Plant, Cell and Environment (2008) 31, 575–585 doi: 10.1111/j.1365-3040.2008.01783.

Significance of terpenoids in induced indirect plant

defence against herbivorous arthropods
ROLAND MUMM1, MAARTEN A. POSTHUMUS2 & MARCEL DICKE1

1
Laboratory 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 2006). The volatile fraction of terpenoids predominantly

consists of the hemiterpene isoprene (C5), monoterpenes
Many plants respond to herbivory by arthropods with an
(C10) and sesquiterpenes (C15) and their derivatives such as
induced emission of volatiles such as green leaf volatiles
homoterpenes (C11 and C16). Monoterpenes and sesquiter-
and terpenoids. These herbivore-induced plant volatiles penes are synthesized by the condensation of two or three
(HIPVs) can attract carnivores, for example, predators and isoprene units, respectively (Cheng et al. 2007). Because of
parasitoids. We investigated the significance of terpenoids their physicochemical properties, such as volatility, reactiv-
in attracting herbivores and carnivores in two tritrophic ity, toxicity and aroma, many protective functions against
systems where we manipulated the terpenoid emission by abiotic and biotic factors have been ascribed to terpenoids
treating the plants with fosmidomycin, which inhibits one of (Holopainen 2004). Terpenoids are involved in plant–
the terpenoid biosynthetic pathways and consequently ter- pollinator interactions and have important functions in
penoid emission. plant defence against herbivores (Dicke 1994; Paré &
In the ‘lima bean’ system, volatiles from spider-mite- Tumlinson 1999; Pichersky & Gershenzon 2002; Dudareva
infested fosmidomycin-treated plants were less attractive et al. 2006; Hilker & Meiners 2006; Keeling & Bohlmann
to the predatory mite Phytoseiulus persimilis than from 2006; Mumm & Hilker 2006;Van Schie, Haring & Schuurink
infested control plants. In the ‘cabbage’ system, fosmidomy- 2006; Cheng et al. 2007).
cin treatment did not alter the attractiveness of Brussels It has been demonstrated in numerous studies that the
sprouts to two Pieris butterflies for oviposition. The para- egg deposition or feeding by herbivorous arthropods, such
sitoid Cotesia glomerata did not discriminate between as insects and mites, can induce alterations in the emission
the volatiles of fosmidomycin-treated and water-treated of volatile blends in many plant species (reviewed by Dicke
caterpillar-infested cabbage. Both P. persimilis and C. & van Loon 2000; Dicke & Van Poecke 2002; D’Alessandro
glomerata preferred volatiles from infested plants to unin- & Turlings 2006; Hilker & Meiners 2006; Mumm & Hilker
fested ones when both were treated with fosmidomycin. 2006).These herbivore-induced changes in the volatile com-
Chemical analysis showed that terpenoid emission was position can be exploited by natural enemies of the herbi-
inhibited more strongly in infested lima bean plants than in vores, such as predators and parasitoids, to locate plants
Brussels sprouts plants after fosmidomycin treatment. infested with their prey/host species. The attraction of
This study shows an important role of terpenoids in the natural enemies to herbivore-induced volatiles is accepted
indirect defence of lima bean, which is discussed relative to to be an important mechanism on how plants can indirectly
the role of other HIPVs. defend themselves (e.g. Dicke & van Loon 2000; Arimura,
Kost & Boland 2005; Hilker & Meiners 2006). Induced
Key-words: Brassica oleracea; Phaseolus lunatus; Tetrany- plant volatiles can also act directly against herbivores by
chus urticae; (E)-4,8-dimethyl-1,3,7-nonatriene; headspace repelling them, but alternatively, herbivores can also use
volatile trapping; homoterpenes; monoterpenes; windtun- these to locate suitable host plants (e.g. De Moraes,
nel; Y-tube olfactometer. Mescher & Tumlinson 2001; Shiojiri et al. 2002; Horiuchi
et al. 2003; Bruinsma et al. 2007).
INTRODUCTION The herbivore-induced changes in the volatile blends can
be quantitative, that is, volatiles that are also present in
Plants produce a plethora of volatile compounds, which are non-induced plants are emitted in larger total amounts,
involved in various interactions with their environment. their relative abundance changes, or both (e.g. Mumm et al.
Terpenoids (isoprenoids) represent the largest and the most 2003; Bukovinszky et al. 2005). On the other hand, her-
diverse group of volatiles that are emitted by plants, some- bivory can also induce de novo production of compounds in
times in substantial amounts (Pichersky & Gershenzon many plant species, resulting in qualitative changes in the
2002; Dudareva & Pichersky 2006; Rodríguez-Concepcion composition of the emitted blend (Turlings et al. 1998;
Correspondence: R. Mumm. Fax: 0031317484821; e-mail: Dicke et al. 1999; Krips et al. 1999; Leitner, Boland &
roland.mumm@wur.nl Mithöfer 2005). Typical volatile plant compounds that are
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd 575
576 R. Mumm et al.

