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Significance of terpenoids in induced indirect plant

defence against herbivorous arthropods

ROLAND MUMM1, MAARTEN A. POSTHUMUS2 & MARCEL DICKE1

1Laboratory of Entomology, Wageningen University, PO Box 8031, 6700 EH Wageningen, the Netherlands and 2Laboratory

of Organic Chemistry, Wageningen University, PO Box 8026, 6700 EG Wageningen, the Netherlands

ABSTRACT

Many plants respond to herbivory by arthropods with an

induced emission of volatiles such as green leaf volatiles

and terpenoids. These herbivore-induced plant volatiles

(HIPVs) can attract carnivores, for example, predators and

parasitoids. We investigated the significance of terpenoids

in attracting herbivores and carnivores in two tritrophicsystems where we manipulated the terpenoid emission by

treating the plants with fosmidomycin, which inhibits one of

the terpenoid biosynthetic pathways and consequently ter-

penoid emission.

In the lima bean system, volatiles from spider-mite-

infested fosmidomycin-treated plants were less attractive

to the predatory mite Phytoseiulus persimilis than from

infested control plants. In the cabbage system, fosmidomy-

cin treatment did not alter the attractiveness of Brussels

sprouts to two Pieris butterflies for oviposition. The para-

sitoid Cotesia glomerata did not discriminate between

the volatiles of fosmidomycin-treated and water-treated

caterpillar-infested cabbage. Both P. persimilis and C.glomerata preferred volatiles from infested plants to unin-

fested ones when both were treated with fosmidomycin.

Chemical analysis showed that terpenoid emission was

inhibited more strongly in infested lima bean plants than in

Brussels sprouts plants after fosmidomycin treatment.

This study shows an important role of terpenoids in the

indirect defence of lima bean, which is discussed relative to

the role of other HIPVs.

Key-words: Brassica oleracea; Phaseolus lunatus; Tetrany-chus urticae; (E)-4,8-dimethyl-1,3,7-nonatriene; headspacevolatile trapping; homoterpenes; monoterpenes; windtun-nel; Y-tube olfactometer.

INTRODUCTION

Plants produce a plethora of volatile compounds, which areinvolved in various interactions with their environment.Terpenoids (isoprenoids) represent the largest and the mostdiverse group of volatiles that are emitted by plants, some-times in substantial amounts (Pichersky & Gershenzon2002; Dudareva & Pichersky 2006; Rodrguez-Concepcion

2006). The volatile fraction of terpenoids predominantlyconsists of the hemiterpene isoprene (C5), monoterpenes(C10) and sesquiterpenes (C15) and their derivatives such ashomoterpenes (C11 and C16). Monoterpenes and sesquiter-penes are synthesized by the condensation of two or threeisoprene units, respectively (Cheng et al. 2007). Because of

their physicochemical properties, such as volatility, reactiv-ity, toxicity and aroma, many protective functions againstabiotic and biotic factors have been ascribed to terpenoids(Holopainen 2004). Terpenoids are involved in plantpollinator interactions and have important functions inplant defence against herbivores (Dicke 1994; Par &Tumlinson 1999; Pichersky & Gershenzon 2002; Dudarevaet al. 2006; Hilker & Meiners 2006; Keeling & Bohlmann2006; Mumm & Hilker 2006;Van Schie,Haring & Schuurink2006; Cheng et al. 2007).

It has been demonstrated in numerous studies that theegg deposition or feeding by herbivorous arthropods, suchas insects and mites, can induce alterations in the emission

of volatile blends in many plant species (reviewed by Dicke& van Loon 2000; Dicke & Van Poecke 2002; DAlessandro& Turlings 2006; Hilker & Meiners 2006; Mumm & Hilker2006).These herbivore-induced changes in the volatile com-position can be exploited by natural enemies of the herbi-vores, such as predators and parasitoids, to locate plantsinfested with their prey/host species. The attraction ofnatural enemies to herbivore-induced volatiles is acceptedto be an important mechanism on how plants can indirectlydefend themselves (e.g. Dicke & van Loon 2000; Arimura,Kost & Boland 2005; Hilker & Meiners 2006). Inducedplant volatiles can also act directly against herbivores byrepelling them, but alternatively, herbivores can also use

these to locate suitable host plants (e.g. De Moraes,Mescher & Tumlinson 2001; Shiojiri et al. 2002; Horiuchiet al. 2003; Bruinsma et al. 2007).

The herbivore-induced changes in the volatile blends canbe quantitative, that is, volatiles that are also present innon-induced plants are emitted in larger total amounts,their relative abundance changes, or both (e.g. Mumm et al.2003; Bukovinszky et al. 2005). On the other hand, her-bivory can also induce de novo production of compounds inmany plant species, resulting in qualitative changes in thecomposition of the emitted blend (Turlings et al. 1998;Dicke et al. 1999; Krips et al. 1999; Leitner, Boland &Mithfer 2005). Typical volatile plant compounds that are

Correspondence: R. Mumm. Fax: 0031317484821; e-mail:

[email protected]

Plant, Cell and Environment (2008) 31, 575585 doi: 10.1111/j.1365-3040.2008.01783.x

2008 The AuthorsJournal compilation 2008 Blackwell Publishing Ltd 575
mailto:[email protected]:[email protected]


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induced by herbivory are C6-alcohols, -aldehydes and-acetates (so-called green leaf volatiles, GLVs); methylsalicylate (MeSA); phenylpropanoids; and various monoter-penes and sesquiterpenes, as well as two homoterpenes,(E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) and (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT)(Arimura

et al. 2005;Dudareva et al. 2006).GLVs arefatty acid deriva-tives resulting from the conversion of linolenic and linoleicacid released from damaged plasmamembranes through thelipoxygenase pathway (Arimura et al. 2005; DAuria et al.2007).All terpenoids aresynthesized via the cytosolic meva-lonate (MVA) pathway or the methylerythritol 4-phosphate(MEP) pathway, which is located in the plastids (Aharoni,Jongsma & Bouwmeester 2005; Rodrguez-Concepcion2006; Cheng et al. 2007). Both terpenoid pathways cansynthesize isopentenyl diphosphate (IPP) and its isomerdimethylallyl diphosphate (DMAPP), which are the centralintermediates for the biosynthesis of terpenes. In a widerange of cyclization and rearrangement steps, precursors are

converted into the parent skeletons of the respective terpe-nes. Finally, the parent skeletons are converted into themyriad of different terpenoids by a big variety of transfor-mations like oxidations, isomerizations and conjugations(Gershenzon & Kreis 1999). Monoterpenes and diterpenesare synthesized via the MEP pathway, whereas sesquiterpe-nes are produced by the MVA pathway. Recently, it wasdemonstrated that there is some exchange of IPP andDMAPP between the two pathways, indicating that thepathways are not strictly separated (Rodrguez-Concepcion2006).

