Recruitment of entomopathogenic
nematodes by insect-damaged
maize roots
Sergio Rasmann
1
, Tobias G. Ko
¨
llner
2
,Jo
¨
rg Degenhardt
2
, Ivan Hiltpold
1
, Stefan Toepfer
3
, Ulrich Kuhlmann
3
, Jonathan Gershenzon
2
& Ted C. J. Turlings
1
1
University of Neucha
ˆ
tel, Institute of Zoology, Laboratory of Animal Ecology and Entomology, CP 2, CH-2007 Neucha
ˆ
tel, Switzerland
2
Max Planck Institute for Chemical Ecology, Hans-Kno
¨
ll-Strasse 8, D-07745 Jena, Germany
3
CABI Bioscience Switzerland Centre, Rue des Grillons 1, 2800 Dele
´
mont, Switzerland
Plants under attack by arthropo d herbivores often emit volatile compounds from their leaves that attract natural enemies of the
herbivores. Here we report the first identification of an insect-induced belo wground plant signal, (E )-
b
-caryophyllene, which
strongly attracts an entomopathogenic nematode. Maize roots release this sesquiterpe ne in response to feeding by larvae of the
beetle Diabrotica virgifera virgifera, a maize pest that is currently invading Europe. Most North American maize lines do not release
(E )-
b
-caryophyllene, whereas European lines and the wild maize ancestor, teosinte, readily do so in response to D. v. virgifera
attack. This difference was consistent with striking differences in the attractiveness of representative lines in the laboratory. Field
experiments showed a fivefold higher nematode infection rate of D. v. virgifera larvae on a maize variety that produces the signal
than on a variety that does not, whereas spiking the soil near the latter variety with authentic (E)-
b
-caryophyllene decreased the
emergence of adult D. v. virgifera to less than half. North American maize lines must have lost the signal during the breeding
process. Development of new varieties that release the attractant in adequate amounts should help enhance the efficacy of
nematodes as biological control agents against root pests like D. v. virgifera.
Plants are not simply passive victims of attacking herbivores; they
have evolved an arsenal of physical and chemical defences to protect
themselves. Often these defences are mobilized only in response
to herbivory
1,2
. Among the proposed inducible defences is the
production and release of volatile chemicals that could serve as
signals to attract natural enemies of the herbivores
3–5
. Manipulating
these signals can help increase the effectiveness of these natural
enemies as control agents
6–8
. The induced emission of chemical
signals is not limited solely to aboveground plant parts. The
entomopathogenic nematode Heterorhabditis megidis was found
to be attracted to exudates emitted by plant roots after damage by
weevil larvae
9,10
, but the nature of the attractants involved is
unknown. Here we show that maize roots damaged by larvae of
the economically important coleopteran pest Diabrotica virgifera
virgifera LeConte are attractive to entomopathogenic nematodes,
and we identify the chemical compound responsible for the attrac-
tion. D. v. virgifera or Western corn rootworm (WCR) is a voracious
pest of maize that is responsible for the use of the bulk of pesticides
applied in the cultivation of this crop in the USA
11
. The recent
introduction and rapid spread of WCR into Europe has caused
major concern for maize production on this continent and has
stimulated the search for new methods of maize protection
12,13
. The
use of nematodes to control WCR is an ecologically sound
option
14,15
, especially if researchers can optimize their efficacy at
finding and killing WCR.
Attraction of nematodes by WCR-damaged roots
To determine whether or not WCR-infested maize plants would
attract nematodes, three glass pots each containing one 10-day-old
maize plant (var. Delprim) were attached to the arms of a custom-
made six-arm olfactometer filled with moist (10% water) sand
(Fig. 1a). The plants had been grown on clean sand in the pots,
starting 5 days after seed germination. Three additional pots,
containing only sand, were attached to the remaining three arms
of the olfactometer. Four such olfactometers, each containing three
plants plus three sand controls, were prepared on a given day. One
plant of each set of three received four second-instar or third-instar
WCR larvae, the roots of a second plant were damaged daily by
stabbing them five times with a metal corkborer 7 mm in diameter,
and the third plant was left unharmed. On day 3 after initial damage,
about 2,000 Heterorhabditis megidis nematodes were released in the
centre of each olfactometer, where they were free to enter the arms
until their passage was blocked by an ultra-fine metal screen (see
Fig. 1a and Methods). One day after release, the number of
nematodes in each arm was recorded. Significantly more nematodes
were recovered from arms connected to the pots with the WCR-
damaged plants than from the arms connected to the other treat-
ments or controls (Fig. 1b), indicating that damage by WCR induces
maize roots to release a nematode attractant.
