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The prospect of applying chemical
elicitors and plant strengtheners to
enhance the biological control of
crop pests
rstb.royalsocietypublishing.org
Research
Cite this article: Sobhy IS, Erb M, Lou Y,
Turlings TCJ. 2014 The prospect of applying
chemical elicitors and plant strengtheners to
enhance the biological control of crop pests.
Phil. Trans. R. Soc. B 369: 20120283.
http://dx.doi.org/10.1098/rstb.2012.0283
One contribution of 16 to a Discussion Meeting
Issue ‘Achieving food and environmental
security: new approaches to close the gap’.
Subject Areas:
ecology, behaviour, biotechnology,
plant science
Keywords:
crop protection, biological control, plant
strengtheners, elicitors, plant volatiles,
parasitoids
Author for correspondence:
Ted C. J. Turlings
e-mail: [email protected]
Islam S. Sobhy1,2,3, Matthias Erb1,4, Yonggen Lou5 and Ted C. J. Turlings1
1
Laboratory of Fundamental and Applied Research in Chemical Ecology, Institute of Biology, University of
Neuchâtel, Rue Emile-Argand 11, CP158, Neuchâtel 2009, Switzerland
2
Department of Plant Protection, Public Service Center of Biological Control (PSCBC), Faculty of Agriculture,
Suez Canal University, Ismailia 41522, Egypt
3
Biological Chemistry and Crop Protection (BCCP) Department, Rothamsted Research, Harpenden,
Herts AL5 2JQ, UK
4
Root-Herbivore Interactions Group, Max Planck Institute for Chemical Ecology, Hans-Knöll-Strasse 8,
Jena 07745, Germany
5
State Key Laboratory of Rice Biology, Institute of Insect Science, Zhejiang University, Hangzhou 310058,
People’s Republic of China
An imminent food crisis reinforces the need for novel strategies to increase
crop yields worldwide. Effective control of pest insects should be part of
such strategies, preferentially with reduced negative impact on the environment and optimal protection and utilization of existing biodiversity.
Enhancing the presence and efficacy of native biological control agents
could be one such strategy. Plant strengthener is a generic term for several
commercially available compounds or mixtures of compounds that can be
applied to cultivated plants in order to ‘boost their vigour, resilience and
performance’. Studies into the consequences of boosting plant resistance
against pests and diseases on plant volatiles have found a surprising and
dramatic increase in the plants’ attractiveness to parasitic wasps. Here, we
summarize the results from these studies and present new results from
assays that illustrate the great potential of two commercially available resistance elicitors. We argue that plant strengtheners may currently be the best
option to enhance the attractiveness of cultivated plants to biological control
agents. Other options, such as the genetic manipulation of the release of
specific volatiles may offer future solutions, but in most systems, we still
miss fundamental knowledge on which key attractants should be targeted
for this approach.
1. Introduction
As repeatedly stressed by the Food and Agricultural Organization of the United
Nations, the biggest contemporary challenge for humanity is to safeguard food
security for current and future generations. A growing demand and a steady
increase of the world population requires that food production per area of cultivated land will have to increase drastically, preferentially with minimal
impact on natural ecosystems [1 –4]. One way to achieve higher yields will be
the reduction of crop losses caused by pathogens and pests, which still
amount to an estimated 25–40% of total production. To this end, new and preferably sustainable and environment-friendly strategies for crop protection have
to be developed. Effective biological control of arthropod pests is generally
accepted as such a sustainable and ecologically sound approach to reduce
crop damages. Increasing the effectiveness of biological control agents, such
as predators and parasitoids, not only involves increasing their numbers in
agro-ecosystems, but also enhancing their foraging success.
& 2014 The Author(s) Published by the Royal Society. All rights reserved.
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In most cases, it is still far from clear which specific volatiles
are most essential for attraction, in particular where it concerns
parasitoids [37,38]. Yet, several compounds are of obvious
importance in some specific systems. One of those is methyl
salicylate, which, for instance, has been found to be particularly important for the attraction of predatory mites [39,40].
This compound is produced via the salicylic acid (SA) pathway, in contrast to the octadecanoid pathway (involving
jasmonic acid) that is responsible for the production of volatiles that are typically induced by caterpillar feeding [41].
