Agriculture, Ecosystems and Environment 233 (2016) 352–360

Contents lists available at ScienceDirect

Agriculture, Ecosystems and Environment

journal homepage: www.elsevier.com/locate/agee

Plant-plant interactions affect the susceptibility of plants to oviposition

by pests but are disrupted by ozone pollution
Patricia S. Girón-Calva* , Tao Li, James D. Blande
Department of Environmental and Biological Sciences, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland

A R T I C L E I N F O A B S T R A C T

Article history:
Received 22 June 2016 Plant volatiles convey information about the physiological status of a plant to other organisms, including
Received in revised form 9 September 2016 neighboring plants and herbivores searching for a suitable oviposition site. Tropospheric air pollutants,
Accepted 22 September 2016 such as ozone, can impair plant–plant interactions, e.g. by degrading volatile signals. In this study, field
Available online 3 October 2016 and laboratory experiments were conducted to assess whether plant–plant interactions between
damaged and undamaged cabbage (Brassica oleracea var. capitata) plants provide receivers with a
Keywords: competitive advantage by altering their susceptibility to oviposition by the specialist herbivores Pieris
Brassica oleracea brassicae and Plutella xylostella. We tested the phenomenon in oviposition choice tests under ambient and
Volatile organic compounds
enriched ozone conditions. We also investigated the effect of receiving a signal from a different Brassica
Air pollution
oleracea cultivar group, in this study broccoli (Brassica oleracea var italica), on the susceptibility to
Pieris brassicae
Plutella xylostella oviposition of cabbage plants. Cabbage plants exposed to damaged cabbage neighbors under ambient
ozone were less susceptible to oviposition by P. brassicae in the field, but not in the laboratory. At ambient
ozone levels P. xylostella preferred to oviposit on cabbage plants exposed to undamaged conspecifics in
both the field and laboratory. At enriched ozone levels in the field, P. xylostella showed no oviposition
preference for cabbage exposed to damaged or undamaged cabbage neighbors. In multiple choice tests in
the laboratory, P. xylostella preferred to oviposit on cabbage plants exposed to damaged broccoli plants at
ambient ozone over plants exposed to undamaged broccoli plants at ambient ozone and damaged and
undamaged broccoli plants at enriched ozone. We conclude that plant–plant interactions and ozone
interplay when influencing the susceptibility of plants to oviposition by herbivores.
ã 2016 Elsevier B.V. All rights reserved.

1. Introduction
Plants are continuously exposed to multiple biotic and abiotic
stressors and respond by altering their emission of volatile organic
compounds (VOCs) (Holopainen and Gershenzon, 2010). VOCs play
several important roles in the ecology of plants. It is well known
that they function as direct plant defenses by repelling or
inhibiting the growth of detrimental herbivores and pathogens
(De Moraes et al., 2001; Egigu et al., 2010; Quintana-Rodriguez
et al., 2015), and they also mediate indirect defenses by attracting
natural enemies of herbivores such as predatory arthropods and
parasitic wasps, which may reduce damage caused by herbivores
(Bruinsma et al., 2009; Turlings et al., 1995, 1990). In addition, VOCs
function as volatile signals between and within plants (Heil and
Silva Bueno, 2007; Heil and Karban, 2010; Kost and Heil, 2006). The
volatile signals convey information on the physiological condition
* Corresponding author.
E-mail address: sarai.gironcalva@uef.fi (P.S. Girón-Calva).
of the emitter and neighboring receivers can respond to the signals
by adjusting their defenses accordingly (Kost and Heil, 2006;
Quintana-Rodriguez et al., 2015; Yi et al., 2009).
Volatile-mediated interactions between plants can have either
active or passive mechanisms. Active plant–plant interactions
occur when the volatile signal elicits a response by the receiver
(Sugimoto et al., 2014; Zebelo et al., 2012) such as the induction of a
genetic and/or physiological change (Arimura et al., 2000, 2001;
Ton et al., 2007; Yi et al., 2009). Active plant–plant interactions
often result in receiver plants having primed defenses, which allow
them to respond faster and stronger once attacked (Heil and Silva
Bueno, 2007; Ton et al., 2007). Passive plant–plant interactions
occur when the volatile chemicals adsorb to the surface of
neighboring plant foliage (Himanen et al., 2010, 2015; Li and
Blande, 2015). Receiver plants with a chemically-altered surface
may benefit by becoming less attractive to detrimental organisms
such as herbivores (Himanen et al., 2010). Passive interactions,
however, have also been shown to increase the susceptibility of
receivers to oviposition by herbivores (Li and Blande, 2015).