Identification of the attractant
Maize leaves had previously been shown to emit a mixture of volatile
compounds in response to damage by caterpillars
4
. To determine
whether WCR damage induces similar changes in plant volatiles, the
leaves and roots from WCR-damaged (3 days) and healthy maize
plants were ground and volatiles collected by solid-phase micro-
extraction (SPME) were analysed by gas chromatography–mass
spectrometry (GC–MS). A marked difference between the treat-
ments was that the sesquiterpene (E)-
b
-caryophyllene was present
in roots damaged by WCR but was completely absent from
undamaged roots (Fig. 1c). The damaged roots contained small
amounts of
a
-humulene and caryophyllene oxide as well. To a
smaller extent, the WCR-induced increase in (E)-
b
-caryophyllene
content was also apparent in the leaves (Fig. 1d). To test whether
(E)-
b
-caryophyllene was indeed attractive to H. meg idis,an
authentic standard (Sigma-Aldrich, more than 98% pure) was tested
in the olfactometer. For this purpose the system was entirely filled
with clean moist sand and a 0.2-
m
l dose of (E)-
b
-caryophyllene was
Published in Nature 434, 732-737, 2005
which should be used for any reference to this work
1
injected in the centre of one of the pots, whereas the five remaining
pots received no such treatment. Nematodes were released in the
middle of the olfactometer and on the next day nematodes were
recovered from the six arms. The arm attached to the pot that had
received (E)-
b
-caryophyllene contained almost three times as many
nematodes as the average control arm (Fig. 2a). Using a much lower
dose of three injections with 200 ng of (E)-
b
-caryophyllene in
pentane produced very similar results. In an additional experiment,
a 10-day-old healthy maize plant (var. Delprim) was placed in each
of two opposing pots of the olfactometer and the other four pots
contained sand only. One of the pots with a plant was spiked with a
0.2-
m
l dose of (E)-
b
-caryophyllene and nematodes were released in
the olfactometer centre. On the next day, the arm with the
caryophyllene-spiked plant contained on average almost fourfold
as many nematodes as the control arms, whereas there was no
statistical difference between the plant without (E)-
b
-caryophyllene
and the control pots (Fig. 2b). We tested several other synthetic
compounds that are commonly released from caterpillar-damaged
maize leaves in three choice tests, always including (E)-
b
-caryo-
phyllene as one of the choices (data not shown). These compounds
either were not attractive (linalool) or were significantly less
attractive than (E)-
b
-caryophyllene ((Z)-3-hexenyl acetate, methyl
salicylate, (E)-
b
-farnesene,
a
-humulene and (E)-nerolidol) at a
0.2-
m
l dose per pot.
Loss of signal in North American maize genotypes
The very limited number of compounds in the volatile blend
obtained from WCR-induced roots is in striking contrast to what
is emitted from maize leaves in response to caterpillar feeding, a
complex mixture of different terpenoids, aromatic compounds
and green-leaf volatiles
4,16,17
. Many of the maize lines that we have
screened in the past for caterpillar-induced leaf volatiles do not emit
(E)-
b
-caryophyllene in detectable amounts. This is particularly
characteristic for most varieties that originate from North American
breeding programmes
18
. We tested whether this difference also
holds true for the roots by measuring WCR-induced (E)-
b
-caryo-
phyllene in six inbred lines selected from a study on caterpillar-
induced leaf emissions
18
: three that emitted large amounts (E)-
b
-
caryophyllene from their leaves (Du101, F2 and F268) and three
lines that released no or very little (E)-
b
-caryophyllene (F584, A654
and F7001). In this experiment we also included the closest wild
ancestor of maize, teosinte (Zea mays parvig lumis
19,20
), which is
known to release relatively large amounts of (E)-
b
-caryophyllene
from its leaves in response to caterpillar feeding
17
. Ten-day-old
plants were subjected to 3 days of WCR feeding, after which (E)-
b
-
caryophyllene levels were measured in the roots as above. Teosinte
roots were found to release moderate amounts of (E)-
b
-caryophyl-
lene in response to WCR damage (Fig. 3a). The experiment also
confirmed a correlation between the levels of (E)-
b
-caryophyllene
induced in the leaves and the roots (Fig. 3a): Du101, F2 and F268
emitted considerable amounts of (E)-
b
-caryophyllene from the
roots after WCR attack and F584, A654 and F7001 emitted barely
detectable amounts. These differences offered an excellent oppor-
tunity to test whether (E)-
b
-caryophyllene is a key compound for
nematode attraction, because non-emitting varieties should be far
less attractive than emitting varieties. This was tested with repre-
sentative lines of commercial maize for which we had information
on caterpillar-induced (E)-
b
-caryophyllene releases
17
.