Indeed, methyl salicylate helps predatory mites to distinguish
between mite-damaged and caterpillar-damaged plants. Yet,
(b) Field studies
There is still limited, but convincing field evidence for a role of
plant volatiles in increasing predation and parasitism in the
field. In each case, hormonal pathways leading to volatile
emissions were manipulated. In a first such study, Thaler
[55] applied the plant defence hormone jasmonic acid to
tomato plants and found that this increased parasitism of sentinel caterpillars placed near the treated plants twofold. In a
2
Phil. Trans. R. Soc. B 369: 20120283
(a) What volatiles should we target?
some studies suggest that the response of the predatory
mites is not genetically fixed, but is learned by association
[42], as is the case for many parasitoids [43,44]. Field studies
by James and co-workers [45–48] confirmed a role of methyl
salicylate in mediating natural enemy recruitment. They
placed methyl salicylate-releasing dispensers in vineyards
and hopyards and found an increased presence of arthropod
predators and certain parasitoids. However, as the authors
recognize, this positive effect may not (only) be an effect of
direct attraction, but could also have been owing to the
effect of the plant hormone on the direct and indirect plant
defence responses. A field study with different maize inbred
lines suggested that parasitism by the parasitoid Cotesia marginiventris may be positively influenced by the emission of
methyl salicylate [49]. On the other hand, attraction of the
parasitoid Diadegma semiclausum is negatively affected by
methyl salicylate [11], again indicating that methyl salicylate
is not of universal importance to attract all natural enemies.
The most common compounds that are specifically
induced in plants by herbivore feeding are terpenoids [50].
This has led to the assumption that they are key to the attraction of natural enemies. Indeed, some of the earliest volatiles
implicated in the attraction of natural enemies are terpenoids,
such as (E)-ocimene, which is prominently released by miteinfested plants [5]. Because of their dominancy in attractive
odour blends of caterpillar-infested plants [41], terpenoids
were also thought to be of key importance for the attraction
of generalist parasitoids, but evidence is accumulating that
this may not necessarily be the case [51]. Indeed, genetic transformation of Arabidopsis plants with typical maize terpene
synthase genes revealed that dominant sesquiterpenes might
mainly be of importance after parasitoids learn to associate
them with caterpillar presence [18,52]. The innate responses
of parasitoids seem to depend on minor compounds that
remain to be identified [38].
One particularly promising terpenoid is (E)-b-farnesene.
This sesquiterpene serves as an alarm pheromone for many
aphid species, which release it upon attack by predators,
causing neighbouring aphids to disperse [53]. (E)-b-Farnesene also has a repellent effect on aphids in search of a host
plant and can attract and arrest aphid natural enemies
[19,54]. Beale et al. [19] successfully transformed Arabidopsis
thaliana plants with a gene from peppermint to constitutively
release (E)-b-farnesene, making the plants repellent to aphids
and attractive to an aphid parasitoid. Field trials with
similarly transformed wheat plants are underway.
Overall, very few examples exist of specific compounds that
can be enhanced to attract a broad range of beneficial arthropods. As long as this is the case, it might therefore be more
advantageous to pursue a general increase of herbivore-induced
volatiles. Several field studies demonstrate that plant attractiveness to natural enemies of herbivores can indeed be increased
and that this leads to higher herbivore mortality.
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The natural enemies of herbivores commonly make use of
plant-produced volatiles in order to locate their victims. More
specifically, upon herbivore attack, plants emit specific
blends of volatiles that attract natural enemies of the herbivores, a phenomenon that was first shown for predatory
mites [5] and parasitoids of caterpillars [6]. The importance
of herbivore-induced plant volatiles as foraging cues has
since been demonstrated in many systems, not only as attractants for natural enemies [7–9], but also, in certain cases, as
repellents for herbivores [10,11]. These effects have prompted
the notion that inducible volatile signals can be enhanced in
crop plants in order to improve the biological control of
insect pests [9,12–16]. Indeed, genetic manipulation has
been used successfully to alter the emissions of volatile compounds, and thereby enhance the attraction of natural
enemies [17–21].
The approach has also been tested in the field, yielding
some very promising results (see below). However, certain
inducible plant volatiles that attract beneficial insects may
also attract arthropod pests [22 –24]. This is not necessary a
problem, because, if appropriately exploited in pest control
strategies, this latter phenomenon can be used to develop
trap crops, for instance, in push –pull approaches [25,26], as
described below. It is also desirable that the manipulated
emissions are inducible by the pest, so that their natural enemies are offered a reliable signal and are attracted only when
they are likely to encounter prey or hosts on the crop [27,28].