http://dx.doi.org/10.1016/j.agee.2016.09.028
0167-8809/ã 2016 Elsevier B.V. All rights reserved.
 P.S. Girón-Calva et al. / Agriculture, Ecosystems and Environment 233 (2016) 352–360
Plant volatiles can be used as olfactory cues by herbivores
during searching for suitable plants as hosts for their eggs (Hilker
and Fatouros, 2015). Plant volatiles can potentially provide
information to gravid females about the plant nutritional quality,
infestation history, and presence of other insects or attractiveness
to natural enemies (Bruce and Pickett, 2011; Choh et al., 2008;
Shiojiri et al., 2002). Assessment and final acceptance of a host
plant for oviposition is based on these factors, although other
factors, such as egg load of the female, can also influence
oviposition-site selection (Javois and Tammaru, 2006; Soler
et al., 2010). Recent studies under laboratory conditions have
shown that volatiles from neighboring plants can strongly affect
the attractiveness of an individual plant to ovipositing herbivores.
Broccoli plants (Brassica oleracea var italica (cv. Lucky)) exposed to
volatiles from Rhododendron tomentosum, for instance, were less
susceptible to oviposition by P. xylostella females (Himanen et al.,
2015). However, Li and Blande (2015) observed under laboratory
conditions that broccoli plants exposed to herbivore-damaged
conspecifics were more susceptible to oviposition by P. xylostella.
These results show that volatile-mediated plant–plant interactions
can modify the attractiveness of receiver plants to herbivorous
insects. Oviposition represents a threat to the plant since eggs will
develop into voracious feeding larvae. Although plants can trigger
aggressive defense responses against deposited insect eggs
(Reymond, 2013), perception of a volatile signal from neighboring
plants that alters their phenotype to become less susceptible to
subsequent oviposition, would provide them with a competitive
advantage and reduce the risk of future herbivory.
It is important to note that most studies to have assessed the
benefits to plants of exposure to volatile chemicals from
neighboring plants have been conducted at small-scales in
laboratories, and thus may not fully reflect the scenario under
field conditions (Kaplan, 2012). In the field, plants are exposed to
multiple biotic and abiotic factors that may potentially interfere
with volatile-mediated interactions (Holopainen and Gershenzon,
2010; Blande et al., 2014). Several studies in the last few years have
linked oxidizing and phytotoxic air pollutants, such as ozone, to
reductions in the effectiveness or durability of a range of
interactions (Blande et al., 2014; McFrederick et al., 2009). Ozone
is naturally present in the atmosphere, however its concentration
in the troposphere has increased and is projected to rise in the
coming decades given climate change (Wilkinson et al., 2012).
Fuentes et al. (2013) showed that tropospheric ozone significantly
reduces the ability of striped cucumber beetles (Acalymma
vittatum) to find their host plant. Ozone has also been shown to
affect plant–plant interactions; Blande et al. (2010) showed that
the distance over which plant–plant signaling occurs under
laboratory conditions is significantly reduced by ozone. Similarly,
Li and Blande (2015) showed that disruption of plant–plant
interactions by ozone led to a decrease in the associational
susceptibility of broccoli plants mediated by the passive adsorp-
tion of sesquiterpenes. Tropospheric ozone has the potential to
impact on plant–plant interactions at different junctures, firstly, it
can induce or reduce plant volatile emissions depending on the
exposure regime (Himanen et al., 2009; Vuorinen et al., 2004).
Secondly, ozone degrades several VOCs travelling in the air, such as
green leaf volatiles (GLVs) (Pinto et al., 2007a) and terpenes (Pinto
et al., 2007b), which are among the key compounds to have been
identified as mediators of plant–plant signaling (Zebelo et al.,
2012). Ozone may also impair the ability of the receiver plants to
detect the volatile signals, or degrade passively adsorbed volatiles
on the leaf surface or in the leaf boundary layer (Li and Blande,
2015).
In this study, we used a system comprising cabbage (Brassica
oleracea var capitata), broccoli (Brassica oleracea var italica), the
large cabbage white butterfly (Pieris brassicae) and the
353
diamondback moth (Plutella xylostella) to investigate if
plant–plant interactions mediated by herbivore-induced VOCs
lead to receiver plants gaining resistance to oviposition by
herbivores. We conducted experiments under field and laboratory
conditions and also investigated the effects of ozone pollution on
the interactions. Specifically, we addressed the following
questions: 1) do undamaged cabbage (receiver plants) gain
resistance to oviposition by P. brassicae after exposure to P.
brassicae-damaged cabbage (emitter plant) neighbors? 2) Does
ozone exposure have an impact on interactions between cabbage
plants, and by doing so affect the oviposition choices of P.
brassicae? 3) Do plant–plant interactions mediated by VOCs
induced by one herbivore species (P. brassicae) have an effect on
the oviposition choices of a second herbivore species (P. xylostella)?
And 4) does elevated ozone affect that interaction? We further
addressed the direct role of ozone exposure on the susceptibility of
plants to oviposition. And finally asked 5) does exposing cabbage
receiver plants to P. brassicae-damaged broccoli plants, result in a
difference to the oviposition choices by P. xylostella? This study was
structured around two central hypotheses: first, that plants
exposed to VOCs from damaged neighbors gain resistance to
oviposition by specialist herbivores, and second that enriched
ozone levels eliminate the resistance to oviposition.
2. Materials and methods
2.1. Plants and insects
Brassica oleracea L. var. capitata cv. Lennox (white cabbage)
(Bejo Zaden b.v. Trambaan 1, 1749 CZ Warmenhuizen, Holland) and
B. oleracea L. var. italica cv. Lucky (broccoli) (Bejo Zaden b.v.
Trambaan 1, 1749 CZ Warmenhuizen, Holland) seeds were sown in
individual 0.8-L pots filled with a mix of peat, soil and sand (3: 1:
1). Seeds were germinated and grown in a greenhouse for
experiments 1 and 2, and in a controlled-environment room
(20
C, 70 mmol m2 s1 photosynthetically active radiation (PAR),
16L: 8D) for experiments 3, 4 and 5. Plants were watered daily and
fertilized twice per week with 80 mL of a solution containing
nitrogen, phosphorus and potassium (19-4-20) 0.1% (Taimi-
Superex, Kekkilä Oyj, Eurajoki As, Finland). Experiments were
started five weeks after seeds were sown and plants had at least
four fully expanded secondary leaves.
Pieris brassicae were reared full cycle on B. oleracea var.