Much attention has been paid to the role of monoter-penes in plant defence against herbivores, because this

group of terpenes is the most abundant among volatileterpenoids. Many plants, such as conifers, show a high con-stitutive emission of monoterpenes; others, however, showa strong induced emission of monoterpenes after her-bivory. Constitutive and inducible defences are supposedto be negatively correlated (Dicke & Van Poecke 2002;Koricheva, Nyknen & Gianoli 2004). Terpenes may act asfeeding deterrents for insects, but also, the ovipositionbehaviour of herbivorous arthropods can be influenced bythe presence or absence of terpenes (Aharoni et al. 2003;Tripathi et al. 2003; Isman 2006). Furthermore, it has beenshown that terpenes can attract predators and parasitoids.Many of these studies used single terpenes or mixtures,

which are then applied to some sort of dispensing mate-rial. Although this approach might work well in somecases, in others, it failed. One reason is that by applyingsingle synthetic compounds, one neglects that backgroundodours of the plants play an important role in bringingterpenes into the right context (Pettersson 2001; Petters-son, Birgersson & Witzgall 2001; Buitenhuis et al. 2005;Mumm & Hilker 2005). Other studies showed that terpe-nes can mask the attractiveness of other plant volatiles toherbivorous insects and parasitoids (Yamasaki, Sato &Sakoguchi 1997; Turlings & Fritzsche 1999). This showsthat many questions regarding the role of terpenes in theinterplay with other plant volatiles in direct and indirect

plant defence against herbivorous arthropods still remainto be answered.

Here, we address the role of terpenes in induced indirectdefence by applying fosmidomycin, an inhibitor of theMEP pathway, to plants. The antibiotic fosmidomycinblocks 1-deoxy-D-xylulose 5-phosphate reductoisomerase,

an enzyme catalysing an early step in the MEP pathway(Zeidler et al. 1998). Fosmidomycin has been shown toeffectively inhibit the biosynthesis and emission of volatilemonoterpenes and partly also sesquiterpenes (Jux,Gleixner& Boland 2001; Copolovici et al. 2005; Hampel, Mosandl &Wst 2005; Bartram et al. 2006).This makes it an interestingtool to study the role of monoterpenes in direct and indirectplant defence.

We used two tritrophic model systems to study theeffect of treating plants with fosmidomycin on the behav-iour of herbivores and their natural enemies. In the limabean system, lima bean plants, Phaseolus lunatus(Fabaceae), are used as host plants for the herbivorous

two-spotted spider mite Tetranychus urticae (Acari, Tet-ranychidae). The predatory mite Phytoseiulus persimilis(Acari, Phytoseiidae) was used as carnivorous species. Thecabbage system consisted of Brussels sprouts plants,Brassica oleracea (Brassicaceae), which are frequentlyattacked by the specialized large cabbage white butterflyPieris brassicae and the small cabbage white butterflyPieris rapae (Lepidoptera, Pieridae). P. brassicae and P.rapae caterpillars are parasitized by the larval parasitoidCotesia glomerata (Hymenoptera, Braconidae), a gregari-ous endoparasitoid that prefers to parasitize young larvalinstars of particularly P. brassicae (Geervliet et al. 2000).

It has previously been demonstrated that C. glomerata is

attracted to caterpillar-infested Brussels sprouts, and P. per-similis to volatiles from spider-mite-infested lima bean,respectively (Dicke et al. 1990a; Mattiacci, Dicke & Posthu-mus 1994; Geervliet 1997; De Boer 2004). In both plantspecies, volatiles are induced after herbivory. However, inBrussels sprouts, the changes in terpene emission afterherbivore feeding are mainly quantitative (Blaakmeeret al. 1994; Mattiacci et al. 1994; Bukovinszky et al. 2005),whereas in lima bean plants, spider mites induce a distinctde novo production of terpenoids such as (E)-b-ocimene,linalool, DMNT and TMTT (Dicke et al. 1990b, 1999; DeBoer, Posthumus & Dicke 2004; Shimoda et al. 2005).

Recently, Smid et al. (2002) showed that the antennae of

C. glomerata respond to limonene, a monoterpene that wasidentified in the headspace ofP. brassicae-infested Brusselssprouts. Furthermore, the predatory mite P. persimilis isknown to behaviourally respond to terpenoids, but MeSA,which is also strongly induced in lima bean by spider mitefeeding, is also strongly attractive to the predatory mites,making it difficult to estimate the relative contribution ofterpenoids (Dicke et al. 1990b; De Boer & Dicke 2004a,b).

By applying fosmidomycin, we expected that particularlythe emission of monoterpenes and the homoterpene TMTTis inhibited. Other inducible compounds, like GLVs andMeSA, should not be affected by the fosmidomycin treat-ment. We expected that if terpenoids play a significant role

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in indirect plant defence,the infested fosmidomycin-treatedplants should be less attractive to predators and parasitoidsthan the untreated ones. On the other hand, herbivores areexpected to prefer to lay eggs on plants with less terpenoidemission. We also collected the headspace volatiles frominfested Brussels sprouts and lima bean plants after fosmi-

domycin treatment to assess treatment effects on thechemical changes in headspace composition for the twoplant species.

MATERIAL AND METHODS

Plants and arthropods

Plants

Lima bean plants, P. lunatus L. cv. Sieva (Fabaceae), andBrussels sprouts plants, B. oleracea var. gemmifera L. cv.Cyrus (Brassicaceae), were grown from seed in separategreenhouse compartments in plastic pots (11 11 11 cm)

at 24

4 C, 60

20% relative humidity (RH) and a 16 hlight/8 h dark photoperiod. Lima bean plants were used inexperiments when the primary leaves had fully expanded,which was 1216 d after sowing. Experiments were con-ducted with 6- to 8-week-old Brussels sprout plants.

Herbivores

A colony of the two-spotted spider mite T. urticae Koch(Acari,Tetranychidae) was maintained on lima bean plantsin another greenhouse compartment under the same con-ditions as described for the lima bean culture.A continuousrearing of the large cabbage white,Pieris brassicae L.(Lepi-

doptera, Pieridae), and the small cabbage white, Pierisrapae L. (Lepidoptera, Pieridae), was maintained onBrussels sprouts in a climatized room at 21 1 C,60 10% RH and a 16 h light/8 h dark photoperiod.

Carnivores

A colony of the predatory mite P. persimilis Athias-Henriot(Acari, Phytoseiidae) was kept on spider-mite-infested limabean leaves in a climate cabinet at 23 1 C, 60 10%RH and continuous light. The parasitoid C. glomerata L.(Hymenoptera, Braconidae) was reared on P. brassicae cat-erpillars feeding on Brussels sprouts under similar environ-

mental conditions as the uninfested cabbage plants. Forexperiments, C. glomerata pupae were collected and kept ina cage in a climate cabinet (23 1 C, 60 10% RH and a16 h light/8 h dark photoperiod). Emerging wasps were pro-vided with water and honey. Male and female wasps werekept together until the experiment.

Behavioural bioassays

Olfactometer and windtunnel tests

AY-tube olfactometerset-up similar to theone described byTakabayashi & Dicke (1992) was used to investigate the

olfactory choice of predatory mites to two odour sources.Ametal wire was positioned at the middle of the olfactometertube.The side arms were both connected to 5 L Duran glassjars(Duran,Mainz,Germany) containingthe odoursources.Pressurized air was filtered over activated charcoal, andwas led into the jars at the top, and the jars were left at the

bottom towards the olfactometer. An airflow in each armof 4 L min-1 was controlled by a flowmeter (Brooks Instr.,Veenendaal, the Netherlands).Air was extracted at the baseof the Y-tube at 8 L min-1. Experiments were conducted at23 2 C and 60 5 mmol m-2 s-1 photosynthetically activeradiation (PAR). In order to correct for unforeseen asym-metry in the set-up, the position of the odour sources wasswitched after five tested predators. After testing 10 preda-tors, the odour sources were replaced with new ones. Everyexperiment was replicated on four different days.