Transgenic approaches show great promise [4], but are
subjected to continuous criticism. Scepticism and fear are
being maintained by controversial, often flawed, but highly
publicized studies that reinforce the notion that transgenic
plants may cause environmental and health problems
[29 –31]. It is therefore unlikely that a transgenic approach
to enhance the attractiveness of crop plants to beneficial
insects will be adopted in the near future, especially not in
Europe. It may, however, be possible to manipulate and
enhance the existing indirect defence capabilities of plants
by switching on relevant genes via the application of chemical elicitors [32]. Several of such elicitors have been shown to
greatly enhance resistance in plants, in particular against
pathogens [33–36]. With this in mind, we and others have
studied the possibility to alter volatile emissions with the
use of chemical elicitors, in particular, plant strengtheners
that are already being used to improve crop performance.
In this paper, we review the research in this area and present
a series of laboratory experiments that show long-time persistence of enhanced parasitoid attraction after treating
maize plants with two such plant strengtheners.
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(c) Plant strengtheners: the case of maize
Rostás & Turlings [61] studied how parasitoid attraction to
maize plants is effected by treating the plants with benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), a
chemical mimic of SA that has been used successfully to
induce resistance to a wide range of diseases on field crops
[62,63]. It was assumed that due to negative cross talk [64]
the activation of the SA pathway would suppress jasmonic
acid activation, and therewith herbivore-induced volatile
emissions. Surprisingly, BTH-treated maize plants, after they
have been infested by caterpillars, were highly attractive to
females of the braconid parasitoid Microplitis rufiventris,
much more so than untreated plants [61]. As expected, BTH
treatment did not increase the emission of volatiles, but, in
fact, it significantly reduced the emission rates of two volatile
compounds (indole and (E)-caryophyllene) in maize seedlings. Apparently, this was not the case for key compounds
responsible for parasitoid attraction and possibly the reduced
release of several less relevant compounds may have increased
the attractiveness of such key attractants. These findings support the conclusion from work by D’Alessandro et al. [38]
that the dominating compounds in the herbivore-induced
volatile emissions from maize plants are not essential for parasitoid attraction and may, in fact, reduce attractiveness by
masking more important parasitoid attractants.
The initial study by Rostás & Turlings [61] was followed up
by Sobhy et al. [65], who tested BTH, as well as another chemical plant elicitor, laminarin, which induces the accumulation of
phytoalexins and expression of a set of pathogenesis-related
proteins [66] through the activation of the SA pathway [67].
The aim was to find out whether treatment with these plant
enhancers has a general positive effect on the attractiveness of
plants to parasitoids. As before, it was found that, compared
with control plants, both BTH- and laminarin-treated plants
released lower amounts of volatiles, in particular, indole and
some sesquiterpenes, and again this increased the attractiveness
of maize plants to parasitic wasps. This effect of treatment was
found for all three of the tested parasitoid species [65]. Overall,
the results imply that maize plants that produce less of the
dominating volatiles show increased attractiveness to a wide
range of parasitoids. Most promising, was the fact that BTH
treatment not only increased wasp attraction, but also increases
the direct defence against larvae of Spodoptera littoralis after
2 days feeding.
The promising results from laboratory studies prompted a
field study in which treatment with BTH in maize plants as
3
Phil. Trans. R. Soc. B 369: 20120283
to the pest and unsuitable as a host. Initially, within the plot,
maize was intercropped with the repellent molasses grass,
which further reduced maize infestation by the stemborers
[25]. Molasses grass was eventually replaced by Desmodium
spp., legumes that are not only repellent to the pests, but
also contribute to soil fertility and stability as well as effectively
suppressing the plant parasitic Striga weed [58,59].
The rice example shows that an effective push –pull strategy can also be obtained with a transgenic approach, at least
in the case of the rice brown planthopper [21]. However, for
leaf-feeding pests, the transgenic approach appears to be
more complicated, as leaf-volatile blends induced by chewers
are more complex, making it difficult to pinpoint the key
compounds that are involved in the attraction of predators
and parasitoids [50,60].
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natural system involving the wild tobacco plant Nicotiana
attenuata, Halitschke et al. [22] silenced two key enzymes of
the octadecanoid defence pathway, NaLOX3 and NaHPL, in
order to attenuate plant defences, including the release of volatile organic compounds. They tested these silenced plants
alongside their non-transformed counterparts and found that
green leaf volatiles as well as terpenoids attract natural enemies of herbivores but also the herbivores themselves. In
recent follow-up work, the same research group [56] could
demonstrate that when tobacco plants are impaired in the
production of volatiles that are attractive to predatory bugs
they are subjected to increased herbivory and produce fewer
flowers, which is correlated with a loss in seed production.