gemmifera (Brussels sprouts) under controlled conditions of
20
C and a light: dark cycle of 16L: 8D. Third instar larvae were
used to induce VOC emissions in the emitter plants in both
laboratory and field experiments. Female and male adults of P.
brassicae were placed together in a cage to allow mating and
provided with water and a 30% honey solution, both soaked into
cotton wool, for four days. Female adults were used in the
oviposition assays.
Plutella xylostella were reared full cycle on broccoli plants. Only
P. xylostella adults were used for the experiments. Adults were
collected together in a cage to allow mating and were provided
with water and a 30% honey solution, both soaked into cotton wool
for three days. The adults used in the oviposition tests were
assumed to include equal proportions of females and males.
2.2. Ozone—Free Air Concentration Enrichment (FACE) facility
Experiments in the field were conducted at the ozone FACE
facility (62
130 N, 27
350 E, 80 m s l.) of the Kuopio campus of the
University of Eastern Finland (UEF). The FACE facility allows the
enrichment of ozone levels in a specific area relative to real-time
recorded ambient values (Blande et al., 2007). The facility has four
control plots with ambient ozone concentrations and four plots
354 P.S. Girón-Calva et al. / Agriculture, Ecosystems and Environment 233 (2016) 352–360
with enriched ozone concentrations. Each plot includes two
subplots. Ambient ozone levels were measured at a central point in
the control plots and then ozone levels in enriched plots were
enriched to 1.5 times the ambient level (Karnosky et al., 2007;
Pinto et al., 2008). An ozone generator (Fisher OZ 500, Meck-
enheim, Bonn, Germany) was used to produce ozone from pure
oxygen, which was infused into the plots through vertical vent
pipes during daylight hours (08:00–22:00). Ozone concentrations
were continuously monitored and logged with a UV photometric
ozone analyzer (Model O342 Module, Environment S.A., Poissy,
France) (see Fig. 1; for other environmental data such as wind
speed, wind direction, temperature, photosynthetically active
radiation (PAR), precipitation and relative humidity (RH) see
Fig. SM1 in the Supplementary material). During the field
experiments in the summer of 2012, ozone enrichment did not
occur due to technical failure.
Fig. 1. Daily mean ozone concentrations for daylight hours (08:00–22:00) for the
exposure periods (A) 2012, (B) 2014 and (C) 2015. Ozone concentrations are given in
parts per billion (ppb). Due to technical failure, data logging and enrichment of
ozone did not occur in 2012. The data presented in (A) is the ambient ozone data
obtained from the Finnish Meteorological Institute Kasarmipuisto weather station
in Kuopio (the institute is located 3 km from the field site) and can be freely accessed
at www.ilmanlaatu.fi (Air Quality in Finland).
2.3. Controlled environment chambers and ozone exposure system
Experiments were conducted in controlled-environment cham-
bers (120 cm length (L)  75 cm width (W)  130 cm height (H));
Weiss Bio 1300, Weiss Umwelttechnik Gmbh, Reiskirchen-
Lindenstruth, Germany), which had a running programme of
16L:8D, 23
C/18
C day/night, 80/60% RH day/night with a 30-min
gradual shift between day and night conditions. The chambers had
a horizontal laminar air flow with a mean flow speed of 0.4 m s1.
Four chambers were used concurrently for the experiments. In two
chambers, the ozone levels were elevated using an ozone system
fitted to the chambers. Ozone was generated from room air with an
ozone generator (Biltema, Art. 39120; Biltema Nordic Services AB)
and then passed through Teflon tubing to a custom-built control
unit. Ozone flow into each chamber was controlled with valves, the
opening and closing of which was regulated by the control unit
based on internal ozone measurements logged with an ozone
analyzer (Environnement SA; O342 M; Poissy, France). Ozone
concentrations were independently controlled in each chamber.
Enriched ozone levels were maintained at 80 parts per billion (ppb)
during the daylight hours (08:00–22:00), dropping to 30 ppb at
night. In the other two chambers, the room ozone level
(approximately 10 ppb) was used (Blande et al., 2010).
2.4. Experimental design
Experiments were conducted in two phases: (1) VOC-exposure,
where undamaged receiver cabbage plants were exposed to an
emitter plant, with exposures conducted under ambient ozone and
enriched ozone conditions in the field and laboratory; and (2)
oviposition preference tests for differently treated receiver plants
with mated P. brassicae females or P. xylostella adults in the field
and laboratory. There is some variation in the methodologies used
for different experiments, which was necessary to accommodate
for several unforeseen circumstances, including a technical
malfunction in one field experiment, low availability of P. brassicae
larvae, and weather conditions that were on occasion unsuitable
for oviposition assays. Each experiment is presented in detail
below, with a summary of key methodological points in Table 1.
2.4.1. Experiment 1—do cabbage plants gain resistance to oviposition
by P. brassicae after receiving a volatile signal from damaged
neighbors in the field?
Phase 1: Plant exposure in the field—in 2012 (7–Jul–19–Jul)—Ten
cabbage plants were randomly assigned to each of two subplots
inside each of the four enriched-ozone and four ambient ozone
FACE plots. Potted plants were used to avoid root contact between
emitter and receiver plants, which were placed in pairs with a
distance of 30 cm between the plant stems. One plant in each pair
was assigned as a VOC-emitter and the other as a VOC-receiver.
One subplot in each plot was assigned to having herbivore-infested
VOC-emitter plants which were subjected to a combination of
herbivore-damage (six larvae of P. brassicae) and mechanical injury
(due to limited insect availability the four biggest leaves of each
plant were crushed 6 times with forceps). No damage was done to
the VOC-emitter plants in the second subplot, which was a control.
VOC-receiver plants remained undamaged in all experiments.
From here on, undamaged plants exposed to volatiles from
damaged plants are denoted as ir-VOC plants, while undamaged
plants exposed to undamaged plants (controls) are denoted as cr-
VOC plants (see Fig. SM2 and SM3 in the Supplementary material
for a representative chromatogram of the constitutive VOCs
emitted by cabbage and the VOC profile emitted by P. brassicae-
infested cabbage plants, respectively). After three days of VOC
exposure, the VOC-receiver plants were used in oviposition
preference tests. Due to technical failure, ozone concentrations
 P.S. Girón-Calva et al. / Agriculture, Ecosystems and Environment 233 (2016) 352–360