induced by herbivory are C6-alcohols, -aldehydes and plant defence against herbivorous arthropods still remain
-acetates (so-called green leaf volatiles, GLVs); methyl to be answered.
salicylate (MeSA); phenylpropanoids; and various monoter- Here, we address the role of terpenes in induced indirect
penes and sesquiterpenes, as well as two homoterpenes, defence by applying fosmidomycin, an inhibitor of the
(E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) and (E,E)- MEP pathway, to plants. The antibiotic fosmidomycin
4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT) (Arimura blocks 1-deoxy-D-xylulose 5-phosphate reductoisomerase,
et al. 2005; Dudareva et al. 2006). GLVs are fatty acid deriva- an enzyme catalysing an early step in the MEP pathway
tives resulting from the conversion of linolenic and linoleic (Zeidler et al. 1998). Fosmidomycin has been shown to
acid released from damaged plasma membranes through the effectively inhibit the biosynthesis and emission of volatile
lipoxygenase pathway (Arimura et al. 2005; D’Auria et al. monoterpenes and partly also sesquiterpenes (Jux, Gleixner
2007).All terpenoids are synthesized via the cytosolic meva- & Boland 2001; Copolovici et al. 2005; Hampel, Mosandl &
lonate (MVA) pathway or the methylerythritol 4-phosphate Wüst 2005; Bartram et al. 2006). This makes it an interesting
(MEP) pathway, which is located in the plastids (Aharoni, tool to study the role of monoterpenes in direct and indirect
Jongsma & Bouwmeester 2005; Rodríguez-Concepcion plant defence.
2006; Cheng et al. 2007). Both terpenoid pathways can We used two tritrophic model systems to study the
synthesize isopentenyl diphosphate (IPP) and its isomer effect of treating plants with fosmidomycin on the behav-
dimethylallyl diphosphate (DMAPP), which are the central iour of herbivores and their natural enemies. In the ‘lima
intermediates for the biosynthesis of terpenes. In a wide bean’ system, lima bean plants, Phaseolus lunatus
range of cyclization and rearrangement steps, precursors are (Fabaceae), are used as host plants for the herbivorous
converted into the parent skeletons of the respective terpe- two-spotted spider mite Tetranychus urticae (Acari, Tet-
nes. Finally, the parent skeletons are converted into the ranychidae). The predatory mite Phytoseiulus persimilis
myriad of different terpenoids by a big variety of transfor- (Acari, Phytoseiidae) was used as carnivorous species. The
mations like oxidations, isomerizations and conjugations ‘cabbage’ system consisted of Brussels sprouts plants,
(Gershenzon & Kreis 1999). Monoterpenes and diterpenes Brassica oleracea (Brassicaceae), which are frequently
are synthesized via the MEP pathway, whereas sesquiterpe- attacked by the specialized large cabbage white butterfly
nes are produced by the MVA pathway. Recently, it was Pieris brassicae and the small cabbage white butterfly
demonstrated that there is some exchange of IPP and Pieris rapae (Lepidoptera, Pieridae). P. brassicae and P.
DMAPP between the two pathways, indicating that the rapae caterpillars are parasitized by the larval parasitoid
pathways are not strictly separated (Rodríguez-Concepcion Cotesia glomerata (Hymenoptera, Braconidae), a gregari-
2006). ous endoparasitoid that prefers to parasitize young larval
Much attention has been paid to the role of monoter- instars of particularly P. brassicae (Geervliet et al. 2000).
penes in plant defence against herbivores, because this It has previously been demonstrated that C. glomerata is
group of terpenes is the most abundant among volatile attracted to caterpillar-infested Brussels sprouts, and P. per-
terpenoids. Many plants, such as conifers, show a high con- similis to volatiles from spider-mite-infested lima bean,
stitutive emission of monoterpenes; others, however, show respectively (Dicke et al. 1990a; Mattiacci, Dicke & Posthu-
a strong induced emission of monoterpenes after her- mus 1994; Geervliet 1997; De Boer 2004). In both plant
bivory. Constitutive and inducible defences are supposed species, volatiles are induced after herbivory. However, in
to be negatively correlated (Dicke & Van Poecke 2002; Brussels sprouts, the changes in terpene emission after
Koricheva, Nykänen & Gianoli 2004). Terpenes may act as herbivore feeding are mainly quantitative (Blaakmeer
feeding deterrents for insects, but also, the oviposition et al. 1994; Mattiacci et al. 1994; Bukovinszky et al. 2005),
behaviour of herbivorous arthropods can be influenced by whereas in lima bean plants, spider mites induce a distinct
the presence or absence of terpenes (Aharoni et al. 2003; de novo production of terpenoids such as (E)-b-ocimene,
Tripathi et al. 2003; Isman 2006). Furthermore, it has been linalool, DMNT and TMTT (Dicke et al. 1990b, 1999; De
shown that terpenes can attract predators and parasitoids. Boer, Posthumus & Dicke 2004; Shimoda et al. 2005).
Many of these studies used single terpenes or mixtures, Recently, Smid et al. (2002) showed that the antennae of
which are then applied to some sort of dispensing mate- C. glomerata respond to limonene, a monoterpene that was
rial. Although this approach might work well in some identified in the headspace of P. brassicae-infested Brussels
cases, in others, it failed. One reason is that by applying sprouts. Furthermore, the predatory mite P. persimilis is
single synthetic compounds, one neglects that background known to behaviourally respond to terpenoids, but MeSA,
odours of the plants play an important role in bringing which is also strongly induced in lima bean by spider mite
terpenes into the right ‘context’ (Pettersson 2001; Petters- feeding, is also strongly attractive to the predatory mites,
son, Birgersson & Witzgall 2001; Buitenhuis et al. 2005; making it difficult to estimate the relative contribution of
Mumm & Hilker 2005). Other studies showed that terpe- terpenoids (Dicke et al. 1990b; De Boer & Dicke 2004a,b).
nes can mask the attractiveness of other plant volatiles to By applying fosmidomycin, we expected that particularly
herbivorous insects and parasitoids (Yamasaki, Sato & the emission of monoterpenes and the homoterpene TMTT
Sakoguchi 1997; Turlings & Fritzsche 1999). This shows is inhibited. Other inducible compounds, like GLVs and
that many questions regarding the role of terpenes in the MeSA, should not be affected by the fosmidomycin treat-
interplay with other plant volatiles in direct and indirect ment. We expected that if terpenoids play a significant role
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 575–585
 Significance of terpenoids in induced indirect plant defence 577

