymenophyllum dentatum sampled from a common environment and hypothesized that lower canopy species respond more strongly to desiccation and rewatering. In all species, rates of photosynthesis and LOX volatile emission decreased with progression of desiccation, but LOX emission decreased with a slower rate than photosynthesis. Rewatering first led to an emission burst of LOX volatiles followed by methanol, indicating that the oxidative burst was elicited in the symplast and further propagated to cell walls. Changes in LOX emissions were more pronounced in the upper‐canopy species that had a greater photosynthetic activity and likely a greater rate of production of photooxidants. We conclude that rewatering induces the most severe stress in filmy ferns, especially in the upper canopy species.     

Variability and stability of tea weevil‐induced volatile emissions from tea plants with different weevil densities,photoperiod and infestation duration

Abstract After herbivore attack, many plants emit herbivore‐induced plant volatiles (HIPVs). HIPVs can attract carnivores and/or repel herbivores, thereby mediating tritrophic plant–herbivore–carnivore interactions. HIPVs act as chemical information between organisms; hence, their variability and stability are vital. In the present study, variations in the volatile emissions, from the tea plant Camellia sinensis (O. Ktze) damaged by the tea weevil Myllocerinus aurolineatus (Voss) (Coleoptera: Curculionidae), with weevil densities, photoperiod and infestation duration, were investigated. The volatiles induced by high‐density weevils were more abundant in composition and amount than those induced by low‐density weevils, whether at noon, night or after weevil removal. The induced volatile emissions were similar on the second and third day after infestation, and the emissions of the major induced compounds displayed diurnal cycles. Linalool, (E,E)‐α‐farnesene, and benzyl nitrile were emitted mainly at noon, whereas 1,3,8‐p‐menthatriene and (E)‐β‐ocimene were maximally emitted at night. Given the different emission dynamics, significant differences were found between noon‐ and night‐induced volatiles. In summary, tea plants damaged by different weevil densities emitted a relatively stable signal at a particular time. This stability could be attributed to the similarities under the two densities of the main induced volatile compounds, their relative ratios and the emission dynamics of the induced volatiles.     

Elevated volatile concentrations in high‐nutrient plants: do insect herbivores pay a high price for good food?

1. A tritrophic perspective is fundamental for understanding the drivers of insect–plant interactions. While host plant traits can directly affect insect herbivore performance by either inhibiting or altering the nutritional benefits of consumption, they can also have an indirect effect on herbivores by influencing rates of predation or parasitism. 2. Enhancing soil nutrients available to trees of the genus Eucalyptus consistently modifies plant traits, typically improving the nutritional quality of the foliage for insect herbivores. We hypothesised that resulting increases in volatile essential oils could have an indirect negative effect on eucalypt‐feeding herbivores by providing their natural enemies with stronger host/prey location cues. 3. Eucalyptus tereticornis Smith seedlings were grown under low‐ and high‐nutrient conditions and the consequences for the release of volatile cues from damaged plants were examined. The influence of 1,8‐cineole (the major volatile terpene in many Eucalyptus species) on rates of predation on model caterpillars in the field was then examined. 4. It was found that the emission of cineole increased significantly after damage (artificial or herbivore), but continued only when damage was sustained by herbivore feeding. Importantly, more cineole was emitted from high‐ than low‐nutrient seedlings given an equivalent amount of damage. In the field, predation was significantly greater on model caterpillars baited with cineole than on unbaited models. 5. These findings are consistent with the hypothesis that any performance benefits insect herbivores derive from feeding on high‐nutrient eucalypt foliage could be at least partially offset by an increased risk of predation or parasitism via increased emission of attractive volatiles.     