To enhance the responsiveness of predatory mites toplant volatiles, all predators had been starved for 2 h priorto release in the olfactometer, by confining them individu-

ally in Eppendorf tubes (Sarstedt, Nmbrecht, Germany).The tubes with the predators were placed into the experi-mental room to acclimatize to the new environment. Preda-tors were individually released on the iron wire in the basaltube, and their behaviour was observed for 10 min. Preda-tory mites that reached a gauze mesh at the middle of theside arm within this period were recorded as having made achoice. Mites that did not make a choice were recorded asno choice. Each predator was used only once.

Behavioural choice experiments with the parasitoid C.glomerata were carried out with a windtunnel set-up(25 1 C, 60 10% RH and 35 mmol m-2 s-1 PAR) asdescribed by Geervliet,Vet & Dicke (1994). The wind speed

was adjusted to 0.2 m s-1

. Two-choice experiments wereconducted by placing a treated leaf and a respective controlleaf at the upwind end of the tunnel.

Nave C. glomerata females were separated from males3 h prior to the experiment and were transferred to anothercage, which was placed in the experimental room to accli-matize to the new environment. The wasps were individu-ally introduced into the windtunnel on an infested cabbageleaf piece from which the caterpillars and their productshad been carefully removed. The wasps were allowed towalk onto the leaf pieces themselves.The leaf piece with thewasp was placed at the middle of the release cylinder,whichwas 60 cm downwind from the two odour sources. As soon

as the parasitoid had left the leaf for some seconds, the leafwas carefully removed using tweezers without disturbingthe parasitoid. The flight behaviour of the wasps wasobserved. Flights that resulted in a landing on one of thetwo odour sources were recorded as a choice. Parasitoidsthat did not leave the release cylinder or landed on otherparts of the windtunnel within 10 min were recorded as nochoice. Every parasitoid was used only once. In order tocorrect for unforeseen asymmetry in the set-up,the positionof the odour sources was switched after five tested parasi-toids. After testing 10 parasitoids, the odour sources werereplaced with new ones. Every experiment was replicatedon four different days.

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Oviposition preference test

Freshly emerged P. brassicae and P. rapae adults were eachtransferred to a large cage (67 100 75 cm) in a green-house compartment at 24 4 C, 60 20% RH and a 16 hlight/8 h dark photoperiod. Butterflies were provided with a10% sucrose solution. Three to five days after emergence,

one male and one female butterfly were transferred to ovi-position cages (67 50 75 cm) in the same greenhousecompartment.In addition to natural daylight,the cages wereilluminatedby sodium vapour lamps(SON-T,500 W,Philips,Eindhoven, the Netherlands) from 1000 to 1600 h. At 48 hprior to the experiment, a single untreated Brussels sproutsleaf wasplaced in each cage as an oviposition substrate.After6 h,the leaf was removed.On the experimental day between0900 and 1000 h in the morning, a fosmidomycin-treatedBrussels sprouts leaf and a respective control leaf wereintroduced into every cage.The treated and the control leafalways originated from the same plant to minimize intraspe-cificvariationbetween treatmentand control.Each butterfly

couple was also provided with a 10% sucrose solution. Theleaves were placed in an upright position approximately40 cm apart from each other. P. brassicae was allowed to layeggs on the two leaves for 24 h, whereas P. rapae could layeggs for 6 h. The leaves were then removed, and the eggslayed were counted. P. rapae lays individual eggs, which aredistributed over the leaves. In general, P. rapae starts todeposit eggs soon after being exposed to leaves. Therefore,6 h wassufficient to obtainsufficient eggs.On theotherhand,P. brassicae lays eggbatches, which can last forseveral hours.Therefore, we chose to confine P. brassicae for 24 h with thecabbage leaves.The experiments were conducted in severalcages at the same time and on 45 d per treatment.

Plant treatments

For all experiments, lima bean leaves or Brussels sproutsleaves were cut under water. When leaves are cut underwater, a small water droplet normally forms around thepetiole, and prevents air invading the vascular systemthrough the petiole. In this way, the leaves could be trans-ferred in glass vials filled either with water or a 50 mmaqueous fosmidomycin solution. Fosmidomycin was pur-chased from Invitrogen Molecular Probes (Breda, theNetherlands).The openings of the glass vials with the leaveswere thoroughly covered with Parafilm (Pekhiney Plastic

Packing, Chicago, IL, USA). Only leaves that were in visu-ally good condition after the treatment period were usedfor the experiments.

Lima bean leaves were treated either with a 50 mmaqueous fosmidomycin solution or with water, and thenwere either immediately infested with 20 adult T. urticae orleft uninfested. Subsequently, the lima bean plants werekept in a climate chamber for 48 h at 23 1 C, 60 5%RH and a 16 h light/8 h dark photoperiod before being usedin the experiment. The leaves used for the behaviouralexperiments with C. glomerata were treated with fosmido-mycin or with water. The fosmidomycin-treated and water-treated leaves were then immediately either infested with

50 L1 P. brassicae caterpillars or were left uninfested.The leaves were kept in a climate chamber at 23 1 C,60 5% RH and a 16 h light/8 h dark photoperiod.For oviposition experiments with butterflies, bothfosmidomycin-treated and control (water-treated) cabbageleaves were kept for 24 h in the greenhouse compartment at

24

4 C, 60

20% RH and a 16 h light/8 h dark photo-period before being used in the bioassay.

Collection of headspace volatiles

Volatiles emitted from fosmidomycin-treated lima beanand Brussels sprouts leaves were collected using a dynamicheadspace collection system. The leaves of one treatmentwere transferred to a 5 L Duran glass jar (Duran). The jarwas tightly closed with a glass lid that was pressed on the jarwith a metal clamp with a viton O-ring in between. The lidhad an air inlet and an air outlet. Pressurized air was filteredover activated charcoal and was led into the jar with a

constant flow of 70 mL min-1

. Air was sucked out of the jarwith 50 mL min-1 by passing through a glass tube filled with90 mg Tenax TA (Grace-Alltech, Deerfield, IL, USA) con-nected to the air outlet of the jar. The slight overpressurewas created to prevent that unfiltered air could invade thesystem. The system was purged for 30 min with cleaned airbefore the volatiles were trapped onto the Tenax. The flowthrough the jar was controlled by flowmeters (BrooksInstr.). Teflon tubing was used for all connections. Head-space collections were made in a climate chamber at23 1 C, 60 5% RH, and 90 5 mmol m-2 s-1 PAR. Thevolatiles of one treatment and its respective control werecollected simultaneously. Lima bean plants were infested

with 20 spider mites per plant. Brussels sprouts wereinfested with 50 L1 P. brassicae caterpillars per leaf. Boththe infested lima bean leaves and Brussels sprouts plantswere then either placed in vials containing fosmidomycin orwater as described previously. The volatiles emitted fromlima bean leaves were trapped for 1 h, and for Brusselssprouts leaves, trapping time was 5 h.