Pactol is a commercial maize variety that releases no detectable
amounts of (E)-
b
-caryophyllene from its leaves in response to
caterpillar feeding, whereas Graf releases relatively large amounts,
significantly more than the variety Delprim, which was used in the
first experiments
17
. Root extracts from WCR-damaged plants con-
firmed the presence of (E)-
b
-caryophyllene in Graf and Delprim
Figure 1 Attraction of entomopathogenic nematodes to a WCR-induced root signal.
a, Drawing of a newly designed belowground six-arm olfactometer in which nematode
attraction was tested. b, Choices between plants: the average number of nematodes
recovered from olfactometer arms that were connected to pots holding either a maize
plant with WCR-damaged roots, mechanically damaged roots or undamaged roots
(n ¼ 12). For each replicate, the total number of nematodes that went to the three
control pots (only moist sand) were summed and divided by three. c, Typical
chromatographic traces obtained from the roots of a healthy plant and of a
WCR-damaged plant. The labelled peaks are as follows: 1, unknown sesquiterpene;
2, (E )-
b
-caryophyllene; 3,
a
-humulene; 4, caryophyllene oxide. d, Quantification of
(E )-
b
-caryophyllene in roots and leaves from healthy and WCR-damaged maize plants
(n ¼ 6). Letters above bars indicate significant differences. Error bars indicate
standard errors.
2
roots and its absence from Pactol roots (Fig. 3b). Next, individual
plants of these three varieties were tested simultaneously in the
olfactometer by letting four third-instar WCR larvae feed on their
roots for 3 days and then releasing nematodes from the olfactometer
centre as before. The numbers of nematodes recovered from the
olfactometer arms revealed strong attraction to Graf and Delprim
and no attraction to Pactol (Fig. 3c). The importance of (E)-
b
-
caryophyllene for this difference in attractiveness was confirmed in
a nearly identical experiment with the three varieties, except that on
the third day of WCR feeding 0.2
m
lof(E)-
b
-caryophyllene was
added to the sand in the pot with the Pactol plant. After this
treatment the Pactol plant was as attractive to the nematode as the
two other plants (Fig. 3d).
Attractiveness in the field
To verify the importance of (E)-
b
-caryophyllene as an attractant for
H. megidis under realistic conditions, we conducted two types of
field experiment in Hungary, where WCR is already an established
pest. For each experiment, six maize plants were planted at an equal
distance from each other in circles 1 m in diameter. For the first
experiment, three of the plants in each of 33 circles were of the
variety Graf, alternated with three plants of the variety Pactol
(Fig. 4a). Eight weeks after planting each plant was infested with
six second-instar WCR larvae. Seven days after this infestation we
released about 10,000 H. megidis, three times at 2-day intervals, in
the centre of each circle. Larval infection rate by nematodes was
determined by collecting the roots with larvae for 15 circles at 3 days
after the last nematode release. For the remaining 18 circles, larvae
were left to pupate and sleeve cages were placed around the plants at
least 1 week before expected adult emergence. In circles with
nematode release, the infection rate for larvae on Graf (43.6% of
the recovered larvae) was more than fivefold that for larvae on
Pactol (8.3% of the recovered larvae; Fig. 4b). This nematode effect
was also evident from a significantly lower emergence of adults from
Graf roots (Fig. 4c).