Table 1
Summary of treatments. For a complete description of the methodology followed in each experiment see the Section Materials and methods.
Experi-ment Emitter Damage agent Receiver
plant plant
1 Cabbage P. brassicae + mechanical Cabbage
wounding
2 Cabbage P. brassicae Cabbage
3 Cabbage P. brassicae Cabbage
4 Cabbage P. brassicae Cabbage
5 Broccoli P. brassicae Cabbage
a
ppb, parts per billion.
b
Daily mean ozone concentrations in the field are depicted in Fig. 1.
c
All laboratory experiments were performed at ambient conditions (ambient ozone levels 10 ppb), while in the field, oviposition experiments were performed in the same
location where the exposure to VOCs took place, in either ambient or enriched ozone levels.
were not enriched in this experiments. Therefore, the whole
experiment was considered to have been conducted at ambient
ozone levels and all receiver plants were used during phase 2.
Phase 2: Oviposition preference of P. brassicae—cr-VOC and ir-VOC
cabbage plants from phase 1 were paired in polycarbonate cages
(60 cm L  33 cm W  33 cm H) in a controlled environment room
(light: dark cycle 16L: 8D and 20
C). Plants were paired in each
cage, cr-VOC vs ir-VOC. Ten P. brassicae females were released
inside each cage for 24 h. The number of eggs, the number of
clutches and the number of eggs per clutch laid during that time
were recorded. The experiment was conducted using 33 plant
pairs.
2.4.2. Experiment 2—do cabbage plants also gain resistance to
oviposition by P. brassicae after receiving VOCs from damaged
neighbors in the laboratory? Does ozone impact on that interaction?
Phase 1: Plant exposures in controlled-environment chambers
under ambient and elevated ozone levels—Four controlled-environ-
ment chambers were used in this experiment, two with the
ambient ozone level of the laboratory (10 ppb) and two with an
enriched ozone level of 80 ppb. One chamber for each ozone level
had herbivore-infested emitter plants and one had undamaged
control emitters. Cabbage plants in each chamber were placed in
plastic trays (56 cm L  36 cm W  7 cm H) on upturned dishes and
arranged in 2 parallel rows of six plants with a 30 cm distance
between stems. Water was added to the tray to avoid the
movement of caterpillars from emitter to receiver plants. Air
passed from right to left in the chambers, plants placed on the right
side of the chamber were VOC-emitters while plants on the left
were VOC-receivers. At the start of the experiment VOC-emitter
plants for the herbivore-infested treatment were infested with 6 P.
brassicae larvae at both ozone levels. No damage was done to any of
the VOC-emitter cabbage plants of the control treatments. VOC-
receiver cabbage plants (cr-VOC and ir-VOC) remained undamaged.
This phase was conducted five times.
Phase 2: Oviposition preference of P. brassicae—cr-VOC and ir-VOC
cabbage plants from ambient and elevated ozone chambers (from
here on amb- denotes plants from experiments conducted at
ambient ozone levels and ozo- denotes plants from experiments
conducted at enriched ozone levels) were transferred to a
controlled environment room (light: dark cycle of 16L: 8D and
20
C) and paired in polycarbonate cages (60 cm L  33 cm W  33
cm H) in the following combinations: amb-cr-VOC vs amb-ir-VOC
and ozo-cr-VOC vs ozo-ir-VOC. Five P. brassicae females were
released inside each cage for 24 h oviposition assays. The number
of eggs laid on each plant was recorded. The experiment was
conducted in five batches, giving a total of 25 pairs of plants for the
355
Exposure Ozone levels tested Oviposition tests Ovipositing herbivore
(ppb)a locationc
Field Ambientb Laboratory P. brassicae
Laboratory Ambient (10 ppb) Laboratory P. brassicae
Enriched (80 ppb)
Field Ambientb Field P. xylostella
Enrichedb
Laboratory Ambient (10 ppb) Laboratory P. xylostella
Enriched (80 ppb)
Laboratory Ambient (10 ppb) Laboratory P. xylostella
Enriched (80 ppb)
ambient ozone treatment and 27 pairs for the elevated ozone
treatment.
2.4.3. Experiment 3—do plant–plant interactions mediated by VOCs
induced by one herbivore species (P. brassicae) have an effect on the
oviposition choices of a second herbivore species (P. xylostella)?
Phase 1: Plant exposures in the field under ambient and elevated
ozone levels—in 2014 (8-Aug-13-Sep) and 2015 (10-Jul-25-Jul)—Two
plastic trays filled with soil were placed in each of three ambient
and three enriched ozone plots in the ozone FACE field. Two potted
cabbage plants were placed 30 cm from each other in each plastic
tray (56 cm L  36 cm W  7 cm H). Trays in the plots were sepa-
rated by a minimum distance of 3 m. Trays were randomly assigned
to have control or herbivore-infested emitter plants. One plant in
each tray was assigned as either a VOC-emitter or a VOC-receiver.
Herbivore-infested emitter plants were infested with 6 P. brassicae
larvae. No larvae were added to the VOC-emitter plant of the
control tray. All VOC-receivers remained undamaged (cr-VOC and
ir-VOC). All trays were covered with a mesh cage (56 cm L  36
cm W  36 cm H; mesh size 1 mm2) to avoid the escape of P.
brassicae larvae and damage by snails. Cages had a mesh screen
between the plants to prevent P. brassicae moving between emitter
and receiver plants. After three days of VOC exposure, emitter
plants were removed from the field and VOC-receiver plants were
used in oviposition preference tests. This phase was conducted four
times.
Phase 2: Oviposition preference of P. xylostella—After phase 1,
amb-cr-VOC and amb-ir-VOC cabbage plants from each plot were
paired inside a tray filled with soil and covered with a mesh cage
(without a mesh screen in the middle). Ozo-cr-VOC and ozo-ir-VOC
plants were paired similarly. Fifty P. xylostella adults were released
inside each mesh cage, they were provided with water and a 30%
honey solution, both soaked in cotton wool. After 48 h the
experiment was terminated and the number of eggs laid on each
plant was recorded. A total of 11 oviposition tests were conducted
for each ozone treatment in four batches.
2.4.4. Experiment 4 – is the susceptibility of receiver plants to
oviposition sensitive to ozone pollution?
Phase 1: Plant exposures in controlled-environment chambers
under ambient and elevated ozone levels—Phase 1 was conducted
similarly to phase 1 of experiment 2 (see Section 2.4.2) using
cabbage as VOC-emitter plants, however instead of using trays
filled with water to avoid the movement of caterpillars, trays of
each treatment were covered with mesh cages (56 cm L  36 cm
W  36 cm H; mesh 1 mm2). No damage was done to the VOC-
emitter cabbage plants of the control treatment. VOC-receiver
356 P.S. Girón-Calva et al. / Agriculture, Ecosystems and Environment 233 (2016) 352–360