Hylobius abietis L. feeding on the novel host Pinus brutia Ten. increases emission of volatile organic compounds

Plants respond to feeding by herbivorous insects by producing volatile organic chemicals, which mediate interactions between herbivores and plants. Yet, few studies investigated whether such plant responses to herbivory differ between historical host and novel plants. Here, we investigated whether herbivory by the pine weevil Hylobius abietis causes a release of volatile organic chemicals from a novel tree Pinus brutia and compared the relative amounts of volatiles released from herbivore's historical hosts and P. brutia. We collected volatiles emitted from P. brutia seedlings that were either subjected to feeding by H. abietis or no feeding. Our results indicated that feeding increased emission of volatile compounds, composed of monoterpenes and sesquiterpenes, and that the emission was several fold higher in the damaged seedlings than in undamaged seedlings. In particular, emission of monoterpenes and sesquiterpenes increased by 4.4‐and 10‐fold in the damaged plants, respectively. Strikingly, individual monoterpenes and sesquiterpenes showed much greater dissimilarity between damaged and undamaged seedlings. Furthermore, several minor monoterpenes showed negative relationships with the weevil gnawed area. We discussed these results with the results of previous studies focused on historical host plants of H. abietis and hypothesized the ecological relevance and importance of our results pertaining relevance to the plant–herbivory interactions.     

Exploiting scents of distress: the prospect of manipulating herbivore-induced plant odours to enhance the control of agricultural pests

In response to feeding by arthropods, plants actively and systemically emit various volatile substances. It has been proposed that these herbivore-induced volatiles (HIPVs) can be exploited in agricultural pest control because they might repel herbivores and because they serve as attractants for the enemies of the herbivores. Indeed, recent studies with transgenic plants confirm that odour emissions can be manipulated in order to enhance the plants' attractiveness to beneficial arthropods. An additional advantage of manipulating HIPV emissions could be their effects on neighbouring plants, as a rapidly increasing number of studies show that exposure to HIPVs primes plants for augmented defence expression. Targeting the right volatiles for enhanced emission should lead to ecologically and economically sound ways of combating important pests.     

Leaf mechanics and herbivory defence: How tough tissue along the leaf body deters growing insect herbivores

Research on herbivory defence often focuses on leaf chemistry but less on how plant mechanical properties like leaf veins deter herbivores. Herbivores often eat tough, complex plant tissue, yet how mechanical properties affect feeding performance as the consumer grows is unclear. We measured the toughness and strength of five types of leaf tissue – the midrib, the secondary and marginal veins and the lamina inside (inner) and outside (outer) the marginal vein – in mature Eucalyptus viminalis and Eucalyptus ovata leaves with punch tests. Leaf veins were, on average, 6.2 times tougher than lamina. Marginal veins were uniformly strong and tough along the leaf body, while midribs were less strong and secondary veins less tough toward leaf tips. We correlated the force required to puncture leaf tissue with the feeding performance of a chewing insect herbivore (the spiny leaf insect, Extatosoma tiaratum (Phasmida)) across four instar stages to explore the role of tough leaf veins as potential feeding barriers. Larvae more often ate less tough leaf tips and tougher tissue as they grew. However, younger larvae were capable of penetrating the tough marginal vein when starved. We suggest tough leaf veins and consumer position along the leaf body influence insect herbivore feeding performance over their lifetime.     

Caterpillars of Euphydryas aurinia (Lepidoptera: Nymphalidae) feeding on Succisa pratensis leaves induce large foliar emissions of methanol

A major new discovery made in the last decade is that plants commonly emit large amounts and varieties of volatiles after damage inflicted by herbivores, and not merely from the site of injury. However, analytical methods for measuring herbivore-induced volatiles do not usually monitor the whole range of these compounds and are complicated by the transient nature of their formation and by their chemical instability. Here we present the results of using a fast and highly sensitive proton transfer reaction-mass spectrometry (PTR-MS) technique that allows simultaneous on-line monitoring of leaf volatiles in the pptv (pmol mol(-1)) range. The resulting on-line mass scans revealed that Euphydryas aurinia caterpillars feeding on Succisa pratensis leaves induced emissions of huge amounts of methanol--a biogeochemically active compound and a significant component of the volatile organic carbon found in the atmosphere--and other immediate, late and systemic volatile blends (including monoterpenes, sesquiterpenes and lipoxygenase-derived volatile compounds). In addition to influencing neighboring plants, as well as herbivores and their predators and parasitoids, these large emissions might affect atmospheric chemistry and physics if they are found to be generalized in other plant species.     