in indirect plant defence, the infested fosmidomycin-treated olfactory choice of predatory mites to two odour sources. A
plants should be less attractive to predators and parasitoids metal wire was positioned at the middle of the olfactometer
than the untreated ones. On the other hand, herbivores are tube. The side arms were both connected to 5 L Duran glass
expected to prefer to lay eggs on plants with less terpenoid jars (Duran, Mainz, Germany) containing the odour sources.
emission. We also collected the headspace volatiles from Pressurized air was filtered over activated charcoal, and
infested Brussels sprouts and lima bean plants after fosmi- was led into the jars at the top, and the jars were left at the
domycin treatment to assess treatment effects on the bottom towards the olfactometer. An airflow in each arm
chemical changes in headspace composition for the two of 4 L min-1 was controlled by a flowmeter (Brooks Instr.,
plant species. Veenendaal, the Netherlands).Air was extracted at the base
of the Y-tube at 8 L min-1. Experiments were conducted at
23 ⫾ 2 °C and 60 ⫾ 5 mmol m-2 s-1 photosynthetically active
MATERIAL AND METHODS radiation (PAR). In order to correct for unforeseen asym-
Plants and arthropods metry in the set-up, the position of the odour sources was
switched after five tested predators. After testing 10 preda-
Plants tors, the odour sources were replaced with new ones. Every
Lima bean plants, P. lunatus L. cv. Sieva (Fabaceae), and experiment was replicated on four different days.
Brussels sprouts plants, B. oleracea var. gemmifera L. cv. To enhance the responsiveness of predatory mites to
Cyrus (Brassicaceae), were grown from seed in separate plant volatiles, all predators had been starved for 2 h prior
greenhouse compartments in plastic pots (11 ¥ 11 ¥ 11 cm) to release in the olfactometer, by confining them individu-
at 24 ⫾ 4 °C, 60 ⫾ 20% relative humidity (RH) and a 16 h ally in Eppendorf tubes (Sarstedt, Nümbrecht, Germany).
light/8 h dark photoperiod. Lima bean plants were used in The tubes with the predators were placed into the experi-
experiments when the primary leaves had fully expanded, mental room to acclimatize to the new environment. Preda-
which was 12–16 d after sowing. Experiments were con- tors were individually released on the iron wire in the basal
ducted with 6- to 8-week-old Brussels sprout plants. tube, and their behaviour was observed for 10 min. Preda-
tory mites that reached a gauze mesh at the middle of the
side arm within this period were recorded as having made a
Herbivores ‘choice’. Mites that did not make a choice were recorded as
A colony of the two-spotted spider mite T. urticae Koch ‘no choice’. Each predator was used only once.
(Acari, Tetranychidae) was maintained on lima bean plants Behavioural choice experiments with the parasitoid C.
in another greenhouse compartment under the same con- glomerata were carried out with a windtunnel set-up
ditions as described for the lima bean culture. A continuous (25 ⫾ 1 °C, 60 ⫾ 10% RH and 35 mmol m-2 s-1 PAR) as
rearing of the large cabbage white, Pieris brassicae L. (Lepi- described by Geervliet, Vet & Dicke (1994). The wind speed
doptera, Pieridae), and the small cabbage white, Pieris was adjusted to 0.2 m s-1. Two-choice experiments were
rapae L. (Lepidoptera, Pieridae), was maintained on conducted by placing a treated leaf and a respective control
Brussels sprouts in a climatized room at 21 ⫾ 1 °C, leaf at the upwind end of the tunnel.
60 ⫾ 10% RH and a 16 h light/8 h dark photoperiod. Naïve C. glomerata females were separated from males
3 h prior to the experiment and were transferred to another
cage, which was placed in the experimental room to accli-
Carnivores matize to the new environment. The wasps were individu-
ally introduced into the windtunnel on an infested cabbage
A colony of the predatory mite P. persimilis Athias-Henriot
leaf piece from which the caterpillars and their products
(Acari, Phytoseiidae) was kept on spider-mite-infested lima
had been carefully removed. The wasps were allowed to
bean leaves in a climate cabinet at 23 ⫾ 1 °C, 60 ⫾ 10%
walk onto the leaf pieces themselves. The leaf piece with the
RH and continuous light. The parasitoid C. glomerata L.
wasp was placed at the middle of the release cylinder, which
(Hymenoptera, Braconidae) was reared on P. brassicae cat-
was 60 cm downwind from the two odour sources. As soon
erpillars feeding on Brussels sprouts under similar environ-
as the parasitoid had left the leaf for some seconds, the leaf
mental conditions as the uninfested cabbage plants. For
was carefully removed using tweezers without disturbing
experiments, C. glomerata pupae were collected and kept in
the parasitoid. The flight behaviour of the wasps was
a cage in a climate cabinet (23 ⫾ 1 °C, 60 ⫾ 10% RH and a
observed. Flights that resulted in a landing on one of the
16 h light/8 h dark photoperiod). Emerging wasps were pro-
two odour sources were recorded as a ‘choice’. Parasitoids
vided with water and honey. Male and female wasps were
that did not leave the release cylinder or landed on other
kept together until the experiment.
parts of the windtunnel within 10 min were recorded as ‘no
choice’. Every parasitoid was used only once. In order to
Behavioural bioassays correct for unforeseen asymmetry in the set-up, the position
of the odour sources was switched after five tested parasi-
Olfactometer and windtunnel tests toids. After testing 10 parasitoids, the odour sources were
A Y-tube olfactometer set-up similar to the one described by replaced with new ones. Every experiment was replicated
Takabayashi & Dicke (1992) was used to investigate the on four different days.
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 575–585
578 R. Mumm et al.