Chemical analysis of headspace samples

Headspace samples were analysed with a Varian 3400 gaschromatograph (GC) (Varian, Palo Alto, CA, USA) con-nected to a Finnigan 95 mass spectrometer (MS) (Thermo

Scientific, Waltham, MA, USA). The collected volatileswere released from the Tenax by heating the trap in a Ther-modesorption Cold Trap Unit (Chrompack, Middelburg,the Netherlands) at 250 C for 10 min, and flushing withhelium at 14 mL min-1.The released compounds were cryo-focused in a cold trap 0.52 mm [inner diametre (ID)] deac-tivated fused silica at a temperature of -85 C. By ballisticheating of the cold trap to 220 C, the volatiles were trans-ferred to the analytical column (60 m 0.25 mm ID,0.25 mm film thickness, DB-5 ms J&W, Folsom, CA, USA).The temperature programme started at 40 C (4 min hold)and rose at the rate of 4 C min-1 (lima bean) or 5 C min-1

(Brussels sprouts) to 280 C (4 min hold). The column

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effluent was ionized by electron impact ionization at 70 eV.Mass scanning was carried out from 24 to 300 m/z with ascan time of 0.7 s/d and an interscan delay of 0.2 s. Thecompounds were identified by comparison of the massspectra with those in the Wiley library and in the Wagenin-gen Mass Spectral Database of Natural Products, and by

checking the retention index.One peak area unit representsapproximately 0.17 0.05 ng.

Statistical analyses

A two-sided binomial test was used to analyse whether thebehavioural choices of predatory mites and parasitoids dif-fered from a 50:50 distribution over the two odour sources.Predatory mites and parasitoids that did not make a choicewere excluded from the statistical analysis. Most individualsof both Pieris species laid eggs on both the control and thefosmidomycin-treated leaves. The number of eggs on eachtreatment per individual was considered as a paired sampleand was analysed with the Wilcoxon signed ranks test.Amounts of headspace volatiles trapped were analysed onthe basis of normalized peak area units, as determined byGC-MS analysis. MannWhitney U-tests were applied totest for differences between the fosmidomycin-treated andcontrol plants for groups of compounds. P values wereadjusted by the sequential Bonferroni method to correct forthe family wise error rate (Holm 1979).

RESULTS

Behavioural response of predatory mites andparasitoids to fosmidomycin-treated plants

The predatory mite P. persimilis significantly preferred thevolatiles from spider-mite-infested lima bean leaves, which

where not treated with fosmidomycin, to those from spider-mite-infested fosmidomycin-treated lima bean leaves(Fig. 1). Thus, treatment with fosmidomycin reduces theattractiveness of spider-mite-induced lima bean volatilesto predatory mites. Yet, predatory mites were stronglyattracted to the volatiles from fosmidomycin-treated,

spider-mite-infested lima bean when offered against thevolatiles from fosmidomycin-treated uninfested lima beanplants (Fig. 1). This shows that although fosmidomycinreduces the attraction of predators to spider-mite-infestedlima bean leaves, it does not eliminate the emission ofattractive volatiles altogether.

The parasitoid C. glomerata did not discriminate betweenthe volatiles from fosmidomycin-treated and P. brassicae-infested cabbage leaves, and the volatiles from controlleaves, that is, water-treated P. brassicae-infested cabbageleaves (Fig. 2). Thus, fosmidomycin treatment did notchange the attractiveness of caterpillar-induced cabbagevolatiles to those from the untreated cabbage. C. glome-

rata females significantly preferred the volatiles fromfosmidomycin-treated P. brassicae-infested leaves to thosefrom fosmidomycin-treated uninfested plants (Fig. 2). Thisshows that the volatiles that are used by the parasitoids todiscriminate between infested and uninfested plants are stillpresent after the treatment with fosmidomycin.

Oviposition preference of cabbage whitebutterflies after application of fosmidomycin

The average number of eggs deposited by the large cab-bage white butterfly P. brassicae was not significantly differ-ent between fosmidomycin-treated cabbage leaves and

untreated controls (Wilcoxon signed ranks test, Z= -0.812,P> 0.05,n = 22)(Fig. 3).Neither didthe small cabbage white

0 5050100 100

Predatory mite choices (%)

***

Fos**

Fos

Fos

H2O 2%

0%

Figure 1. Effect of fosmidomycin treatment of lima bean plants on the response of Phytoseiulus persimilis to lima bean volatiles. Upperpanel: response to spider-mite-infested lima bean treated with 50 mm fosmidomycin solution (grey bar) or spider-mite-infested lima bean(white bar) n = 117. Lower panel: response to spider-mite-infested lima bean treated with 50 mm fosmidomycin solution (grey bar) oruninfested lima bean treated with 50 mm fosmidomycin solution (white bar) n = 80. The number behind each bar is the percentage ofpredatory mites that did not make a choice. Choices between odour sources were analysed with a two-sided binomial test (**P< 0.01,***P< 0.001).




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butterfly P. rapae discriminate between fosmidomycin-treated and untreated cabbage leaves (Wilcoxon signedranks test, Z= -0.975, P> 0.05, n = 23) (Fig. 3). Thedifferences in the average number of eggs laid by the twospecies probably result from the differences in the oviposi-tion behaviour and the differences in time the butterflieswere allowed to oviposit.

Analysis of headspace volatiles

In the headspace of caterpillar-infested Brussels sproutsplants, 31 compounds were detected, and 12 compoundswere identified in the headspace of spider-mite-infestedlima bean leaves. Both plant species were treated eitherwith fosmidomycin or with water as control (Table 1). Allcompounds that were emitted by fosmidomycin-treatedleaves were also detected in the headspace of controlleaves. Therefore, the compounds that were detected in atleast half of the control samples are depicted in Table 1.Herbivore-induced volatiles of excised leaves of lima beanand Brussels sprouts resembled those emitted by wholeplants (Bukovinszky et al. 2005; Pinto et al. 2007).

The emission of the monoterpene hydrocarbons (Z)- and(E)-b-ocimene, the oxygenated monoterpene linalool andthe homoterpene TMTT was completely inhibited byfosmidomycin in lima bean (Table 1, Fig. 4). The secondhomoterpene DMNT was emitted in significantly loweramounts by fosmidomycin-treated lima bean leaves com-pared with the water-treated controls (Fig. 4, Table 1).The only sesquiterpene, that is, (E)-b-caryophyllene, wasemitted in lower amounts by fosmidomycin-treated limabean leaves than by water-treated leaves, but this was notstatistically significant.The emission of GLVs (hexanal, (Z)-3-hexen-1-ol, (Z)-3-hexen-1-ol acetate) and MeSA was notsignificantly affected by fosmidomycin treatment (Fig. 4,Table 1).

In contrast to the situation in lima bean, fosmidomycindid not inhibit the emission of monoterpenes in Brusselssprouts leaves significantly (Fig. 5, Table 1). However,fosmidomycin-treated Brussels sprouts leaves emittedlower amounts of monoterpenes than the water-treatedleaves. Furthermore, the emission of GLVs was reduced infosmidomycin-treated plants although there was a strongvariability in the emission of these compounds (Fig. 5,

0 5050100 100

Wasps choices (%)

Fos

n.s.

***

18%

17%

Fos

Fos

H2O

Figure 2. Effect of fosmidomycin treatment of Brussels sprouts on the response of Cotesia glomerata to Brussels sprouts odours. Upperpanel: response to caterpillar-infested Brussels sprouts treated with 50 mm fosmidomycin solution (grey bar) or caterpillar-infestedBrussels sprouts (white bar) n = 45. Lower panel: response to caterpillar-infested Brussels sprouts treated with 50 mm fosmidomycinsolution (grey bar) or uninfested Brussels sprouts treated with 50 mm fosmidomycin solution (white bar) n = 36. The number behind eachbar is the percentage of parasitoids that did not make a choice. Choices between odour sources were analysed with a two-sided binomialtest (n.s., P> 0.05; ***P< 0.001).

Figure 3. Oviposition ofPieris brassicaeand Pieris rapae on Brussels sprout plantseither treated with 50 mm fosmidomycin(black bars) or left untreated (whitebars). Mean number of eggs perfemale + standard error (n.s., P> 0.05,Wilcoxon signed ranks test).