Effects of feeding Spodoptera littoralis on lima bean leaves. II. Continuous mechanical wounding resembling insect feeding is sufficient to elicit herbivory-related volatile emission

Herbivore feeding elicits defense responses in infested plants, including the emission of volatile organic compounds that can serve as indirect defense signals. Until now, the contribution of plant tissue wounding during the feeding process in the elicitation of defense responses has not been clear. For example, in lima bean (Phaseolus lunatus), the composition of the volatiles induced by both the insect caterpillar Spodoptera littoralis and the snail Cepaea hortensis is very similar. Thus, a mechanical caterpillar, MecWorm, has been designed and used in this study, which very closely resembles the herbivore-caused tissue damage in terms of similar physical appearance and long-lasting wounding period on defined leaf areas. This mode of treatment was sufficient to induce the emission of a volatile organic compound blend qualitatively similar to that as known from real herbivore feeding, although there were significant quantitative differences for a number of compounds. Moreover, both the duration and the area that has been mechanically damaged contribute to the induction of the whole volatile response. Based on those two parameters, time and area, which can replace each other to some extent, a damage level can be defined. That damage level exhibits a close linear relationship with the accumulation of fatty acid-derived volatiles and monoterpenes, while other terpenoid volatiles and methyl salicylate respond in a nonlinear manner. The results strongly suggest that the impact of mechanical wounding on the induction of defense responses during herbivore feeding was until now underestimated. Controlled and reproducible mechanical damage that strongly resembles the insect's feeding process represents a valuable tool for analyzing the role of the various signals involved in the induction of plant defense reactions against herbivory.     

Epirrita autumnata induced VOC emission of silver birch differ from emission induced by leaf fungal pathogen

The production of volatile organic compounds (VOCs) through the activation of different signal-transduction pathways may be induced in various biotic and abiotic stress situations having importance e.g. in insect and disease resistance. We compared the emission of VOCs emitted from silver birch Betula pendula Roth (clones 4 and 80) twigs damaged either by larvae of Epirrita autumnata, or infected with pathogenic leaf spot causing fungus Marssonina betulae. We also analysed whether local herbivore damage can systemically induce the release of VOCs from the undamaged top of same sapling. The emissions of methylsalicylate (MeSA), (Z)-ocimene, (E)-β-ocimene, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) and linalool were induced from the twigs after 72 h feeding damage by E. autumnata larvae. However, 48 h feeding damage did not induce rapid systemic release of VOCs from undamaged top leaves of the same twigs. Pathogen-infected birch twigs had significantly greater emission of (Z)-ocimene and (E)-β-ocimene than intact control twigs. The emission of DMNT was not significantly induced and MeSA was not found at all after pathogen infection, both being significantly different from herbivore damaged twigs. According to our results leaf fungal pathogen induces VOC emission profile differs from that of arthropod herbivore-damaged leaves, suggesting that birch is able to transmit parasite-specific information via VOC emissions to conspecifics and natural enemies of herbivores. Handling editor: Yvan Rahbé     

Emission of constitutive isoprene,induced monoterpenes,and other volatiles under high temperatures in Eucalyptus camaldulensis: A 13C labelling study

Eucalypts are major emitters of biogenic volatile organic compounds (BVOCs), especially volatile isoprenoids. Emissions and incorporation of ¹³C in BVOCs were measured in Eucalyptus camaldulensis branches exposed to rapid heat stress or progressive temperature increases, in order to detect both metabolic processes and their dynamics. Isoprene emission increased and photosynthesis decreased with temperatures rising from 30°C to 45°C, and an increasing percentage of unlabelled carbon was incorporated into isoprene in heat‐stressed leaves. Intramolecular labelling was also incomplete in isoprene emitted by heat‐stressed leaves, suggesting increasing contribution of respiratory (and possibly also photorespiratory) carbon. At temperature above 45°C, a drop of isoprene emission was mirrored by the appearance of unlabelled monoterpenes, green leaf volatiles, methanol, and ethanol, indicating that the emission of stored volatiles was mainly induced by cellular damage. Emission of partially labelled acetaldehyde was also observed at very high temperatures, suggesting a double source of carbon, with a large unlabelled component likely transported from roots and associated to the surge of transpiration at very high temperatures. Eucalypt plantations cover large areas worldwide, and our findings may dramatically change forecast and modelling of future BVOC emissions at planetary level, especially considering climate warming and frequent heat waves.     