Oviposition preference test 50 L1 P. brassicae caterpillars or were left uninfested.

The leaves were kept in a climate chamber at 23 ⫾ 1 °C,
Freshly emerged P. brassicae and P. rapae adults were each
60 ⫾ 5% RH and a 16 h light/8 h dark photoperiod.
transferred to a large cage (67 ¥ 100 ¥ 75 cm) in a green-
For oviposition experiments with butterflies, both
house compartment at 24 ⫾ 4 °C, 60 ⫾ 20% RH and a 16 h
fosmidomycin-treated and control (water-treated) cabbage
light/8 h dark photoperiod. Butterflies were provided with a
leaves were kept for 24 h in the greenhouse compartment at
10% sucrose solution. Three to five days after emergence,
24 ⫾ 4 °C, 60 ⫾ 20% RH and a 16 h light/8 h dark photo-
one male and one female butterfly were transferred to ovi-
period before being used in the bioassay.
position cages (67 ¥ 50 ¥ 75 cm) in the same greenhouse
compartment. In addition to natural daylight, the cages were
illuminated by sodium vapour lamps (SON-T, 500 W, Philips, Collection of headspace volatiles
Eindhoven, the Netherlands) from 1000 to 1600 h. At 48 h
Volatiles emitted from fosmidomycin-treated lima bean
prior to the experiment, a single untreated Brussels sprouts
and Brussels sprouts leaves were collected using a dynamic
leaf was placed in each cage as an oviposition substrate.After
headspace collection system. The leaves of one treatment
6 h, the leaf was removed. On the experimental day between
were transferred to a 5 L Duran glass jar (Duran). The jar
0900 and 1000 h in the morning, a fosmidomycin-treated
was tightly closed with a glass lid that was pressed on the jar
Brussels sprouts leaf and a respective control leaf were
with a metal clamp with a viton O-ring in between. The lid
introduced into every cage. The treated and the control leaf
had an air inlet and an air outlet. Pressurized air was filtered
always originated from the same plant to minimize intraspe-
over activated charcoal and was led into the jar with a
cific variation between treatment and control. Each butterfly
constant flow of 70 mL min-1. Air was sucked out of the jar
couple was also provided with a 10% sucrose solution. The
with 50 mL min-1 by passing through a glass tube filled with
leaves were placed in an upright position approximately
90 mg Tenax TA (Grace-Alltech, Deerfield, IL, USA) con-
40 cm apart from each other. P. brassicae was allowed to lay
nected to the air outlet of the jar. The slight overpressure
eggs on the two leaves for 24 h, whereas P. rapae could lay
was created to prevent that unfiltered air could invade the
eggs for 6 h. The leaves were then removed, and the eggs
system. The system was purged for 30 min with cleaned air
layed were counted. P. rapae lays individual eggs, which are
before the volatiles were trapped onto the Tenax. The flow
distributed over the leaves. In general, P. rapae starts to
through the jar was controlled by flowmeters (Brooks
deposit eggs soon after being exposed to leaves. Therefore,
Instr.). Teflon tubing was used for all connections. Head-
6 h was sufficient to obtain sufficient eggs. On the other hand,
space collections were made in a climate chamber at
P. brassicae lays egg batches, which can last for several hours.
23 ⫾ 1 °C, 60 ⫾ 5% RH, and 90 ⫾ 5 mmol m-2 s-1 PAR. The
Therefore, we chose to confine P. brassicae for 24 h with the
volatiles of one treatment and its respective control were
cabbage leaves. The experiments were conducted in several
collected simultaneously. Lima bean plants were infested
cages at the same time and on 4–5 d per treatment.
with 20 spider mites per plant. Brussels sprouts were
infested with 50 L1 P. brassicae caterpillars per leaf. Both
Plant treatments
the infested lima bean leaves and Brussels sprouts plants
For all experiments, lima bean leaves or Brussels sprouts were then either placed in vials containing fosmidomycin or
leaves were cut under water. When leaves are cut under water as described previously. The volatiles emitted from
water, a small water droplet normally forms around the lima bean leaves were trapped for 1 h, and for Brussels
petiole, and prevents air invading the vascular system sprouts leaves, trapping time was 5 h.
through the petiole. In this way, the leaves could be trans-
ferred in glass vials filled either with water or a 50 mm
Chemical analysis of headspace samples
aqueous fosmidomycin solution. Fosmidomycin was pur-
chased from Invitrogen Molecular Probes (Breda, the Headspace samples were analysed with a Varian 3400 gas
Netherlands). The openings of the glass vials with the leaves chromatograph (GC) (Varian, Palo Alto, CA, USA) con-
were thoroughly covered with Parafilm (Pekhiney Plastic nected to a Finnigan 95 mass spectrometer (MS) (Thermo
Packing, Chicago, IL, USA). Only leaves that were in visu- Scientific, Waltham, MA, USA). The collected volatiles
ally good condition after the treatment period were used were released from the Tenax by heating the trap in a Ther-
for the experiments. modesorption Cold Trap Unit (Chrompack, Middelburg,
Lima bean leaves were treated either with a 50 mm the Netherlands) at 250 °C for 10 min, and flushing with
aqueous fosmidomycin solution or with water, and then helium at 14 mL min-1. The released compounds were cryo-
were either immediately infested with 20 adult T. urticae or focused in a cold trap 0.52 mm [inner diametre (ID)] deac-
left uninfested. Subsequently, the lima bean plants were tivated fused silica at a temperature of -85 °C. By ballistic
kept in a climate chamber for 48 h at 23 ⫾ 1 °C, 60 ⫾ 5% heating of the cold trap to 220 °C, the volatiles were trans-
RH and a 16 h light/8 h dark photoperiod before being used ferred to the analytical column (60 m ¥ 0.25 mm ID,
in the experiment. The leaves used for the behavioural 0.25 mm film thickness, DB-5 ms J&W, Folsom, CA, USA).
experiments with C. glomerata were treated with fosmido- The temperature programme started at 40 °C (4 min hold)
mycin or with water. The fosmidomycin-treated and water- and rose at the rate of 4 °C min-1 (lima bean) or 5 °C min-1
treated leaves were then immediately either infested with (Brussels sprouts) to 280 °C (4 min hold). The column
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 575–585
 Significance of terpenoids in induced indirect plant defence 579