0

20

40

60

80

averagenumberoflaideggs N=22

n.s.

N=23

n.s.

Pieris brassicae Pieris rapae

580 R. Mumm et al.



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Table 1). The homoterpene DMNT was only detected in

trace amounts in a few samples,and TMTT was not found inthe headspace of Brussels sprouts (Table 1).

DISCUSSION

The treatment with fosmidomycin affected the terpeneemissions in lima bean and to a lesser extent in Brusselssprouts (Figs 4 and 5). Likewise, the effects on carnivorebehaviour were more pronounced in the lima bean than inBrussels sprouts (Figs 1 and 2). We used fosmidomycin toblock the emission of terpenoids that are synthesized viathe plastidial MEP pathway. Therefore, we expected espe-cially the emission of monoterpenes and the homoterpene

TMTT, which is a derivative of the diterpene geranyllina-

lool, to be inhibited (Boland et al. 1998).The concentration of fosmidomycin we used was shownto be effective in inhibiting isoprenoid emission when fedthrough the petiole (Barta & Loreto 2006; Bartram et al.2006). The incubation period with fosmidomycin differedbetween 24 h in Brussels sprouts and 48 h in lima bean,because we knew from previous experiments that parasi-toids and predatory mites are attracted to induced volatilesafter 24 and 48 h of infestation, respectively. It is not likelythat this difference in incubation time is responsible for thedifferences in inhibition because fosmidomycin can effec-tively inhibit the isoprenoid emission already after a fewhours (Loreto & Velikova 2001; Barta & Loreto 2006;

Table 1. List of volatile compounds detected in the headspace of Brussels sprouts and lima bean after different treatments

Brussels sprouts Lima bean

Fosmidomycin (n = 3) Control (n = 5) Fosmidomycin (n = 5) Control (n = 5)

1 1-Penten-3-ol 68 (54103) 105 (21206) nd nd2 3-Pentanone 35 (20207) 131 (27249) nd nd

3 3-Pentanol 56 (40123) 128 (83157) nd nd4 2-Methylbutanal nd nd 0 (03) 5 (08)5 3-Methylbutanal nd nd 9 (612) 14 (620)6 (Z)/(E)-2-Penten-1-ol 2 (14) 7 (414) nd nd7 (Z)-3-Hexenal 12 (649) 34 (3495) nd nd8 Hexanal 8 (79) 12 (416) 1 (04) 8 (28)9 (E)-2-Hexenal 0 (06) 7 (045) nd nd

10 (Z)-3-Hexen-1-ol 237 (203884) 650 (263899) 1 (05) 1 (12)11 2-Methyl-2-cyclopenten-1-one 0 (02) 5 (08) nd nd12 (Z)-2-Penten-1-ol acetate 79 (7185) 117 (71199) nd nd13 Pentyl acetate 3 (23) 4 (05) nd nd14 a-Thujene 110 (67130) 158 (54178) nd nd15 a-Pinene 25 (2037) 44 (1283) nd nd16 3-Ethyl-1,5-octadiene 6 (67) 10 (813) nd nd17 Sabinene 437 (271546) 716 (207841) nd nd18 b-Pinene 27 (1733) 39 (1080) nd nd19 Myrcene 103 (62116) 151 (94201) nd nd20 (Z)-3-Hexen-1-ol acetate 2630 (25002855) 3680 (29305320) 20 (041) 33 (083)21 Hexyl acetate 42 (2148) 36 (3379) nd nd22 Limonene 258 (158284) 343 (269547) nd nd23 b-Phellandrene 4 (24) 10 (512) nd nd24 1,8-Cineole 174 (98181) 170 (104249) nd nd25 (Z)-b-Ocimene nd nd 0 (00) 18 (1459)26 Phenylacetaldehyde 0 (01) 7 (012) nd nd27 (E)-b-Ocimene nd nd 0 (00) 280 (210958)28 Salicylaldehyde 0 (00) 3 (06) nd nd29 g-Terpinene 0 (00) 5 (317) nd nd30 trans-4-Thujanol 0 (05) 11 (019) nd nd31 Linalool nd nd 0 (00) 31 (1132)32 Nonanal 16 (818) 58 (1781) nd nd33 (E)-4,8-dimethyl-1,3,7-nonatriene nd tr 57 (1584) 573 (405820)34 2-Methyl-6-methylene-1,7-octadiene-3-one 0 (02) 5 (013) nd nd35 Methyl salicylate 5 (47) 5 (312) 3 (277) 103 (30123)36 a-Gurjunene 4 (35) 3 (030) nd nd37 Longifolene 7 (68) 6 (010) nd nd38 (E)-b-Caryophyllene nd nd 2 (03) 10 (818)39 (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene nd nd 0 (00) 51 (4267)

Median and interquartile range (in parentheses) of normalized peak area units are given.nd, Compounds were neither detected in fosmidomycin-treated plants nor in the controls; tr, trace amounts in single samples.




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Bartram et al. 2006).In lima bean leaves, the emission of noother compound except for terpenoids was significantlyaffected by fosmidomycin treatment compared with thecontrols, suggesting that the plants did not severely sufferfrom the treatment.

The headspace data for lima bean plants indicate that theherbivore-induced de novo production of monoterpenesand TMTT relies completely on the MEP pathway. Inter-estingly, the allocation of precursors of DMNT (producedthrough the cytosolic MVA pathway) is more plastic(Bartram et al. 2006). When plants are not stressed, precur-sors of mainly the MVA pathway but partly also from the

MEP pathway are assembled into DMNT. If the supply ofprecursors from the MEP pathway was blocked, this didnot reduce the constitutive emission of DMNT because ofan increasing assemblage of MVA-derived precursors(Bartram et al. 2006). However, when the emission of

DMNT is induced in lima bean plants by herbivory or treat-ment with jasmonic acid (JA), the increased demand ofprecursors is covered by the MEP pathway (Piel et al. 1998;Jux et al. 2001). Our results show that in spider-mite-infested lima bean plants, the MVA pathway is not able tofully compensate for a lack of precursors from the MEPpathway after fosmidomycin treatment, resulting in areduced emission of DMNT and (E)-b-caryophyllene(Fig. 4). In turn, this indicates that spider mite feeding onlima bean does not induce the MVA pathway, but particu-larly the MEP pathway, similar to what was demonstratedfor JA treatment. However, Jux et al. (2001) found no

significant reduction in DMNT emission after JA andfosmidomycin treatment,suggesting that the mechanisms interpenoid induction and regulation in response to spider-mite infestation and JA application are similar but not iden-tical (see also Dicke et al. 1999).

Figure 4. Emission of volatilecompound classes by lima bean plantsinfested with 50 Tetranychus urticaecaterpillars and that were either treatedwith 50 mm fosmidomycin (grey bars) orleft untreated (white bars).Weightedmean and standard error of normalizedpeak area units are given. n.s., P> 0.05;*P> 0.05, MannWhitney U-test, Pvalues are corrected by sequentialBonferroni method (Holm 1979).

0

100

200

300

400

500

600

700

Green leaf

volatiles

Monoterpenes Methylbutanal

Norm.peakareaunits

.s.n.s.n n.s.

*

*

* n.s.