More direct evidence for the importance of (E)-
b
-caryophyllene
was obtained with a second experiment with only the Pactol variety
planted in the six-plant circles. Again, all plants were infested with
six WCR larvae. The soil directly next to three of the plants per circle
was spiked on a daily basis with 2
m
lof(E)-
b
-caryophyllene for 5
days (Fig. 5a). One day after the first spiking (7 days after WCR
infestation), about 10,000 nematodes were released in the centre of
each circle; this was repeated twice at 2-day intervals. We recovered
relatively few larvae (18% as opposed to 40% for the Pactol–Graf
experiment) from the 12 circles that had been reserved to measure
infection rates. This was probably due to poor irrigation of these
circles, which could also explain why we did not observe a difference
in infection rate between treatments. However, the results from the
24 circles that were left to measure adult emergence showed a
significant effect of (E)-
b
-caryophyllene, with a more than twofold
decrease in adult emergence for the plants that had been spiked with
the signal (Fig. 5b).
The possibility that there could have been a direct effect of (E)-
b
-
caryophyllene on the WCR larvae or on the quality of the plant
was tested in subsequent laboratory experiments. Equal amounts of
(E)-
b
-caryophyllene to those in the field experiments were injected
in 15 0.5-litre pots each containing a maize plant and five WCR
larvae, whereas 15 other pots each containing a plant and five larvae
Figure 2 Attraction of H. megidis to authentic (E)-
b
-caryophyllene. a, Average number of
nematodes recovered from olfactometer arms connected to a pot spiked with 0.2
m
lof
(E )-
b
-caryophyllene compared with those recovered from arms connected to
untreated pots (n ¼ 12). b, Average number of nematodes recovered from olfactometer
arms connected to a pot with a healthy maize plant and spiked with 0.2
m
lof
(E )-
b
-caryophyllene, an arm connected to a pot with a healthy plant only, and four control
pots with moist sand only (n ¼ 12). For each replicate, the results for control pots were
summed and divided by the number of control pots. Different letters above bars indicate
significant differences. Error bars indicate standard errors.
Figure 3 The absence of the (E)-
b
-caryophyllene signal in certain maize genotypes
renders these plants unattractive to the nematode. a, Average amounts of (E )-
b
-
caryophyllene detected from the WCR-damaged roots of Zea mays parviglumis (teosinte),
of three lines (Du101, F2 and F268) that are known to release (E )-
b
-caryophyllene from
their leaves in response to caterpillar damage and of three lines (F584, A654 and F7001)
that release no detectable amounts of (E )-
b
-caryophyllene from their leaves
18
. b, Average
amount of (E )-
b
-caryophyllene extracted from WCR-damaged roots of the commercial
maize varieties Delprim, Graf and Pactol (n ¼ 6). c, Average number of nematodes
recovered from olfactometer arms connected to pots holding WCR-damaged maize plant
of the varieties Delprim, Graf and Pactol (n ¼ 12). d, Average number of nematodes
recovered from olfactometer arms connected to pots holding WCR-damaged maize plant
of the varieties Delprim, Graf and Pactol, after the pot with Pactol was spiked with 0.2
m
lof
(E )-
b
-caryophyllene (n ¼ 12). Statistical differences are indicated with different letters
above the bars. Error bars indicate standard errors.
3
served as controls. No difference was found in the total number of
adults that emerged from these pots (data not shown), supporting
the hypothesis that nematode attraction to (E)-
b
-caryophyllene was
responsible for the difference observed in the field.
Suitability of (E)-
b
-caryophyllene as a belowground signal
(E)-
b
-Caryophyllene is a common secondary plant compound that
is also emitted from the silk of mature maize plants and has been
shown to be weakly attractive to adult WCR females
21
.This
sesquiterpene is probably not the only attractant for H. megidis,
because some degree of nematode attraction was also found to
healthy and mechanically damaged plants (Fig. 1b), even though
emission of (E)-
b
-caryophyllene from maize leaves and roots has
been detected only after herbivory. Indeed, several plant metabo-
lites, including CO
2
, are known to be attractants for entomopatho-
genic nematodes
22
. Cues that come directly from host larvae might
also guide nematodes
23–26
,butthesehavebeenshowntobe
attractive only over short distances. The overriding importance of
(E)-
b
-caryophyllene as a long-range attractant is best indicated by
its abundance in the root extracts of the most attractive varieties and
the fact that supplementing sand with (E)-
b
-caryophyllene renders
an otherwise unattractive variety highly attractive (Fig. 3d).