It has been frequently proposed that enhancing the
release of volatiles in crop plants may help to improve biological control [9,12–16]. A study on maize has provided a
demonstration that a transgenic manipulation of volatile
releases can indeed help to enhance crop protection. Degenhardt et al. [20] transformed a maize line with a gene from
oregano to restore the emission of (E)-caryophyllene. This
sesquiterpene is naturally emitted by the roots of many
maize varieties and their wild ancestor, teosinte, when the
roots are damaged, thereby attracting entomophagous nematodes, which in turn reduce the abundance of root feeding
herbivores [20]. In field trials, the transformed maize plants,
which constitutively produce (E)-caryophyllene, were found
to be more effective at attracting entomopathogenic nematodes, thereby significantly reducing root damage inflicted
by the western corn rootworm Diabrotica virgifera, a major
belowground pest of maize [20].
The possibility to manipulate the attractiveness of plants to
economically important insects was also evident from a study
on rice volatiles by Xiao et al. [21]. They found that genetically
altering the emissions of the monoterpene alcohol (S)-linalool
and the sesquiterpene (E)-caryophyllene had a significant
effect on the attraction of a major Asian pest of rice, the rice
brown planthopper Nilaparvata lugens, as well as some of its
natural enemies, in particular, an egg parasitoid and spiders.
Depending on the compound that was altered, the composition
of the insect community changed either in favour or the detriment of the pest insect, implying a great application potential
to control the rice brown planthopper and possibly other
pests. The fact that (E)-caryophyllene was not only attractive
to natural enemies, but also to the pest itself complicates the
issue. The same was found for (E)-caryophyllene emitted by
maize roots damaged by western corn rootworm: belowground, this volatile not only attracts entomopathogenic
nematodes [20,27], but also rootworm larvae, which use the
signal to aggregate on maize root systems, but only at intermediate concentrations [23,24]. This phenomenon could be
useful in the development of a belowground push–pull
approach, which obviously would work at much shorter
ranges than aboveground systems, where pests and their natural
enemies move considerably longer distances. Yet, aboveground
(E)-caryophyllene also appears to attract adult herbivores,
including Diabrotica looking for oviposition sites, and could be
used to divert them from non-producing plants [28,57].
The push–pull idea has received a tremendous boost after
its very successful adoption in subsistence maize farming in
Africa [25,26]. In this specific example, maize plots were bordered by Napier grass, which was found to be highly
attractive to stemborers. Most important to the success of this
approach is the fact that this attractive grass is, in fact, toxic
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In the cases discussed so far, known elicitors and plant
enhancers were used and were found to have a positive
effect on natural enemy attraction, but the mechanisms and
further implications of these effects remain largely unknown.
Xin et al. [69] introduced an approach that targets genes
which are specifically involved in the production of volatiles
of known importance, and screened for chemicals that activate these genes. They used this approach on rice plants
and developed a high-throughput chemical genetics screening system based on a herbivore-induced (S)-linalool
synthase promoter fused to a b-glucuronidase reporter construct to test candidate synthetic compounds for their
potential to induce rice defences. (S)-Linalool was chosen,
because it is one of the key volatiles that is emitted by rice
plants in response to attack by brown planthopper
N. lugens [70–74] and is highly attractive to its main egg parasitoid Anagrus nilaparvatae [75]. Of the tested compounds, the
widely used herbicide 2,4-dichlorophenoxyacetic acid (2,4-D)
was found to be particularly active. It induced a strong
defensive reaction and a significant increase in volatile production. Induced plants were more resistant to a caterpillar
pest, but became highly attractive to N. lugens as well as to
its parasitoid A. nilaparvatae. In a subsequent field experiment, 2,4-D application showed great potential to draw
away N. lugens from non-treated plants and turned the treated plants into deadly traps by also attracting large
numbers of parasitoids [69]. As mentioned above, this volatile-mediated trap cropping or push –pull approach has
already shown great potential in subsistence maize farming
in Africa [25,59], and could be used at larger scales with
the use of appropriate elicitors.
(e) Persistence and early (seed) treatment
The highly promising results from our laboratory studies
with maize [61] were not as evident in the field study that
was conducted in Mexico [68]. Our main explanation for
this is that the plants were treated too late and that most of
the plants had already suffered insect attack under high
insect pressures. This will need to be further tested with
additional field trials. We expect that by treating the plants
earlier, even at the seed stage, might yield better results.
This would require, however, that the treatment effect is
long-lasting. To test this, we conducted the following series
of experiments.