Regulation of Floral Terpenoid Emission and Biosynthesis in Sweet Basil (<Emphasis Type="Italic">Ocimum basilicum</Emphasis>)

Past studies have focused on the composition of essential oil of Ocimum basilicum leaves, but data on composition and regulation of its aerial emissions, especially floral volatile emissions, are scarce. We studied the chemical profile, within-flower spatial distribution (sepals, petals, pistils with stamina, and pedicels), diurnal emission kinetics and effects of exogenous methyl jasmonate (MeJA) application on the emission of floral volatiles by dynamic headspace collection, and identification using gas chromatography-mass spectrometry (GC–MS) and proton-transfer reaction mass spectrometry. We observed more abundant floral emissions from flowers compared with leaves. Sepals were the main emitters of floral volatiles among the flower parts studied. The emissions of lipoxygenase compounds and monoterpenoids, but not sesquiterpene emissions, displayed a diurnal variation driven by light. Response to exogenous MeJA treatment of flowers consisted of a rapid stress response and a longer-term acclimation response. The initial response was associated with enhanced emissions of fatty acid derivatives, monoterpenoids, and sesquiterpenoids without variation of the composition of individual compounds. The longer-term response was associated with enhanced monoterpenoid and sesquiterpenoid emissions with profound changes in the emission spectrum. According to correlated patterns of terpenoid emission changes upon stress, highlighted by a hierarchical cluster analysis, candidate terpenoid synthases responsible for observed diversity and complexity of released terpenoid blends were postulated. We conclude that flower volatile emissions differ quantitatively and qualitatively from leaf emissions, and overall contribute importantly to O. basilicum flavor, especially under stress conditions.     

Synergistic interactions between volicitin,jasmonic acid and ethylene mediate insect-induced volatile emission in Zea mays

Plants display differential responses following mechanical damage and insect herbivory. Both caterpillar attack and the application of caterpillar oral secretions (OS) to wounded leaves stimulates volatile emission above mechanical damage alone. Volicitin ( N- 17-hydroxylinolenoyl- l -glutamine), present in beet armyworm (BAW, Spodoptera exigua ) OS, is a powerful elicitor of volatiles in excised maize seedlings ( Zea mays cv. Delprim). We consider some of the mechanistic differences between wounding and insect herbivory in maize by examining the activity of volicitin, changes in jasmonic acid (JA) levels, and volatile emission from both intact plant and excised leaf bioassays. Compared to mechanical damage alone, volicitin stimulated increases in both JA levels and sesquiterpene volatiles when applied to intact plants. In a bioassay comparison, excised leaves were more sensitive and produced far greater volatile responses than intact plants following applications of both volicitin and JA. In the excised leaf bioassay, volicitin applications (10–500 pmol) to wounded leaves resulted in dose dependent JA increases and a direct positive relationship between JA and sesquiterpene volatile emission. Interestingly, volicitin-induced JA levels did not differ between intact and excised bioassays, suggesting a possible interaction of JA with other regulatory signals in excised plants. In addition to JA, insect herbivory is known to stimulate the production of ethylene. Significant increases in ethylene were induced only by BAW herbivory and not by either wounding or volicitin treatments. Using intact plant bioassays, ethylene (at 1 µl l⁻¹ or less) greatly promoted volatile emission induced by volicitin and JA but not mechanical damage alone. For intact plants, wounding, elicitor-induced JA and insect-induced ethylene appear to be important interacting components in the stimulation of insect-induced volatile emission.