plants (cr-VOC and ir-VOC) remained undamaged during the

experiment. Phase 1 was repeated five times.
Phase 2: Oviposition preferences of P. xylostella in multiple choice
assays – Polycarbonate cages (100 cm L  70 cm W  70 cm H) were
used for P. xylostella oviposition preference tests in a controlled
environment room (light: dark cycle of 16L: 8D and 20
C). Two
receiver plants from each treatment were placed inside each cage
2 x (amb-cr-VOC, amb-ir-VOC, ozo-cr-VOC, ozo-ir-VOC). The plants
were positioned in two rows of four plants that were equally
spaced along the cage. For each repeat the plant positions were
altered so that the same treatment was not in the same position
during consecutive replicates. One hundred P. xylostella adults
were released inside each cage from two different points, they
were provided with a honey solution (30%) and water, both soaked
into cotton wool. After 48 h the experiment ended and the number
of eggs laid on each plant was recorded. Phase 2 was conducted 10
times.

2.4.5. Experiment 5—does exposure to emitter plants of a different Fig. 2. Oviposition preference of P. brassicae for undamaged cabbages exposed to
cultivar group of Brassica oleracea have a differential effect on either undamaged (cr-VOC) or herbivore-infested conspecifics (ir-VOC) under field
conditions. Statistical significance of differences between treatments were tested
receiver plants? And does ozone further affect that interaction?
with paired-sample t-test (n = 33). Bars represent mean (SE) percentage of eggs.
Phase 1: Plant exposures in controlled-environment chambers
under ambient and elevated ozone levels—This phase was conducted
(50.4%  6.52%) (Paired-sample t-test; t24 = 0.062, P = 0.951; n = 25;
in a similar way to experiment 4 phase 1 (see Section 2.4.4), but
Fig. 3A). Similarly, at elevated ozone conditions, no significant
broccoli plants were used as VOC-emitters instead of cabbage
difference was found in the proportion of eggs laid by P. brassicae
plants (see Fig. SM4 in the Supplementary material for a
females on ozo-cr-VOC plants (49.31%  6.35%) and on ozo-ir-VOC
representative chromatogram of the constitutive VOCs emitted
plants (50.69%  6.35%) (Paired-sample t-test; t26 = 0.109,
by broccoli plants; and Fig. SM5 for a representative chromatogram
P = 0.914; n = 27; Fig. 3B).
of the VOC profile emitted by P. brassicae-infested broccoli plants).
In this experiment, phase 1 was repeated nine times.
3.3. Experiment 3
Phase 2: Oviposition preference of P. xylostella—Oviposition
preference tests were set-up as in experiment 4 (see Section 2.4.4).
In oviposition experiments in the field at ambient ozone
Phase 2 for this experiment was repeated 17 times.
conditions, P. xylostella females laid more eggs on amb-cr-VOC
plants (58.58%  3.77%) than on amb-ir-VOC plants
2.5. Data analysis
(41.42%  3.77%) (Paired-sample t-test; t10 = 2.277, P = 0.046;
n = 11) (Fig. 4A). At elevated ozone conditions, exposure to VOC
All statistical analyses were performed using SPSS 21 software
emitters had no significant effect on the oviposition preference of P.
(IBM Corp. Armonk, NY). Data was checked for normality with a
xylostella females (44.62%  5.07% of eggs were laid on ozo-cr-VOC,
Shapiro-Wilk test before statistical analysis. The treatment effect
while 55.38%  5.07% were laid on ozo-ir-VOC plants) (paired-
(= VOC exposure) on the percentage of eggs laid by P. brassicae on
sample t-test; t10 = 1.061, P = 0.314; n = 11) (Fig. 4B).
VOC-receiver plants in both field and laboratory experiments, was
analyzed using either paired-sample t-tests or related-samples
3.4. Experiment 4
Wilcoxon Signed Rank tests. The level of significance selected was
a = 0.05. Similarly, the treatment effect on the percentage of eggs Laboratory experiments conducted in large cages provided
laid by P. xylostella on VOC-receiver plants in the field experiment
information on the oviposition preference of P. xylostella females in
was analyzed using paired-sample t-test with a = 0.05. The
an environment with more oviposition-site options, and allowed
treatment effect on the percentage of eggs laid by P. xylostella in
testing the host suitability of plants exposed to VOCs from
multiple-choice experiments in the laboratory was analyzed with a
neighbors with and without the presence of ozone. GLM indicated
logistic regression (generalized linear model, GLM), followed by an
a significant overall effect of the VOC treatment on the oviposition
LSD post hoc test to test for differences among treatments.
preference of P. xylostella (Fig. 5; GLM, Wald chi-square = 9.229,
df = 3, P = 0.026, n = 10). P. xylostella laid more eggs on amb-cr-VOC
3. Results
plants (30.65%  2.18%) than on amb-ir-VOC plants, which was
marginally significant (25.24%  2.28%; LSD post hoc test, P = 0.099).
3.1. Experiment 1
Additionally, P. xylostella females laid significantly more eggs on
amb-cr-VOC plants than on ozo-cr-VOC (22.45%  2.42%) and ozo-
In choice tests, gravid P. brassicae females laid significantly more
ir-VOC (21.66%  2.85%) plants (LSD post hoc test, P = 0.012 and
eggs on cr-VOC plants (58.71%  4.23%) than on ir-VOC plants
P = 0.006, respectively).
(41.29%  4.23%) (Fig. 2; paired-sample t-test; t32 = 2.061, P = 0.048;
n = 33).
3.5. Experiment 5