4
(a) Plants and insects
Zea mays var. Delprim plants were grown in commercial potting
soil (Ricoter, Aussaaterde, Aarberg, Switzerland) in plastic pots
(11 cm height, 4 cm diameter) at 25 + 28C, 60 + 5% r.h., 16: 8 h
L : D, and 926 mmol m22 s21 in a climate chamber (CLF plant climatics, Percival). Maize plants used for the experiments were
10 – 12 days old and had three fully developed leaves. For each
experiment, we selected plants of approximately the same size.
Eggs of S. littoralis Boisd. (Lepidoptera: Noctuidae) were supplied by Syngenta (Stein, Switzerland). Newly hatched larvae
were reared in transparent plastic boxes on a wheat germbased artificial diet until they had reached the second instar, at
which point they were used for experiments. The larval endoparasitoids, M. rufiventris Kok., Cotesia marginiventris (Cresson;
Braconidae: Hymenoptera) and Campoletis sonorensis (Cameron;
Ichneumonidae: Hymenoptera) were reared as described in
Sobhy et al. [65]. Thirty minutes before the bioassays, cages
with 2 – 4 day old parasitoid adults were transferred to the
laboratory for acclimatization.
(b) Application of plant strengtheners
BTH (BION) was obtained from Syngenta, Switzerland, as a
water-dispersible granular formulation containing 50% active
ingredient. Laminarin (IODUS 40) was obtained from Stähler,
Switzerland, as a soluble liquid formulation containing 3.5%
active ingredient. To evaluate the use of both plant strengtheners
in an agricultural context, we tested two modes of application:
first, we applied the plant strengtheners at different time points
(6 days and 2 days) before the bioassays. In order to test plants
at the same age, they were sprayed 6 and 10 days old after
germination, respectively. The chemicals were applied at concentrations of 0.15 g l21 (BTH) and 20 ml l21 (Laminarin) [65]. These
concentrations correspond to the recommended doses by the
manufacturers for application in agriculture. Control plants
were not sprayed at all. In a second experiment, maize seeds
were soaked in chemical solutions for 12 h until complete
absorption of the liquid [76]. Laminarin treatment was performed using a solution of 20 ml, using a concentration of
0.02% (v/v) in distilled water. BTH treatment was performed
using a solution of 0.15 g ml21 dissolved in distilled water [77].
A control treatment was performed using seeds soaked in distilled water. Treated and control seeds were sown separately
and used for experiments after 10 – 12 days.
(c) Olfactometer bioassays
A series of experiments using a six-arm olfactometer was conducted to evaluate the effect of the plant enhancers on the
attractiveness of maize plants to parasitic wasps. For all experiments, 3 – 5 days old mated naive female wasps were used. For
the choice bioassays, six female wasps were removed from
their cage with an aspirator and released into the central choice
chamber of the olfactometer. The wasps moved up to the top
of the chamber attracted by the diffuse light coming from
above. Depending on the attractiveness of the different odour
sources, they then walked into one of the six arms connected
to the central chamber. The central choice chamber was connected via a Tygon tube to a water-filled glass U-tube that
served as a pressure gauge to balance incoming and outgoing
air, minimizing pressure differences with the outside. Each
group of wasps was given 30 min to make a choice. Wasps that
did not enter an arm after this time were removed from the central part of the olfactometer and considered as individuals that
made ‘no choice’. Five groups of six wasps were tested on each
experimental day. Each olfactometer experiment was repeated
Phil. Trans. R. Soc. B 369: 20120283
(d) Identifying the ideal elicitor: a genetic screening
approach
2. Material and methods
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well as treatment with methyl jasmonate (MeJA) was tried out in
a subtropical region of Mexico with high insect pressures [68].
As in the laboratory, application of BTH slightly reduced volatile emission in maize, whereas MeJA increased the emission
compared with control treatments. However, these changes in
volatile emissions had no consistent effects on infestation by
Spodoptera frugiperda larvae, the main insect pest found on the
maize seedlings in this region, and the treatments only marginally effected parasitism rates. These somewhat disappointing
results may be explained by the severe biotic and abiotic conditions during the time of the experiment, which may have
masked the effects of the treatments [68]. To test whether this
is indeed the case, further field experiments, using early (seed)
treatments, before pest infestations, will be needed.