Caryophyllene DMNT TMTT Methy l sali cy late

Figure 5. Emission of volatilescompound classes by Brussels sproutsplants infested with 50 Pieris brassicaecaterpillars and that were either treatedwith 50 mm fosmidomycin (grey bars) orleft untreated (white bars).Weightedmean and standard error of normalizedpeak area units are given. n.s., P> 0.05,MannWhitney U-test, P values arecorrected by sequential Bonferronimethod (Holm 1979).

0

200

400

600

800

1000

Green leafvolatiles

Methyl salicylate

Norm.peakareaunits

n.s.

n.s. n.s. n.s. n.s.

Monoterpenes Sesquiterpenes Others

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The lima bean system

The predatory mite P. persimilis significantly preferred thevolatiles of water-treated, infested lima bean plants to thoseof fosmidomycin-treated ones (Fig. 1). On the other hand,the volatiles from spider-mite-infested plants were stillmore attractive to predatory mites compared with the unin-

fested ones when both were treated with fosmidomycin(Fig. 1).This shows that the absence of certain terpenoids inthe headspace makes infested lima bean clearly less attrac-tive to predatory mites, but still other chemical cues thanterpenoids are apparently used to discriminate betweeninfested and uninfested lima beans. It has been shown thatMeSA plays a crucial role in the attraction of predatorymites (Dicke et al. 1999; De Boer & Dicke 2004a,b). MeSAis induced after spider-mite infestation (e.g. Dicke et al.1999), but as expected,fosmidomycin did not affect its emis-sion (Fig. 4). De Boer et al. (2004) showed that offeringMeSA as an alternative odour source reduced the prefer-ence of predatory mites to spider-mite-infested lima bean

volatiles.In addition, when MeSA was added to the volatileblends induced by JA or feeding by Spodoptera exigua,which were similar to spider-mite-induced volatiles butlacking MeSA, they became more attractive to predatorymites than those of spider-mite-induced plants. The attrac-tive effect of MeSA was thereby based on qualitative dif-ferences rather than quantitative changes (De Boer &Dicke 2004a,b, 2005; De Boer et al. 2004).

Despite the presence of MeSA, predatory mites pre-ferred the volatiles of untreated lima bean leaves tofosmidomycin-treated ones, thus demonstrating the rela-tive importance of terpenoids for the predatory mites.P. persimilis and other predatory mites are attracted

to several synthetic terpenoids, such as linalool, (E)-b-ocimene or DMNT, when offered individually (e.g. Dickeet al. 1990b; Shimoda et al. 2005). Interestingly, TMTT,although not attractive to predatory mites as a pure syn-thetic compound, did affect the attractiveness of a mixtureof herbivore-induced lima bean plants (De Boer et al.2004). This suggests that the role of terpenoids in indirectplant defence depends on the presence and composition ofcertain background volatiles as was also demonstrated forother tritrophic systems. For example, Mumm & Hilker(2005) showed that the egg parasitoid Chrysonotomyiaruforum (Hymenoptera, Eulophidae) only responded tothe combination of the sesquiterpene (E)-b-farnesene and

background volatiles of pines.The parasitoids response wasdependent on the concentration of (E)-b-farnesene, sug-gesting that this parasitoid uses the contrast between (E)-b-farnesene and the background odour, that is, when thecompound was experienced in the right chemical context(Mumm & Hilker 2005, 2006; Hilker & Meiners 2006).

The cabbage system

In oviposition choice experiments, we tested whether P.brassicae or P. rapae avoided or preferred intact Brusselssprout leaves treated with fosmidomycin to water-treated

control leaves. Both P. brassicae and P. rapae did notdiscriminate between fosmidomycin-treated and water-treated cabbage leaves (Fig. 3). Neither did the treatmentwith fosmidomycin reduce the attractiveness ofP. brassicae-induced cabbage volatiles to C. glomerata (Fig. 2). In con-trast to lima bean plants, the emission of terpenoids in

cabbage leaves was not significantly inhibited by fosmido-mycin. Thus, a role of terpenoids in the direct or indirectdefence of Brussels sprouts against Pieris butterflies cannotbe excluded by this study. Future studies should elucidatethe regulation of constitutive and induced terpenoid pro-duction, for example, by applying combinations of differentinhibitors, for example, fosmidomycin and cerivastatin.

CONCLUSION

In conclusion, this study demonstrates that inhibitorslike fosmidomycin can be used to investigate the role ofterpenoid infochemicals in plant defence mechanisms

against herbivores. In comparison with compounds induc-ing plant defence responses, such as coronalon or jas-monates (Schler, Mithfer & Baldwin 2004; Wasternacket al. 2006), inhibitors have been applied far less in studiesaddressing indirect plant defence. Although many of theinhibitors are specific for a certain biosynthetic pathway,they may not specifically inhibit particular chemical com-pounds as biochemical pathways generally have more thanone final product. In the case of fosmidomycin, one shouldbe aware that not only volatile terpenoids are inhibited butalso other essential terpenoids, which are important mem-brane components, photosynthetic pigments, or antioxi-dants, such carotenoids, sterols and gibberellins, might be

affected as well (Owen & Peuelas 2005).Therefore, experi-ments studying the significance of volatile terpenoidsshould use rather short incubation times.

Future studies need to elucidate what the role of particu-lar compounds is in attracting carnivorous arthropods. Oneapproach would be to combine ecological and moleculartools by using plants that have been genetically modified inthe emission of certain terpenoids.This new field of ecoge-nomics has been successfully developed in the last few yearsand provides promising opportunities on the way to under-stand how indirect plant defence mechanisms function(Dicke, van Loon & de Jong 2004; Dicke 2006; Ouborg &Vriezen 2007; Snoeren, De Jong & Dicke 2007).

ACKNOWLEDGMENTS

We would like to thank Rieta Gols and GabriellaBukovinszkineKiss for the help with the experiments.Many thanks to Leo Koopman, Frans van Aggelen andAndr Gidding for culturing the insects and mites, and theexperimental farm of Wageningen University (Unifarm) forrearing the Brussels sprout plants. We also thank twoanonymous reviewers for their highly valuable comments.The study was financially supported by the EuropeanCommission contract MC-RTN-CT-2003-504720 ISONETand by aVICI grant (nr 865.03.002) from the Earth and Life




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Sciences Foundation, which is subsidized by the Nether-lands Organization for Scientific Research.

REFERENCES

Aharoni A., Giri A.P., Deuerlein S., Griepink F., de Kogel W.J.,

Verstappen F.W.A., Verhoeven H.A., Jongsma M.A., Schwab W.& Bouwmeester H.J. (2003) Terpenoid metabolism in wild-typeand transgenic Arabidopsis plants. The Plant Cell15, 28662884.

Aharoni A., Jongsma M.A. & Bouwmeester H.J. (2005) Volatilescience? Metabolic engineering of terpenoids in plants. Trends inPlant Science 10, 594602.

Arimura G., Kost C. & Boland W. (2005) Herbivore-induced,indirect plant defences. Biochimica et Biophysica Acta 1734,91111.

Barta C. & Loreto F. (2006) The relationship between the methyl-erythritol phosphate pathway leading to emission of volatileisoprenoids and abscisic acid content in leaves. Plant Physiology141, 16761683.

Bartram S., Jux A., Gleixner G. & Boland W. (2006) Dynamicpathway allocation in early terpenoid biosynthesis of stress-

induced lima bean leaves. Phytochemistry 67, 16611672.Blaakmeer A., Geervliet J.B.F., Van Loon J.J.A., Posthumus M.A.,Van Beek T.A. & De Groot A. (1994) Comparative headspaceanalysis of cabbage plants damaged by two species of Pieriscaterpillars: consequences for in-flight host location by Cotesiaparasitoids. Entomologia Experimentalis et Applicata 73, 175182.