3.2. Experiment 2
GLM indicated a significant overall effect of the exposure
treatment on the oviposition preference of P. xylostella (Fig. 6; GLM,
In the laboratory, oviposition experiments with receiver plants
Wald chi-square = 10.737, df = 3, P = 0.013, n = 17). However, in this
exposed under ambient ozone conditions showed no significant
laboratory experiment, plant–plant interactions between broccoli
difference in the proportion of eggs laid by P. brassicae females on
and cabbage plants resulted in a significantly higher oviposition
amb-cr-VOC plants (49.6%  6.52%) and on amb-ir-VOC plants
 P.S. Girón-Calva et al. / Agriculture, Ecosystems and Environment 233 (2016) 352–360 357

Fig. 3. Oviposition preference of P. brassicae under laboratory conditions for undamaged cabbages exposed under ambient ozone (A) and enriched ozone levels (B) to either
undamaged (amb-cr-VOC and ozo-cr-VOC, respectively) or herbivore-infested conspecifics (amb-ir-VOC and ozo-ir-VOC, respectively). Statistical significance of differences
between treatments were tested with paired-sample t-test (n = 25 for treatments under ambient ozone levels and n = 27 for treatments under enriched ozone levels). Bars
represent mean ( SE) percentage of eggs.

Fig. 4. Oviposition preference of P. xylostella under field conditions for undamaged cabbages exposed under ambient ozone (A) and enriched ozone levels (B) to either
undamaged (amb-cr-VOC and ozo-cr-VOC, respectively) or herbivore-infested conspecifics (amb-ir-VOC and ozo-ir-VOC, respectively). Statistical significance of differences
between treatments were tested with paired-sample t-test (n = 11 for each treatment under both ambient and enriched ozone levels). Bars represent mean ( SE) percentage
of eggs.

rate on amb-ir-VOC (26.09%  1.46%) than on amb-cr-VOC
(20.6%  1.55%; LSD post hoc test, P = 0.016) cabbage plants.
Similarly, P. xylostella females laid significantly more eggs on
ozo-cr-VOC (25.01%  1.72%) and ozo-ir-VOC (28.3%  1.81%) than
on amb-cr-VOC plants (LSD post hoc test, P = 0.054 and P = 0.002,
respectively).
receiver plant-herbivore interactions, and that the mechanisms are
sensitive to ozone pollution and dependent upon the identity of
the emitter plant.
4.1. Plant-plant interactions reduce the susceptibility of receivers to
oviposition by P. brassicae