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(a)
5
induction
treatment
(2)
(1)
Cotesia marginiventris
O
60
68.3
a
a
18.9
21.1
a
response
a
a
28.3
7.2
70.5
64.5
a
40
b
b
b
20
c
b
c
(i)
0
80
response
response
60
33.9
a
a
60
36.1
18.3
a
52.8
75.1
ab
1
40
response
11.
no. attracted wasps
BTH
6.6
mechanical
damage and
regurgitant
6.1
SCH3
a
b
a
b
a
c
20
c
b
(ii)
0
OH
n OH
OH
a
a
68.9
ab
b
40
b
b
20
c
c
c
0
mechanical
damage and
regurgitant
no. attracted wasps
response
response
80
2.2
m
3.9
78.9
b
.1
21
a
60
.7
21
a
76.7
response
a
a
a
.7
OH
b-1,3
b-1,6
laminarin
22.2
a
71.1
16
O
empty
response
6.1
O
6 days control
(i)
CH2
OH
ab
a
2 days
response
2.8
2
60
empty
.2
6.1
O
O
6 days control
response
80
no. attracted wasps
CH2OH
2 days
8.9
8.9
Spodoptera
littoralis
empty
17
2 days 6 days control
(b)
10
73.3
72.2
b
40
a
b
20
b
c
(ii)
c
0
2 days 6 days control
empty
2 days
6 days control
empty
2 days
6 days control
empty
Figure 1. (a,b) Responses of naive female parasitoid wasps tested in a six-arm olfactometer. Values shown represent the number of parasitoids attracted to a
particular odour. Wasps were allowed to choose between odours of plants sprayed with strengtheners 2 or 6 days before infesting the plants with caterpillars
(i) or simulating herbivory by mechanically damaging the plants and treating them with caterpillar regurgitant (ii); control, untreated maize plants; empty,
empty control vessel (mean value of three vessels). Pie charts indicate percentages of female wasps (dark grey, females choose the empty bottles; light grey,
non-responding females; white, responding females). Different letters indicate significant differences between treatments ( p , 0.05).
six times on different experimental days, each time with a new
set of treated plants as odour sources and with new wasps.
The position of the odour source was changed clockwise after
each day of testing to avoid position effects. All bioassays were
performed between 9.00 and 17.00.
Using the above procedure, three treatment combinations
were tested individually for the three different parasitoid
species. First, plants that had been sprayed with plant strengtheners for 2 or 6 days were infested with 10 second-instar
S. littoralis larvae on the evening before the experiment. After
infestation, plants were kept under laboratory conditions (25 +
28C, 16 L : 8 D h). The following odour sources were then offered
simultaneously to the parasitoids: (i) a maize plant damaged by
S. littoralis caterpillars and treated with a chemical 2 days before,
(ii) a maize plant damaged by S. littoralis and treated with a
chemical 6 days before, (iii) an untreated maize plant damaged
by caterpillars (as control), and (iv) three empty control vessels.
BTH and laminarin were assayed in separate experiments. A
second set of experiments were conducted in the same way,
apart from the fact that plants were induced artificially by
scratching the abaxial side of fully developed leaves (20 mm2)
with a scalpel blade without damaging the midrib and applying
10 ml of S. littoralis larval regurgitant using a micropipette.
Regurgitant had previously been collected with a micropipette
from fourth instar larvae that had been feeding on maize
leaves for at least 24 h and was stored at 2808C until use [78].
The above-mentioned treatment was applied the evening
before and a second time on the morning of each experimental
Phil. Trans. R. Soc. B 369: 20120283
N S
N
response
response
10
.6
no. attracted wasps
80
Microplitis rufiventris
Campoletis sonorensis
10
.6
Spodoptera
littoralis
(3)
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plant
strengthener
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induction
treatment
6
80
response
3.9
no. attracted wasps
Cotesia marginiventris
60
a
a
26.1
70
40
b
c
20
(b)
80
response
Spodoptera
littoralis
60
8.9
no. attracted wasps
Campoletis sonorensis
a
8.9
a
72.2
40
b
20
c
0
(c)
80
no. attracted wasps
Microplitis rufiventris
60
response
a
28.33
6.1
1
a
40
65.56
b
20
c
0
BTH
laminarin
control
chemical treatment
empty
Figure 2. (a – c) Responses of naive females of three species of parasitic wasps tested in a six-arm olfactometer. Values shown are numbers of parasitoids attracted
to a particular odour. Wasps were allowed to choose between odours of BTH: maize plants that germinated from BTH-treated seeds, laminarin: maize plants that
germinated from laminarin-treated seeds, control: maize plants that germinated from distilled water-treated seeds, and empty: empty control vessel (mean value of
three vessels). Pie charts indicate percentages of female wasps (dark grey, non-responding females; light grey, females choose the empty bottles; black, responding
females). Different letters indicate significant differences between treatments ( p , 0.05).
day, about 3 h before the start of bioassays. The third experimental set-up included seed-treated plants that were induced by
adding 10 second-instar larvae of S. littoralis to each plant as
above. The odour sources were: (i) attacked maize plants from
BTH-treated seeds, (ii) attacked maize plants from laminarintreated seeds, and (iii) attacked maize plants from seeds
soaked in distilled water (control treatment), as well as three
empty arms as blank controls.