To test the ability of (E)-
b
-caryophyllene to diffuse in moist sand,
2
m
g of this sesquiterpene were pipetted into one spot in a sand-filled
glass dish. At a distance of 10 cm from this spot a SPME fibre was
inserted into a hole in the sand (see Methods). Every half hour the
compounds adsorbed on the fibre were desorbed and analysed by
GC–MS, starting with the half hour before the addition of (E)-
b
-
caryophyllene. (E)-
b
-Caryophyllene travelled rapidly through the
sand and was already trapped on the fibre during the first half hour
after it had been introduced to the sand. The amount trapped
increased steadily for 2 h, after which it decreased sharply (Fig. 6a).
A similar experiment in a sand-filled olfactometer, with an arm
modified to permit the introduction of a SPME fibre, revealed the
presence of (E)-
b
-caryophyllene in the centre part of an arm 2 h
after injecting 0.2
m
l into a pot connected to that arm (not shown).
To determine whether the rapid decrease in (E)-
b
-caryophyllene
detection was due to evaporation from the sand, an additional
experiment was performed by which a drop containing 1
m
gof(E)-
b
-caryophyllene was placed on the bottom of a beaker, which was
immediately covered by 5 cm of moist sand. The beaker was placed
in a closed-loop volatile-collection system where the headspace
above the sand was continuously sampled at intervals of 30 min. A
very similar time course of (E)-
b
-caryophyllene diffusion was
obtained, with the first detection after 30 min and a peak after 2 h
(Fig. 6b), indicating rapid evaporation. Recovery was more than
90%, which implies that the degradation of (E)-
b
-caryophyllene or
its immobilization to sand particles is not significant under these
conditions. The rapid diffusion of (E)-
b
-caryophyllene in moist
sand and its chemical stability seem to make it exceptionally suitable
as a belowground signal. In the olfactometer assays described above,
the nematodes were released 25 cm from the treatment pots. There-
fore, after detecting the signal they move a distance of more than 250
times their body length within a day.
Figure 4 More WCR larvae were infected with nematodes and fewer adults emerged near
Graf plants than near Pactol plants. a, Design of field circle experiment for which maize
plants of the varieties Pactol and Graf were alternated. The cross marks the spot at
which nematodes were released. b, Mean numbers of larvae per plant that were
healthy (white areas), infected by fungi (grey areas) or infected by nematodes (black
areas). Statistical differences between the proportions of the three larval types are
indicated with different letters. c, The mean number of adults that emerged for each plant
was significantly different for the two varieties (P , 0.01). Error bars indicate standard
errors.
Figure 5 Fewer WCR adults emerged near Pactol plants that were spiked with
(E )-
b
-caryophyllene than near Pactol plants that received no (E )-
b
-caryophyllene.
a, Design of field circle experiment with only plants of the variety Pactol. The
w
signs mark
the sites at which five times 2
m
lof(E )-
b
-caryophyllene was injected into the soil; the
cross marks the spot at which nematodes were released. b, The mean number of adults
that emerged near the spiked plants was significantly lower than for the unspiked plants
(P , 0.0001). Error bars indicate standard errors.
4
Discussion
The failure of most North American maize lines to release (E)-
b
-
caryophyllene suggests that the ability to produce this compound
has been lost during breeding. Indeed, the closest wild ancestor of
maize, Zea mays ssp. parviglumis
19,20
, was also found to release (E)-
b
-caryophyllene from its roots in response to WCR damage
(Fig. 3a). The loss of direct defences to herbivores during plant
domestication has been amply documented
27
. However, to our
knowledge this is the first example of the loss of a signal involved
in indirect defence.
WCR has already caused large economic losses to maize in
Central Europe. Since 2003 it has been detected in almost all
European countries south of Scandinavia, and will inevitably
become a major threat to maize cultivation throughout Europe
28
.