4. Discussion Exposure to damaged conspecific neighbors in the field

Plant susceptibility to oviposition by gravid female Lepidoptera
is a key factor in determining the herbivore pressure to which they
will be subjected. Here we showed that cabbage plants exposed to
damaged conspecific neighbors under ambient field conditions are
less susceptible to oviposition by P. brassicae and P. xylostella than
plants exposed to undamaged neighbors. However, elevated ozone
levels in the field eliminated the reduced susceptibility to P.
xylostella. Furthermore, laboratory tests revealed that exposure to
herbivore-damaged broccoli emitter plants resulted in a different
response, whereby cabbage receiver plants were more susceptible
to oviposition by P. xylostella than receivers exposed to undamaged
control emitters. Taking our results together, we have strong
evidence that volatile-mediated plant–plant interactions affect
rendered receiver cabbage plants less susceptible to oviposition
by P. brassicae in choice tests. Few similar studies have been
conducted, but Zakir et al. (2013) showed in the field that cotton
plants were less susceptible to oviposition by Spodoptera littoralis
in the presence of cotton neighbors undergoing continual damage
by larvae. The authors determined that this observation was most
likely due to VOCs from damaged emitters directly deterring
oviposition (De Moraes et al., 2001; Zakir et al., 2013). In our study,
a direct repellent effect of VOCs from herbivore-damaged
neighbors could not have occurred, due to the emitter plants
not being present during the oviposition choice tests. Therefore, it
is most likely that the oviposition choices of P. brassicae were due to
either an induction of VOC emissions in receiver plants or an
alteration to the surface chemistry of the receiver that could have
358 P.S. Girón-Calva et al. / Agriculture, Ecosystems and Environment 233 (2016) 352–360
Fig. 5. Oviposition preference of P. xylostella under laboratory conditions where
adults were offered plants from four different treatments as options for oviposition.
Undamaged cabbages were exposed to either undamaged (cr-VOC) or herbivore-
infested conspecifics (ir-VOC) under ambient ozone (amb) and elevated ozone
levels (ozo). Results of the LSD post hoc test used for pairwise comparisons among
treatments (P values) are displayed above bars. n = 10. Bars represent mean ( SE)
percentage of eggs.
Fig. 6. Oviposition preference of P. xylostella under laboratory conditions. Adults
were offered plants from four different treatments as options for oviposition.
Undamaged cabbages were exposed to either undamaged (cr-VOC) or herbivore-
infested broccoli plants (ir-VOC) under ambient ozone (amb) and elevated ozone
levels (ozo). Results of the LSD post hoc test used for pairwise comparisons among
treatments (P values) are displayed above bars. n = 17. Bars represent mean ( SE)
percentage of eggs.
been detected by females prior to oviposition (Gomez-Jimenez
et al., 2014; Himanen et al., 2015; Reddy et al., 2004). Previous
studies with brassicaceous plants have shown that exposure to
volatiles can prime plants for VOC emissions and other defense
responses, but do not induce volatile emissions (Li and Blande,
2015; Peng et al., 2011), indicating that an alternative mechanism
could be involved. VOCs from damaged plants have been observed
to adsorb to leaf surfaces of neighboring brassicaceous plants
(Himanen et al., 2010; Li and Blande, 2015), which alters the
receiver plants’ attractiveness to host searching females (Himanen
et al., 2015). It is possible that in our study VOCs from damaged
plants passively adsorbed to undamaged plant leaves, altering the
foliage quality and reducing the susceptibility of receiver plants to
oviposition. Shiojiri et al. (2015) found that Pieris rapae butterflies
strongly preferred to oviposit on uninfested cabbage plants over
plants infested by conspecific larvae, which correlates with
avoidance of intraspecific resource competition, parasitism and
predation (Shiojiri et al., 2015). It is plausible that detection of
herbivore induced volatiles may have elicited similar responses in
our study.
Oviposition preference tests of P. brassicae under laboratory
conditions were conducted to further corroborate the observations
in the field, and to also examine the impact of ozone, which had not
been possible in the field trial conducted earlier. Surprisingly, P.
brassicae females did not discriminate between amb-cr-VOC and
amb-ir-VOC cabbage plants as earlier observed in the field
experiment under ambient ozone levels. One difference, other
than the location, between the field and laboratory experiments is
the method used for damaging the emitter plants. The VOC-emitter
plants in the laboratory were induced by herbivore damage alone,
while emitter plants in the field were induced with a combination
of herbivore-feeding and mechanical damage. The mechanical
damage was included to compensate for small numbers of larvae,
but may have led to a strong emission of GLVs at the beginning of
the exposure (Heil, 2009; Mithöfer et al., 2005; Reymond et al.,
2000), meaning that receiver plants were exposed to a different
VOC blend to plants in the field. In this laboratory experiment, it is
not possible to clearly identify an effect of ozone exposure on
plant–plant signaling and oviposition choices, due to there being
no preference at ambient ozone levels.
4.2. Plant-plant interactions reduce susceptibility of receivers to
oviposition by P. xylostella, but are sensitive to ozone pollution
In the field, plants are often subjected to simultaneous or
sequential attack by different herbivore species over the entire
growing season. The response of plants to one herbivore may affect
their response to another herbivore (Poelman et al., 2008; Shiojiri
et al., 2015). Our studies in the field showed that VOCs from P.
brassicae-damaged cabbage plants also make receivers less
attractive to P. xylostella. In this experiment, we were also able
to observe the effect of enriched ozone levels on the plant
susceptibility to oviposition. Under ambient ozone levels, P.
xylostella females oviposited less on plants exposed to P.
brassicae-damaged cabbage plants, matching the response of P.
brassicae. Himanen et al. (2015) observed that P. xylostella females
also laid fewer eggs on broccoli plants exposed to R. tomentosum.
While our study did not extend to a full elucidation of the
mechanisms involved, the resistance gained by receiver plants
appears to extend beyond the species of herbivore damaging the
emitter plant.
Under enriched ozone levels, gravid P. xylostella females did not