(d) Odour trapping and analysis
Volatiles emitted by the various odour sources were trapped for
3 h during the bioassays on Super Q adsorbent filters (25 mg,
80 – 100 mesh; Alltech Associates, Deerfield, IL, USA) [79].
Before use, traps were washed with 3 ml dichloromethane. In
all experiments, a filter was attached to the horizontal port at
the top of each odour source vessel. Purified air entered the bottles at a rate of 1.1 l min21, and air carrying the volatiles was
pulled through each trap at a rate of 0.7 l min21 (Analytical
Research System, Gainesville, FL, USA). Traps were extracted
with 150 ml dichloromethane (Super solvent; Merck, Dietikon,
Switzerland), and 200 ng of n-octane and n-nonyl acetate
(Sigma, Buchs, Switzerland) in 10 ml dichloromethane were
added to each sample as internal standards. Samples were
either analysed immediately or stored at 2808C in small vials
(Supelco, Amber Vial, 7 ml with solid cap w/PTFE Liner).
Odour samples were analysed using a gas chromatograph (Agilent 7890A) coupled to a mass spectrometer (Agilent 5975C VL
MSD). After injection of 2 ml of sample, the temperature was
maintained at 408C for 3.5 min, and then increased to 1008C at
88C per minute and subsequently to 2008C at 58C per minute followed by a post-run of 5 min at 2508C. Helium at constant flow
(0.9 ml min21) was used as carrier gas. The volatiles were identified by comparing their mass spectra with those of the NIST05
library and by comparing their retention times with those of previous analyses [38,65]. The total emission for each compound
Phil. Trans. R. Soc. B 369: 20120283
0
rstb.royalsocietypublishing.org
(a)
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application of
strengthener
induction
treatment
7
2 days
(a)
6 days
control
N S
N
O
SCH3
BTH
800
600
*
**
400
**
*
*
200
*
*
0
1200
(b)
O
CH2
O
*
O
O
OH
OH
OH
n OH
OH
b-1,3
b-1,6
m
laminarin
mean amount (ng 3 h–1)
1000
CH2OH
800
600
400
**
200
*
*
*
*
*
*
**
*
0
2500
(c)
N S
N
BTH
SCH3
Spodoptera
littoralis
BTH
CH2OH
O
CH
H2
O
*
O
OH
OH
O
OH
OH
n OH
OH
b-1,3
b-1,6
laminarin
m
mean amount (ng 3 h–1)
O
laminarin
control
2000
1500
*
1000
*
*
500
**
*
O
*
H
control
0
Z3
he
xe
n
al
ne e
al ol e te ne ol ne te ate le ate te ne
le esen
en -1- rcen ceta ime alo trie ceta cet ndo anil ceta ylle
u
x
n
a
a
n
i hr a ph
m rn
a l
y l a oc li n
he e
t yl yo
hu -fa
2- -hex m eny E)- (S)- -no thyl thy
n
n
a
r
a )-b
e
a
(
E -3
x
,7 e
yl ger )-ca
)
,3 l-m hen
he
h
(E
t
Z
1
(
e
E
3
l- ny 2-p
(
y
m
Z
h e
et ph
m
i
-d
,8
-4
)
(E
bbe
rg
am
ot
en
e
H
Figure 3. Mean emission (+s.e.; ng) of Spodoptera littoralis induced odours emitted by 11 day old maize seedlings at different time points after plant strengthener
application (a,b) and after seed treatment (c). Asterisks indicate differences between controls and chemically treated plants ( p , 0.05; n ¼ 18).
was estimated as the sum of the amounts for all compounds
released during the collection period (3 h), assuming equal ionization efficiency in the source of the mass spectrometer for the
different compounds.
(e) Statistical analyses
The functional relationship between parasitoid responses and the
different volatile sources offered in the six-arm olfactometer was
examined with a generalized linear model as described earlier
[79]. The model was fitted by maximum quasi-likelihood
estimation in the software package R (R: a language and environment for statistical computing, v. 2.9.0, Zurich, Switzerland,
2009, http://www.R-project.org), and its adequacy was assessed
through likelihood ratio statistics and examination of residuals.