Effective, ecologically sound control methods are needed. Entomo-
pathogenic nematodes could be an option
14,29–31
, but they have not
yet been employed with sufficient efficacy. The results of this study
lead us to speculate that the absence of an attractive signal in many
American maize lines could explain why attempts to control WCR
with nematodes have yielded only mixed results on the North
American continent
32,33
. Reintroduction of this signal in newly
developed maize varieties might aid in effective control of this
voracious pest.
This first identification of an inducible belowground plant signal
that attracts enemies of root-feeding herbivores underscores the
breadth and sophistication of indirect plant defences. With a
growing interest in belowground plant-mediated interactions and
their effects on various trophic levels
34,35
our results should prompt
new studies into the evolutionary history and ecological conse-
quences of multitrophic-level interactions and should lead to the
exploitation of the signal for crop protection. A
Methods
Olfactometer assays
The attraction of nematodes to plant-produced substances was tested in a belowground
olfactometer consisting of a central glass chamber (8 cm in diameter, 11 cm deep) with six
equally distributed side arms at 0.5 cm height with a female (24 mm diameter £ 29 mm
long) connector (Fig. 1a). These arms connected the central chamber with six glass pots
(5 cm in diameter, 11 cm deep) in which plants or other sources of attractants could be
placed. Each pot also had a female connector (29/32) at 0.5 cm height. The connecting
arms consisted of two detachable parts; one was a glass tube with ground-glass connectors
(male, 24/29) on both sides, and the second part, a Teflon connector (24/29 to 29/32) was
used to attach the glass tube to the odour source pot. The custom-made Teflon connectors
(Analytical Research Systems) contained an ultra-fine metal screen (2,300 mesh; Small
Parts Inc.) preventing the nematodes from reaching the odour source pots (Fig. 1a). For
each experiment, the entire system was filled with sterilized white sand (Migros) to
about 5 cm from the rim of the pots. Nematodes were released in a drop of water in the
centre of the central pot. One day after nematode release, the olfactometer was
disassembled and the sand in each detachable glass tube was placed on a separate cotton
filter disk 19 cm in diameter (Hoeschele GmbH). The disk with the sand was placed in a
Bearmann extractor
36,37
, and nematodes in the collection tube were counted on the next
day.
Statistical differences in choices made by the nematodes were determined with
log-linear models on the basis of the assumption that the nematodes would disperse
equally among the arms in the absence of any attraction. The models were adapted to
account for possible overdispersion due to directional biases
38
.
Root analyses
For the analysis of volatile terpenes, roots of WCR-damaged and undamaged maize plants
were washed with water and frozen in liquid nitrogen; they were then pulverized in a
mortar and 0.4 g of root powder was placed in a glass vial with a septum in the lid. A
100-
m
m polydimethylsiloxane (PDMS) SPME (Supelco) fibre was inserted through the
septum and exposed for 60 min at 40 8C. The compounds adsorbed on the fibre were
analysed by GC–MS with an Agilent 6890 Series GC system G1530A coupled to a
quadrupole-type mass-selective detector (Agilent 5973; transfer line 230 8C, source 230 8C,
ionization potential 70 eV). The fibre was inserted manually into the injector port (230 8C)
and desorbed and chromatographed on an apolar column (DB5-MS, 30 m, 0.25 mm
internal diameter, 0.25
m
m film thickness; J & W Scientific). Helium at a constant pressure
of 18.55 lb in
22
(127.9 kPa) was used for carrier gas flow. After fibre insertion, the column
temperature was maintained at 50 8C for 3 min and then increased to 180 8Cat58Cmin
21
followed by a final stage of 3 min at 250 8C. Approximate quantification was performed
with an external standard by performing analyses on 0.4 g of powdered root tissue from
maize line B73 (which produces only traces of (E )-
b
-caryophyllene)
39
spiked with known
amounts (4.5, 9.0, 45 and 90 ng) of this compound.
The (E)-
b
-caryophyllene in the roots was provisionally identified as the
(2)-enantiomer by chromatography on a chiral column using published procedures
for the separation of the two enantiomers
40
. However, the lack of a standard for the
(þ)-enantiomer prevented final confirmation.
Field experiments
Field experiments were conducted at the Plant Health Station in Hodme
´
zo
¨
va
´
sa
´
rhely, in
southern Hungary (468 15.554
0
N, 208 09.743
0
E), from April to October 2004. Six plants
were grown from seed in 1-m-diameter circles and with a 1-m distance between circles.