effluent was ionized by electron impact ionization at 70 eV. where not treated with fosmidomycin, to those from spider-
Mass scanning was carried out from 24 to 300 m/z with a mite-infested fosmidomycin-treated lima bean leaves
scan time of 0.7 s/d and an interscan delay of 0.2 s. The (Fig. 1). Thus, treatment with fosmidomycin reduces the
compounds were identified by comparison of the mass attractiveness of spider-mite-induced lima bean volatiles
spectra with those in the Wiley library and in the Wagenin- to predatory mites. Yet, predatory mites were strongly
gen Mass Spectral Database of Natural Products, and by attracted to the volatiles from fosmidomycin-treated,
checking the retention index. One peak area unit represents spider-mite-infested lima bean when offered against the
approximately 0.17 ⫾ 0.05 ng. volatiles from fosmidomycin-treated uninfested lima bean
plants (Fig. 1). This shows that although fosmidomycin
Statistical analyses reduces the attraction of predators to spider-mite-infested
lima bean leaves, it does not eliminate the emission of
A two-sided binomial test was used to analyse whether the attractive volatiles altogether.
behavioural choices of predatory mites and parasitoids dif- The parasitoid C. glomerata did not discriminate between
fered from a 50:50 distribution over the two odour sources. the volatiles from fosmidomycin-treated and P. brassicae-
Predatory mites and parasitoids that did not make a choice infested cabbage leaves, and the volatiles from control
were excluded from the statistical analysis. Most individuals leaves, that is, water-treated P. brassicae-infested cabbage
of both Pieris species laid eggs on both the control and the leaves (Fig. 2). Thus, fosmidomycin treatment did not
fosmidomycin-treated leaves. The number of eggs on each change the attractiveness of caterpillar-induced cabbage
treatment per individual was considered as a paired sample volatiles to those from the untreated cabbage. C. glome-
and was analysed with the Wilcoxon signed ranks test. rata females significantly preferred the volatiles from
Amounts of headspace volatiles trapped were analysed on fosmidomycin-treated P. brassicae-infested leaves to those
the basis of normalized peak area units, as determined by from fosmidomycin-treated uninfested plants (Fig. 2). This
GC-MS analysis. Mann–Whitney U-tests were applied to shows that the volatiles that are used by the parasitoids to
test for differences between the fosmidomycin-treated and discriminate between infested and uninfested plants are still
control plants for groups of compounds. P values were present after the treatment with fosmidomycin.
adjusted by the sequential Bonferroni method to correct for
the family wise error rate (Holm 1979).
Oviposition preference of cabbage white
butterflies after application of fosmidomycin
RESULTS
The average number of eggs deposited by the large cab-
Behavioural response of predatory mites and
bage white butterfly P. brassicae was not significantly differ-
parasitoids to fosmidomycin-treated plants
ent between fosmidomycin-treated cabbage leaves and
The predatory mite P. persimilis significantly preferred the untreated controls (Wilcoxon signed ranks test, Z = -0.812,
volatiles from spider-mite-infested lima bean leaves, which P > 0.05, n = 22) (Fig. 3). Neither did the small cabbage white

H2O *** Fos 2%

Fos ** Fos 0%

100 50 0 50 100
Predatory mite choices (%)

Figure 1. Effect of fosmidomycin treatment of lima bean plants on the response of Phytoseiulus persimilis to lima bean volatiles. Upper
panel: 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) or
uninfested lima bean treated with 50 mm fosmidomycin solution (white bar) n = 80. The number behind each bar is the percentage of
predatory 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).
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 575–585
580 R. Mumm et al.