BolandW.,GblerA.,GilbertM.&FengZ.F.(1998)BiosynthesisofC-11 and C-16 homoterpenes in higher plants; stereochemistry ofthe C-C-bond cleavage reaction. Tetrahedron 54, 1472514736.

Bruinsma M., Van Dam N.M., Van Loon J.J.A. & Dicke M. (2007)Jasmonic acid-induced changes in Brassica oleracea affect ovipo-sition preference of two specialist herbivores. Journal of Chemi-cal Ecology 33, 655668.

Buitenhuis R., Vet L.E.M.,Boivin G. & Brodeur J. (2005) Foraging

behaviour at the fourth trophic level: a comparative study of hostlocation in aphid hyperparasitoids. Entomologia Experimentaliset Applicata 114, 107117.

Bukovinszky T., Gols R., Posthumus M.A., Vet L.E.M. & vanLenteren J.C. (2005) Variation in plant volatiles and attraction ofthe parasitoid Diadegma semiclausum (Hellen). Journal ofChemical Ecology 31, 461480.

Cheng A.X., Lou Y.G., Mao Y.B., Lu S., Wang L.J. & Chen X.Y.(2007) Plant terpenoids: biosynthesis and ecological functions.Journal of Integrative Plant Biology 49, 179186.

Copolovici L.O., Filella I., Llusi J., Niinemets . & Peuelas J.(2005) The capacity for thermal protection of photosyntheticelectron transport varies for different monoterpenes in Quercusilex. Plant Physiology 139, 485496.

DAlessandro M. & Turlings T.C.J. (2006) Advances and challenges

in the identification of volatiles that mediate interactions amongplants and arthropods. The Analyst 131, 2432.

DAuria J.C., Pichersky E., Schaub A., Hansel A. & Gershenzon J.(2007) Characterization of a BAHD acyltransferase responsiblefor producing the green leaf volatile (Z)-3-hexen-1-yl acetate in Arabidopsis thaliana. Plant Journal 49, 194207.

De Boer J.G. (2004) Bugs in odour space. How predatory mitesrespond to variation in herbivore-induced plant volatiles. PhDthesis, Wageningen University, the Netherlands.

De Boer J.G. & Dicke M. (2004a) The role of methyl salicylate inprey searching behaviour of the predatory mite Phytoseiuluspersimilis. Journal of Chemical Ecology 30, 255271.

De Boer J.G. & Dicke M. (2004b) Experience with methyl salicy-late affects behavioural responses of a predatory mite to blends

of herbivore-induced plant volatiles. Entomologia Experimenta-lis et Applicata 110, 181189.

De Boer J.G. & Dicke M. (2005) Information use by the predatorymite Phytoseiulus persimilis (Acari: Phytoseiidae), a specialisednatural enemy of herbivorous spider mites.Applied Entomologyand Zoology 40, 112.

De Boer J.G., Posthumus M.A. & Dicke M. (2004) Identification of

volatiles that are used in discrimination between plants infestedwith prey or nonprey herbivores by a predatory mite.Journal ofChemical Ecology 30, 22152230.

De Moraes C.M., Mescher M.C. & Tumlinson J.H. (2001)Caterpillar-induced nocturnal plant volatiles repel conspecificfemales. Nature 410, 577579.

Dicke M. (1994) Local and systemic production of volatileherbivore-induced terpenoids: their role in plant-carnivoremutualism. Journal of Plant Physiology 143, 465472.

Dicke M. (2006) Chemical ecology from genes to communities. InChemical Ecology: From Gene to Ecosystem (eds M. Dicke& W. Takken),pp. 175189.Springer,Dordrecht,the Netherlands.

Dicke M. & van Loon J.J.A. (2000) Multitrophic effects ofherbivore-induced plant volatiles in an evolutionary context.Entomologia Experimentalis et Applicata 97, 237249.

Dicke M. & Van Poecke R.M.P. (2002) Signalling in plantinsectinteractions: signal transduction in direct and indirect plantdefence. In Plant Signal Transduction (eds D. Scheel & C.Wasternack),pp. 289316.Oxford University Press, Oxford, UK.

Dicke M., Maas van der K.J., Takabayashi J. & Vet L.E.M. (1990a)Learning affects response to volatile allelochemicals by preda-tory mites. Proceedings of Experimental & Applied Entomology1, 3136.

Dicke M., van Beek T.A., Posthumus M.A., ben Dom N., vanBokhoven H. & de Groot A.E. (1990b) Isolation and identifica-tion of volatile kairomone that affects acarine predatorpreyinteractions. Involvement of host plant in its production.Journalof Chemical Ecology 16, 381396.

Dicke M., Gols R., Ludeking D. & Posthumus M.A. (1999) Jas-monic acid and herbivory differentially induce carnivore-

attracting plant volatiles in lima bean plants.Journal of ChemicalEcology 25, 19071922.Dicke M., van Loon J.J.A. & de Jong P.W. (2004) Ecogenomics

benefits community ecology. Science 305, 618619.Dudareva N. & Pichersky E. (2006) Metabolic engineering of

floral scent of ornamentals. Journal of Crop Improvement 18,325346.

Dudareva N., Negre F., Nagegowda D.A. & Orlova I. (2006) Plantvolatiles: recent advances and future perspectives. Handbook ofEnvironmental Chemistry,Volume 5:Water Pollution 25, 417440.

Geervliet J.B.F. (1997) Infochemical use by insect parasitoids in atritrophic context:comparison of a generalistand a specialist.PhDthesis, Wageningen University, the Netherlands.

Geervliet J.B.F., Vet L.E.M. & Dicke M. (1994) Volatiles fromdamaged plants as major cues in long-range host-searching by

the specialist parasitoid Cotesia rubecula. Entomologia Experi-mentalis et Applicata 73, 289297.

Geervliet J.B.F., Verdel M.S.W., Snellen H., Schaub J., Dicke M. &Vet L.E.M. (2000) Coexistence and niche segregation by fieldpopulations of the parasitoids Cotesia glomerata and C. rubeculain the Netherlands: predicting field performance from laboratorydata. Oecologia 124, 5563.

Gershenzon J. & Kreis W. (1999) Biochemistry of terpenoids:monoterpenes, sesquiterpenes, diterpenes, sterols, cardiac glyco-sides and steroid saponins. In Biochemistry of Plant SecondaryMetabolism (ed. M. Wink), pp. 222299. Sheffield AcademicPress, Sheffield UK.

Hampel D., Mosandl A. & Wst M. (2005) Induction of de novovolatile terpene biosynthesis via cytosolic and plastidial

584 R. Mumm et al.



11/12

pathways by methyl jasmonate in foliage of Vitis vinifera L.Journal of Agricultural and Food Chemistry 53, 26522657.

Hilker M. & Meiners T. (2006) Early herbivore alert: insect eggsinduce plantdefense.Journalof Chemical Ecology 32, 13791397.

Holm S. (1979) A simple sequential rejective multiple test proce-dure. Scandinavian Journal of Statistics 6, 6577.

Holopainen J.K. (2004) Multiple functions of inducible plant vola-

tiles.Trends in Plant Science

9, 529533.Horiuchi J.I., Arimura G.I., Ozawa R., Shimoda T., Takabayashi J.& Nishioka T. (2003) A comparison of the responses of Tetrany-chus urticae (Acari: Tetranychidae) and Phytoseiulus persimilis(Acari: Phytoseiidae) to volatiles emitted from lima bean leaveswith different levels of damage made by T. urticae or Spodopteraexigua (Lepidoptera: Noctuidae). Applied Entomology andZoology 38, 109116.