Two types of circle were formed: one contained the two varieties Zea mays var. Pactol
(Syngenta) and Z. mays var. Graf (Landi) and the other contained only the Pactol variety.
Eight weeks after planting, each plant was infested with six WCR larvae by digging out
5 cm of soil near the base of the plant and dropping the larvae with some potting soil into
the hole. The larvae came from a laboratory colony that had been established with
field-collected adults the year before. At 7, 9 and 11 days after infestation, about 10,000
H. megidis nematodes were released in the centre of the treatment circles at a depth of
about 10 cm. Additionally, half of the plants in the circles with only the Pactol variety were
spiked daily with 2
m
lof(E)-
b
-caryophyllene (more than 98% pure; Sigma-Aldrich) for 5
days, starting on the sixth day after infestation with WCR larvae (1 day before nematode
release).
Two measurements were taken to determine the effect of the treatments on nematode
effectiveness. For 15 of the Pactol–Graf circles and 12 of the Pactol–Pactol circles, the aerial
part of the plants was removed and with a 1-litre core sample the roots and soil around it
were collected. Larvae were extracted by crumbling the soil over a black plastic sheet and
dissecting the roots. Each recovered larva was placed on a moist filter paper in a plastic
Petri dish (5 cm in diameter, 2 cm deep) and stored at 17 8C for 1 month. They were
checked weekly under a microscope for nematode infection, characterized by red
pigmentation resulting from symbiotic bacteria, and for nematode emergence. Infections
by other pathogens were also noted.
In addition, adult emergence was measured in another 18 Pactol–Graf circles and 24
Pactol–Pactol circles. For this, cylindrical sleeve cages (30 cm £ 70 cm, MegaView Science
Education Services Co. Ltd) were fixed on plastic cylinders 20 cm in diameter and 25 cm
deep that were placed about 10 cm in the soil around each plant. The upper part of each
sleeve was tightly attached around the stem of the plant to prevent adults from escaping.
Once a week, from the beginning of July until the end of August, adults in the emergence
cages were counted and collected until no more adults were found. The same log-linear
models as employed for the olfactometer data were used to determine differences between
treatments
38
.
Diffusion measurements
A glass dish (15 cm in diameter, 8 cm deep) was filled with a 5-cm layer of moist (10%
water) sand. With a micropipette, 2
m
g of authentic (E )-
b
-caryophyllene (98% pure;
Aldrich) in 10
m
l of pentane was placed 3 cm deep in the sand at 2 cm from the dish side,
immediately after which the hole was covered. At a distance of 10 cm from this spot, a hole
2 mm wide and 3 cm deep was made with a metal rod and a 100-
m
m PDMS SPME fibre was
placed in the hole. Every 25 min the compounds adsorbed on the fibre were analysed by
GC–MS essentially as described above, except that the mass selective detector was operated
in the selective ion mode, scanning only for the characteristic ions at molecular masses
204, 133 and 93. After the 5-min desorption period, the fibre was placed back in the hole
in the sand for a further 25-min collection. The first collection started 30 min before the
(E)-
b
-caryophyllene sample was added to the sand, and the last collection was 7 h later.
To measure the time course of evaporation from sand, a 10-
m
l drop of dichloromethane
containing 1
m
gof(E)-
b
-caryophyllene was placed on the bottom of a 25-mm diameter
glass beaker and was immediately covered by a 5-cm layer of 30 ml moist sand. The beaker
was placed in a closed-loop volatile collection system consisting of a 1-litre desiccator in
which the headspace above the sand was continuously collected by pulling air through a
75-mg activated charcoal filter at a rate of 2 l min
21
. The filter was extracted with
dichloromethane at intervals of 30-min and the eluate was analysed by GC–MS as
described above.
Figure 6 (E )-
b
-Caryophyllene diffuses readily through sand and then evaporates rapidly
without breakdown or irreversible adsorption. a, Detection of authentic (E )-
b
-
caryophyllene with a SPME fibre in moist sand at 10 cm from a release point, every half
hour after release. b, Detection of (E )-
b
-caryophyllene in the headspace above a beaker
containing 5 cm of moist sand after (E )-
b
-caryophyllene had been placed at the bottom of
the beaker.
5