H2O n.s. Fos 18%

Fos *** Fos

17%

100 50 0 50 100
Wasps choices (%)

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

butterfly P. rapae discriminate between fosmidomycin- The emission of the monoterpene hydrocarbons (Z)- and
treated and untreated cabbage leaves (Wilcoxon signed (E)-b-ocimene, the oxygenated monoterpene linalool and
ranks test, Z = -0.975, P > 0.05, n = 23) (Fig. 3). The the homoterpene TMTT was completely inhibited by
differences in the average number of eggs laid by the two fosmidomycin in lima bean (Table 1, Fig. 4). The second
species probably result from the differences in the oviposi- homoterpene DMNT was emitted in significantly lower
tion behaviour and the differences in time the butterflies amounts by fosmidomycin-treated lima bean leaves com-
were allowed to oviposit. pared with the water-treated controls (Fig. 4, Table 1).
The only sesquiterpene, that is, (E)-b-caryophyllene, was
emitted in lower amounts by fosmidomycin-treated lima
Analysis of headspace volatiles
bean leaves than by water-treated leaves, but this was not
In the headspace of caterpillar-infested Brussels sprouts statistically significant. The emission of GLVs (hexanal, (Z)-
plants, 31 compounds were detected, and 12 compounds 3-hexen-1-ol, (Z)-3-hexen-1-ol acetate) and MeSA was not
were identified in the headspace of spider-mite-infested significantly affected by fosmidomycin treatment (Fig. 4,
lima bean leaves. Both plant species were treated either Table 1).
with fosmidomycin or with water as control (Table 1). All In contrast to the situation in lima bean, fosmidomycin
compounds that were emitted by fosmidomycin-treated did not inhibit the emission of monoterpenes in Brussels
leaves were also detected in the headspace of control sprouts leaves significantly (Fig. 5, Table 1). However,
leaves. Therefore, the compounds that were detected in at fosmidomycin-treated Brussels sprouts leaves emitted
least half of the control samples are depicted in Table 1. lower amounts of monoterpenes than the water-treated
Herbivore-induced volatiles of excised leaves of lima bean leaves. Furthermore, the emission of GLVs was reduced in
and Brussels sprouts resembled those emitted by whole fosmidomycin-treated plants although there was a strong
plants (Bukovinszky et al. 2005; Pinto et al. 2007). variability in the emission of these compounds (Fig. 5,

80 N = 22
average number of laid eggs

n.s.

60
N = 23
n.s.
40
Figure 3. Oviposition of Pieris brassicae
and Pieris rapae on Brussels sprout plants
20 either treated with 50 mm fosmidomycin
(black bars) or left untreated (white
bars). Mean number of eggs per
0 female + standard error (n.s., P > 0.05,
Pieris brassicae Pieris rapae Wilcoxon signed ranks test).

© 2008 The Authors

Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 575–585
 Significance of terpenoids in induced indirect plant defence 581

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 (54–103) 105 (21–206) nd nd

2 3-Pentanone 35 (20–207) 131 (27–249) nd nd
3 3-Pentanol 56 (40–123) 128 (83–157) nd nd
4 2-Methylbutanal nd nd 0 (0–3) 5 (0–8)
5 3-Methylbutanal nd nd 9 (6–12) 14 (6–20)
6 (Z)/(E)-2-Penten-1-ol 2 (1–4) 7 (4–14) nd nd
7 (Z)-3-Hexenal 12 (6–49) 34 (34–95) nd nd
8 Hexanal 8 (7–9) 12 (4–16) 1 (0–4) 8 (2–8)
9 (E)-2-Hexenal 0 (0–6) 7 (0–45) nd nd
10 (Z)-3-Hexen-1-ol 237 (203–884) 650 (263–899) 1 (0–5) 1 (1–2)
11 2-Methyl-2-cyclopenten-1-one 0 (0–2) 5 (0–8) nd nd
12 (Z)-2-Penten-1-ol acetate 79 (71–85) 117 (71–199) nd nd
13 Pentyl acetate 3 (2–3) 4 (0–5) nd nd
14 a-Thujene 110 (67–130) 158 (54–178) nd nd
15 a-Pinene 25 (20–37) 44 (12–83) nd nd
16 3-Ethyl-1,5-octadiene 6 (6–7) 10 (8–13) nd nd
17 Sabinene 437 (271–546) 716 (207–841) nd nd
18 b-Pinene 27 (17–33) 39 (10–80) nd nd
19 Myrcene 103 (62–116) 151 (94–201) nd nd
20 (Z)-3-Hexen-1-ol acetate 2630 (2500–2855) 3680 (2930–5320) 20 (0–41) 33 (0–83)
21 Hexyl acetate 42 (21–48) 36 (33–79) nd nd
22 Limonene 258 (158–284) 343 (269–547) nd nd
23 b-Phellandrene 4 (2–4) 10 (5–12) nd nd
24 1,8-Cineole 174 (98–181) 170 (104–249) nd nd
25 (Z)-b-Ocimene nd nd 0 (0–0) 18 (14–59)
26 Phenylacetaldehyde 0 (0–1) 7 (0–12) nd nd
27 (E)-b-Ocimene nd nd 0 (0–0) 280 (210–958)
28 Salicylaldehyde 0 (0–0) 3 (0–6) nd nd
29 g-Terpinene 0 (0–0) 5 (3–17) nd nd
30 trans-4-Thujanol 0 (0–5) 11 (0–19) nd nd
31 Linalool nd nd 0 (0–0) 31 (11–32)
32 Nonanal 16 (8–18) 58 (17–81) nd nd
33 (E)-4,8-dimethyl-1,3,7-nonatriene nd tr 57 (15–84) 573 (405–820)
34 2-Methyl-6-methylene-1,7-octadiene-3-one 0 (0–2) 5 (0–13) nd nd
35 Methyl salicylate 5 (4–7) 5 (3–12) 3 (2–77) 103 (30–123)
36 a-Gurjunene 4 (3–5) 3 (0–30) nd nd
37 Longifolene 7 (6–8) 6 (0–10) nd nd
38 (E)-b-Caryophyllene nd nd 2 (0–3) 10 (8–18)
39 (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene nd nd 0 (0–0) 51 (42–67)

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.