discriminate between plants exposed to volatiles from damaged
and undamaged neighbors. Ozone can interfere with volatile
mediated interactions in several ways. Firstly, ozone could have
altered the susceptibility of a plant to oviposition by altering
foliage quality (Vuorinen et al., 2004), resulting in gravid females
assessing both ozo-ir-VOC and ozo-cr-VOC plants as similar quality
hosts. Secondly, ozone could have degraded the volatile signal by
reacting with the VOCs transported in the air. It is known that
ozone reduces the distance over which plant–plant signaling
occurs under laboratory conditions, likely by degradation of
signaling compounds (Blande et al., 2010). More recently, Li and
Blande (2015) showed that ozone degrades the volatile blend
emitted by broccoli plants and significantly affects the suscepti-
bility of broccoli plants to oviposition by P. xylostella. However, in
our study, it is not possible to determine whether under enriched
ozone levels the volatile signal was partially or completely absent
or some other reaction products were present. Thirdly, ozone may
also interfere with signal perception of the receiver plants by
altering stomatal conductance (Vahisalu et al., 2010) and restrict-
ing volatile signal flux into the leaves (Sugimoto et al., 2014). Ozone
 P.S. Girón-Calva et al. / Agriculture, Ecosystems and Environment 233 (2016) 352–360
may also affect the capacity of leaves to adsorb compounds by
altering the leaf wax structure and chemical composition. Finally,
since oviposition choice tests were conducted in the field, it is
possible that ozone had a direct effect on the herbivores. It has
been shown that some herbivorous insects do not avoid ozone-
polluted air directly (Fuentes et al., 2013). Therefore, it seems
unlikely that ozone itself impaired the ability of P. xylostella
females to evaluate host quality and choose a suitable host.
Ozone pollution alone seems to significantly affect the
susceptibility of brassicaceous plants to oviposition. When
assessing the susceptibility of cr-VOC and ir-VOC plants to
oviposition with and without elevated ozone during the exposure
period, we observed that amb-ir-VOC plants received fewer eggs
than amb-cr-VOC plants. This finding is consistent with our field
observation, even though the statistical significance was marginal.
However, ozone exposed receiver plants did not significantly differ
in their susceptibility to oviposition, irrespective of whether they
were exposed to damaged or undamaged emitter plants. Both ozo-
cr-VOC and ozo-ir-VOC plants were oviposited on significantly less
than amb-cr-VOC plants. This not only suggests that ozone may
have broken down signaling between plants, but that exposure to
ozone made plants less susceptible to oviposition. This may have
been due to ozone exposed plants being perceived as of a lower
quality than non-exposed plants, but the mechanisms would need
to be fully elucidated in further studies.
4.3. Interactions between plants of different Brassica oleracea cultivar
groups
Plant-plant signaling is mostly considered to benefit receiver
plants, while there is no clear benefit for the emitter plant.
Favorable consequences of plant–plant signaling have been
reported for receivers in conspecific and heterospecific associa-
tions under both laboratory and field conditions (Himanen et al.,
2010, 2015; Karban and Shiojiri, 2009; Karban et al., 2013). We also
observed favorable effects on B. oleracea receiver plants after
receipt of herbivore-induced volatiles from plants of the same
cultivar. However, when broccoli plants were used as the
VOC-emitter, gravid P. xylostella females deposited significantly
more eggs on amb-ir-VOC plants than amb-cr-VOC plants. This
finding is in line with a study by Li and Blande (2015), in which they
showed that broccoli plants exposed to damaged conspecifics were
more susceptible to oviposition by P. xylostella than plants exposed
to undamaged neighbors. The oviposition choices of P. xylostella
were correlated with emission of the sesquiterpenes b-elemene,
(E,E)-a-farnesene and a-selinene, which are herbivore induced
emissions of broccoli and adsorb to the surfaces of neighboring
plants (Li and Blande, 2015). Our study provides fresh evidence
that VOC emissions of broccoli plants act as cues to promote
oviposition by P. xylostella. As observed earlier, ozone appeared to
break down the interaction resulting in no significant differences
in the susceptibility to oviposition of the ozo-ir-VOC and ozo-cr-
VOC plants. However, contrary to the observation with cabbage
emitters, the ozone exposed plants were significantly more
susceptible to oviposition than the amb-cr-VOC plants and did
not differ from the Amb-irVOC plants. This observation suggests a
deeper complexity in the role of ozone on plant susceptibility to
oviposition, which should be elucidated further in more mecha-
nism oriented studies.
5. Conclusions
Plant-plant interactions mediated by VOCs can significantly
alter the susceptibility of a receiver plant to oviposition. Under
field conditions, cabbage plants exposed to P. brassicae-damaged
cabbage neighbors gain resistance to oviposition by both
359
`P. brassicae and P. xylostella. The mechanisms underlying the
observations could be either active or passive in mode and require
further elucidation. The identity of the emitter plant plays an
essential role in influencing whether or not a plant–plant
interaction is favorable to the receiver plant; cabbage emitter
plants lead to increased resistance and broccoli emitter plants to
increased susceptibility in cabbage receivers. The precise mecha-
nisms underlying the opposing responses are unknown, but could
potentially vary in their mode. Tropospheric ozone has a significant
impact on plant–plant interactions and alters these important
ecologically relevant interactions in potentially complex ways. This
implies that ecologically important volatile-mediated interactions
are prone to disturbance under elevated ozone conditions.
Acknowledgments
We thank Marjatta Puurunen and Topi Kuronen for assisting
with plant growing and insect rearing. We thank Timo Oksanen for
the technical support in both laboratory and field ozone experi-
ments. Gaukhar Malikova, Álvaro Cortés Molino and Adedayo
Mofikoya are gratefully acknowledged for their support during the
performance of the experiments. This research was supported by
the Academy of Finland [decision numbers 256050, 251898 and
283122]. PSGC was supported by a personal grant from the Finnish
Cultural Foundation.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.agee.2016.09.028.
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