One-way ANOVAs and Tukey’s post hoc comparisons were performed to compare means of volatile emission results when the
Phil. Trans. R. Soc. B 369: 20120283
mean amount (ng 3 h–1)
1000
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1200
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3. Results
Foliar application of BTH at two different intervals increased
the attractiveness of S. littoralis infested plants to three different parasitoid species. In general, the effect was stronger when
plants had been treated 6 days before elicitation with real or
simulated herbivory (figure 1). Microplitis rufiventris reacted
more strongly to plants induced with S. littoralis, whereas
C. sonorensis was more sensitive to mechanically damaged
BTH-treated plants. Similar to BTH, laminarin increased the
attractiveness of herbivore-induced plants 6 days after application to all three parasitoids, with the exception of
C. sonorensis, which was not significantly attracted to
mechanically induced laminarin-treated plants (figure 1b(ii)).
Similarly, treating maize seeds with either BTH or laminarin
increased the attractiveness of herbivore-infested plants for
all three tested parasitoid species (figure 2). Previous experiments show that neither laminarin nor BTH itself are
attractive to the parasitoids and that they do not significantly
alter the overall larval feeding, although a small, but significant difference was found after 2 days of feeding [61,65].
These results are highly encouraging in the light of an easy
to apply treatment of maize with plant strengtheners for
long-lasting enhanced attractiveness to parasitoids.
(b) Plant strengtheners alter volatile emission
Previous experiments revealed minor changes in constitutive
volatile emission of BTH- or laminarin-treated, undamaged
maize plants, but surprisingly some herbivore-induced volatiles were found to be suppressed after treatment [61,65]. This
study confirms this suppression, both in the case of foliar and
seed application of the plant strengtheners (figure 3). The
most dramatic responses were found for the aromatic compound indole and the sesquiterpene (E)-b-farnesene, both
of which were emitted in lower quantities in plant strengthener-treated plants, irrespective of the timing and mode of
application. The emission of (E)-caryophyllene and b-bergamotene was also reduced, albeit not in all cases. The
emission of the terpenes linalool and (E)-4,8-dimethyl-1,3,7nonatriene and geranyl acetate, on the other hand, was
4. Discussion
The notion that plant volatile emissions can be enhanced or
otherwise manipulated to help combat insect pests has been
around for some time, also in the context of biological control
[80,81]. The targeted manipulation of specific compounds has
already shown promising results in laboratory and greenhouse
assays [17–21]. A few field studies show direct evidence that
engineering volatile emissions can strongly affect the attraction
of biological control agents. For instance, the restoration of a
root-produced signal that attracts entomopathogenic nematodes was found to significantly improve nematode-mediated
protection of maize roots against an exceedingly important
coleopteran pest [20]. Also, first field tests with genetically
altered volatile emissions in rice show promise for the control
for one of the most important rice pests, the rice brown planthopper [21]. In general, however, it remains largely unclear
what compounds should be enhanced, especially to improve
the attraction of parasitic wasps [38]. A transgenic approach
remains controversial and requires major R&D investments
that are rarely cost effective. Therefore, the coincidental discovery [61] that treatment with plant strengtheners can, besides
enhancing the direct resistance of plants against pests and diseases, dramatically increase the attractiveness to a spectrum of
parasitoid species shows great promise for application. Here,
we show that the effect is long-lasting, persisting over weeks,
and would therefore be particularly promising to enhance
parasitoid-mediated protection of plants at an early stage
when the plants are most vulnerable to yield-reducing
caterpillar damage. Particularly promising is the fact that the
effect can also be achieved by soaking the seeds in solutions
with the elicitors. The effect remains unexplained, as for now
we cannot pinpoint the chemical changes that are responsible
for the enhanced parasitoid attraction. Without information
on the illusive compounds that are of key importance for the
attraction of these generalist parasitoids, treatment with the
studied elicitors of resistance seems to be the best option to
improve plant resistance and performance, and simultaneously
improve the biological control of important lepidopteran pests.
Acknowledgements. We thank Thomas Degen and Matthias Held for
their invaluable advice and assistance with the writing of this paper.
Funding statement. Our work on understanding the importance of plant
volatiles in plant–insect interactions and our efforts to exploit such
volatiles for crop protection is supported by the European Science
Foundation (EuroCore project Eurovol) and by the National Centre
of Competence in Research (NCCR) Plant Survival, a research programme of the Swiss National Science Foundation. The work of
M.E. is supported by a Marie Curie Intra-European Fellowship
(grant no. 273107).
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