Table 1). The homoterpene DMNT was only detected in TMTT, which is a derivative of the diterpene geranyllina-
trace amounts in a few samples, and TMTT was not found in lool, to be inhibited (Boland et al. 1998).
the headspace of Brussels sprouts (Table 1). The concentration of fosmidomycin we used was shown
to be effective in inhibiting isoprenoid emission when fed
through the petiole (Barta & Loreto 2006; Bartram et al.
DISCUSSION
2006). The incubation period with fosmidomycin differed
The treatment with fosmidomycin affected the terpene between 24 h in Brussels sprouts and 48 h in lima bean,
emissions in lima bean and to a lesser extent in Brussels because we knew from previous experiments that parasi-
sprouts (Figs 4 and 5). Likewise, the effects on carnivore toids and predatory mites are attracted to induced volatiles
behaviour were more pronounced in the lima bean than in after 24 and 48 h of infestation, respectively. It is not likely
Brussels sprouts (Figs 1 and 2). We used fosmidomycin to that this difference in incubation time is responsible for the
block the emission of terpenoids that are synthesized via differences in inhibition because fosmidomycin can effec-
the plastidial MEP pathway. Therefore, we expected espe- tively inhibit the isoprenoid emission already after a few
cially the emission of monoterpenes and the homoterpene hours (Loreto & Velikova 2001; Barta & Loreto 2006;
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 575–585
582 R. Mumm et al.

700
*
600
Norm. peak area units

500

400
*
Figure 4. Emission of volatile
300 compound classes by lima bean plants
infested with 50 Tetranychus urticae
200 caterpillars and that were either treated
n.s. n.s.
with 50 mm fosmidomycin (grey bars) or
n.s. n.s. * left untreated (white bars). Weighted
100 mean and standard error of normalized
peak area units are given. n.s., P > 0.05;
*P > 0.05, Mann–Whitney U-test, P
0
Green leaf Monoterpenes Caryophyllene DMNT TMTT Methyl salicylate Methylbutanal values are corrected by sequential
volatiles Bonferroni method (Holm 1979).

Bartram et al. 2006). In lima bean leaves, the emission of no DMNT is induced in lima bean plants by herbivory or treat-
other compound except for terpenoids was significantly ment with jasmonic acid (JA), the increased demand of
affected by fosmidomycin treatment compared with the precursors is covered by the MEP pathway (Piel et al. 1998;
controls, suggesting that the plants did not severely suffer Jux et al. 2001). Our results show that in spider-mite-
from the treatment. infested lima bean plants, the MVA pathway is not able to
The headspace data for lima bean plants indicate that the fully compensate for a lack of precursors from the MEP
herbivore-induced de novo production of monoterpenes pathway after fosmidomycin treatment, resulting in a
and TMTT relies completely on the MEP pathway. Inter- reduced emission of DMNT and (E)-b-caryophyllene
estingly, the allocation of precursors of DMNT (produced (Fig. 4). In turn, this indicates that spider mite feeding on
through the cytosolic MVA pathway) is more plastic lima bean does not induce the MVA pathway, but particu-
(Bartram et al. 2006). When plants are not stressed, precur- larly the MEP pathway, similar to what was demonstrated
sors of mainly the MVA pathway but partly also from the for JA treatment. However, Jux et al. (2001) found no
MEP pathway are assembled into DMNT. If the supply of significant reduction in DMNT emission after JA and
precursors from the MEP pathway was blocked, this did fosmidomycin treatment, suggesting that the mechanisms in
not reduce the constitutive emission of DMNT because of terpenoid induction and regulation in response to spider-
an increasing assemblage of MVA-derived precursors mite infestation and JA application are similar but not iden-
(Bartram et al. 2006). However, when the emission of tical (see also Dicke et al. 1999).

n.s.
1000
Norm. peak area units

800

600
Figure 5. Emission of volatiles
compound classes by Brussels sprouts
400 plants infested with 50 Pieris brassicae
n.s. n.s. n.s. n.s.
caterpillars and that were either treated
with 50 mm fosmidomycin (grey bars) or
200 left untreated (white bars). Weighted
mean and standard error of normalized
peak area units are given. n.s., P > 0.05,
0 Mann–Whitney U-test, P values are
Green leaf Monoterpenes Sesquiterpenes Methyl salicylate Others corrected by sequential Bonferroni
volatiles method (Holm 1979).
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 575–585
 Significance of terpenoids in induced indirect plant defence 583