Isman M.B. (2006) Botanical insecticides, deterrents,and repellentsin modern agriculture and an increasingly regulated world.Annual Review of Entomology 51, 4566.

Jux A., Gleixner G. & Boland W. (2001) Classification of terpe-noids according to the methylerythritolphosphate or the meva-lonate pathway with natural 12C/13C isotope ratios: dynamicallocation of resources in induced plants. Angewandte Chemie

International Edition 40, 20912093.Keeling C.I. & Bohlmann J. (2006) Genes, enzymes and chemicalsof terpenoid diversity in the constitutive and induced defence ofconifers against insects and pathogens. New Phytologist 170,657675.

Koricheva J., Nyknen H. & Gianoli E. (2004) Meta-analysis oftrade-offs among plant antiherbivore defenses: are plants jacks-of-all-trades, masters of all? American Naturalist163, E64E75.

Krips O.E., Willems P.E.L., Gols R., Posthumus M.A. & Dicke M.(1999) The response of Phytoseiulus persimilis to spider mite-induced volatiles from gerbera: influence of starvation and expe-rience. Journal of Chemical Ecology 25, 26232641.

Leitner M., Boland W. & Mithfer A. (2005) Direct and indirectdefences induced by piercing-sucking and chewing herbivoresin Medicago truncatula. New Phytologist 167, 597606.

Loreto F. & Velikova V. (2001) Isoprene produced by leavesprotects the photosynthetic apparatus against ozone damage,quenches ozone products, and reduces lipid peroxidation of cel-lular membranes. Plant Physiology 127, 17811787.

Mattiacci L., Dicke M. & Posthumus M.A. (1994) Induction ofparasitoid attracting synomone in Brussels sprouts plants byfeeding ofPieris brassicae larvae: role of mechanical damage andherbivore elicitor. Journal of Chemical Ecology 20, 22292247.

Mumm R. & Hilker M. (2005) The significance of backgroundodour for an egg parasitoid to detect plants with host eggs.Chemical Senses 30, 337343.

Mumm R. & Hilker M. (2006) Direct and indirect chemicaldefence of pine against folivorous insects. Trends in Plant Science11, 351358.

Mumm R., Schrank K., Wegener R., Schulz S. & Hilker M. (2003)

Chemical analysis of volatiles emitted by Pinus sylvestris afterinduction by insect oviposition.Journal of Chemical Ecology 29,12351252.

Ouborg N.J. & Vriezen W.H. (2007) An ecologists guide to ecoge-nomics. Journal of Ecology 95, 816.

Owen S.M. & Peuelas J. (2005) Opportunistic emissions of vola-tile isoprenoids. Trends in Plant Science 10, 420426.

Par P.W. & Tumlinson J.H. (1999) Plant volatiles as a defenseagainst insect herbivores. Plant Physiology 121, 325331.

Pettersson E.M. (2001) Volatile attractants for three pteromalidparasitoids attacking concealed spruce bark beetles. Chemoecol-ogy 11, 8995.

Pettersson E.M., Birgersson G. & Witzgall P. (2001) Syntheticattractants for the bark beetle parasitoid Coeloides bostrichorum

Giraud (Hymenoptera: Braconidae). Naturwissenschaften 88,8891.

Pichersky E. & Gershenzon J. (2002) The formation and functionof plant volatiles: perfumes for pollinator attraction and defense.Current Opinion in Plant Biology 5, 237243.

Piel J., Donath J., Bandemer K. & Boland W. (1998) Mevalonate-independent biosynthesis of terpenoid volatiles in plants:

induced and constitutive emission of volatiles.Angewandte

Chemie-International Edition 37, 24782481.Pinto D.M., Blande J.D., Nykanen R., Dong W.X., Nerg A.M. &

Holopainen J.K. (2007) Ozone degrades common herbivore-induced plant volatiles: does this affect herbivore prey locationby predators and parasitoids? Journal of Chemical Ecology 33,683694.

Rodrguez-Concepcion M. (2006) Early steps in isoprenoid biosyn-thesis: multilevel regulation of the supply of common precursorsin plant cells. Phytochemistry Reviews 5, 115.

Schler G., Mithfer A., Baldwin I.T., et al. (2004) Coronalon: apowerful tool in plant stress physiology.FEBS Letters 563, 1722.

Shimoda T., Ozawa R., Sano K., Yano E. & Takabayashi J. (2005)The involvement of volatile infochemicals from spider mites andfrom food-plants in prey location of the generalist predatory

mite Neoseiulus californicus. Journal of Chemical Ecology 31,20192032.Shiojiri K., Takabayashi J., Yano S. & Takafuji A. (2002) Oviposi-

tion preferences of herbivores are affected by tritrophic interac-tion webs. Ecology Letters 5, 186192.

Smid H.M., Van Loon J.J.A., Posthumus M.A. & Vet L.E.M. (2002)GC-EAG-analysis of volatiles from Brussels sprouts plantsdamaged by two species ofPieris caterpillars: olfactory receptiverange of a specialist and a generalist parasitoid wasp species.Chemoecology 12, 169176.

Snoeren T.A.L., De Jong P.W. & Dicke M. (2007) Ecogenomicapproach to the role of herbivore-induced plant volatiles in com-munity ecology. Journal of Ecology 95, 1726.

Takabayashi J. & Dicke M. (1992) Response of predatorymites with different rearing histories to volatiles of uninfested

plants. Entomologia Experimentalis et Applicata 64, 187193.Tripathi A.K., Prajapati V.,Khanuja S.P.S. & Kumar S. (2003) Effectof d-limonene on three stored-product beetles. Journal of Eco-nomic Entomology 96, 990995.

Turlings T.C.J. & Fritzsche M.E. (1999) Attraction of parasiticwasps by caterpillar-damaged plants. InInsectPlant Interactionsand Induced Plant Defence (eds D.J. Chadwick & J.A. Goode),pp. 2132. Wiley & Sons, Chichester, UK.

Turlings T.C.J., Bernasconi M., Bertossa R., Bigler F., Caloz G. &Dorn S. (1998) The induction of volatile emissions in maize bythree herbivore species with different feeding habits: possibleconsequences for their natural enemies. Biological Control 11,122129.

Van Schie C.C.N., Haring M.A. & Schuurink R.C. (2006) Regula-tion of terpenoid and benzenoid production in flowers. Current

Opinion in Plant Biology 9, 203208.Wasternack C., Stenzel I., Hause B., Hause G., Kutter C., Maucher

H., Neumerkel J., Feussner I. & Miersch O. (2006) The woundresponse in tomato role of jasmonic acid. Journal of PlantPhysiology 163, 297306.

Yamasaki T., Sato M. & Sakoguchi H. (1997) (-)-Germacrene D:masking substance of attractants for the cerambycid beetle,Monochamus alternatus (Hope). Applied Entomology andZoology 32, 423429.

Zeidler J., Schwender J., Mller C., Wiesner J., Weidemeyer C.,Beck E., Jomaa H. & Lichtenthaler H.K. (1998) Inhibition of thenon-mevalonate 1-deoxy-d-xylulose-5-phosphate pathway ofplant isoprenoid biosynthesis by fosmidomycin. Zeitschrift frNaturforschung C53, 980986.




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