The lima bean system control leaves. Both P. brassicae and P. rapae did not
discriminate between fosmidomycin-treated and water-
The predatory mite P. persimilis significantly preferred the treated cabbage leaves (Fig. 3). Neither did the treatment
volatiles of water-treated, infested lima bean plants to those with fosmidomycin reduce the attractiveness of P. brassicae-
of fosmidomycin-treated ones (Fig. 1). On the other hand, induced cabbage volatiles to C. glomerata (Fig. 2). In con-
the volatiles from spider-mite-infested plants were still trast to lima bean plants, the emission of terpenoids in
more attractive to predatory mites compared with the unin- cabbage leaves was not significantly inhibited by fosmido-
fested ones when both were treated with fosmidomycin mycin. Thus, a role of terpenoids in the direct or indirect
(Fig. 1). This shows that the absence of certain terpenoids in defence of Brussels sprouts against Pieris butterflies cannot
the headspace makes infested lima bean clearly less attrac- be excluded by this study. Future studies should elucidate
tive to predatory mites, but still other chemical cues than the regulation of constitutive and induced terpenoid pro-
terpenoids are apparently used to discriminate between duction, for example, by applying combinations of different
infested and uninfested lima beans. It has been shown that inhibitors, for example, fosmidomycin and cerivastatin.
MeSA plays a crucial role in the attraction of predatory
mites (Dicke et al. 1999; De Boer & Dicke 2004a,b). MeSA
is induced after spider-mite infestation (e.g. Dicke et al. CONCLUSION
1999), but as expected, fosmidomycin did not affect its emis- In conclusion, this study demonstrates that inhibitors
sion (Fig. 4). De Boer et al. (2004) showed that offering like fosmidomycin can be used to investigate the role of
MeSA as an alternative odour source reduced the prefer- terpenoid infochemicals in plant defence mechanisms
ence of predatory mites to spider-mite-infested lima bean against herbivores. In comparison with compounds induc-
volatiles. In addition, when MeSA was added to the volatile ing plant defence responses, such as coronalon or jas-
blends induced by JA or feeding by Spodoptera exigua, monates (Schüler, Mithöfer & Baldwin 2004; Wasternack
which were similar to spider-mite-induced volatiles but et al. 2006), inhibitors have been applied far less in studies
lacking MeSA, they became more attractive to predatory addressing indirect plant defence. Although many of the
mites than those of spider-mite-induced plants. The attrac- inhibitors are specific for a certain biosynthetic pathway,
tive effect of MeSA was thereby based on qualitative dif- they may not specifically inhibit particular chemical com-
ferences rather than quantitative changes (De Boer & pounds as biochemical pathways generally have more than
Dicke 2004a,b, 2005; De Boer et al. 2004). one final product. In the case of fosmidomycin, one should
Despite the presence of MeSA, predatory mites pre- be aware that not only volatile terpenoids are inhibited but
ferred the volatiles of untreated lima bean leaves to also other ‘essential’ terpenoids, which are important mem-
fosmidomycin-treated ones, thus demonstrating the rela- brane components, photosynthetic pigments, or antioxi-
tive importance of terpenoids for the predatory mites. dants, such carotenoids, sterols and gibberellins, might be
P. persimilis and other predatory mites are attracted affected as well (Owen & Peñuelas 2005).Therefore, experi-
to several synthetic terpenoids, such as linalool, (E)-b- ments studying the significance of volatile terpenoids
ocimene or DMNT, when offered individually (e.g. Dicke should use rather short incubation times.
et al. 1990b; Shimoda et al. 2005). Interestingly, TMTT, Future studies need to elucidate what the role of particu-
although not attractive to predatory mites as a pure syn- lar compounds is in attracting carnivorous arthropods. One
thetic compound, did affect the attractiveness of a mixture approach would be to combine ecological and molecular
of herbivore-induced lima bean plants (De Boer et al. tools by using plants that have been genetically modified in
2004). This suggests that the role of terpenoids in indirect the emission of certain terpenoids. This new field of ecoge-
plant defence depends on the presence and composition of nomics has been successfully developed in the last few years
certain background volatiles as was also demonstrated for and provides promising opportunities on the way to under-
other tritrophic systems. For example, Mumm & Hilker stand how indirect plant defence mechanisms function
(2005) showed that the egg parasitoid Chrysonotomyia (Dicke, van Loon & de Jong 2004; Dicke 2006; Ouborg &
ruforum (Hymenoptera, Eulophidae) only responded to Vriezen 2007; Snoeren, De Jong & Dicke 2007).
the combination of the sesquiterpene (E)-b-farnesene and
background volatiles of pines.The parasitoid’s response was
dependent on the concentration of (E)-b-farnesene, sug- ACKNOWLEDGMENTS
gesting that this parasitoid uses the contrast between (E)- We would like to thank Rieta Gols and Gabriella
b-farnesene and the background odour, that is, when the Bukovinszkine’Kiss for the help with the experiments.
compound was experienced in the ‘right’ chemical context Many thanks to Leo Koopman, Frans van Aggelen and
(Mumm & Hilker 2005, 2006; Hilker & Meiners 2006). André Gidding for culturing the insects and mites, and the
experimental farm of Wageningen University (Unifarm) for
rearing the Brussels sprout plants. We also thank two
The cabbage system
anonymous reviewers for their highly valuable comments.
In oviposition choice experiments, we tested whether P. The study was financially supported by the European
brassicae or P. rapae avoided or preferred intact Brussels Commission contract MC-RTN-CT-2003-504720 ‘ISONET’
sprout leaves treated with fosmidomycin to water-treated and by a VICI grant (nr 865.03.002) from the Earth and Life
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 575–585
584 R. Mumm et al.

Sciences Foundation, which is subsidized by the Nether- of herbivore-induced plant volatiles. Entomologia Experimenta-
lands Organization for Scientific Research. lis et Applicata 110, 181–189.
De Boer J.G. & Dicke M. (2005) Information use by the predatory
mite Phytoseiulus persimilis (Acari: Phytoseiidae), a specialised
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Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 575–585