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DOI: 10.1111/eea.


Root damage to apple plants by cockchafer larvae

induces a change in volatile signals below- and aboveground
John Abraham*, Valentino Giacomuzzi & Sergio Angeli
Faculty of Science and Technology, Free University of Bozen-Bolzano, Piazza Universita 5, 39100 Bolzano, Italy
Accepted: 2 June 2015

Key words: intra-plant communication, plantplant communication, systemic VOCs, priming,

talking trees, cry for help, eavesdropping, Rosaceae, Melolontha melolontha, Coleoptera, Scarabaeidae


Volatile organic compounds (VOCs) mediate communication between plants and insects. Plants
under insect herbivore attack release VOCs either at the site of attack or systemically, indicating
within-plant communication. Some of these VOCs, which may be induced only upon herbivore
attack, recruit parasitoids and predatory insects to feed on the attacking insects. Moreover, some
plants are able to eavesdrop on herbivore-induced plant volatiles (HIPVs) to prime themselves
against impending attack; such eavesdropping exemplifies plantplant communication. In apple
orchards, the beetle Melolontha melolontha L. (Coleoptera: Scarabaeidae) is an important insect pest
whose larvae live and feed on roots for about 4 years. In this study, we investigated whether the feeding activity of M. melolontha larvae (1) alters the volatile profile of apple roots, (2) induces the release
of HIPVs systemically in the leaves, and (3) whether infested plants communicate to neighbouring
non-infested conspecifics through HIPVs. To answer these questions, we collected constitutive VOCs
from intact M9 roots as well as M. melolontha larvae-damaged roots using a newly designed rhizobox, to collect root-released volatiles in situ, without damaging the plant root system. We also collected VOCs from the leaf-bearing shoots of M9 whose roots were under attack by M. melolontha
larvae and from shoots of neighbouring non-infested conspecifics. Gas chromatography-mass spectrometry analysis showed that feeding activity of M. melolontha larvae induces the release of specific
HIPVs; for instance, camphor was found in the roots only after larvae caused root damage. Melolontha
melolontha also induced the systemic release of methyl salicylate and (E,E)-a-farnesene from the
leaf-bearing shoots. Methyl salicylate and (E,E)-a-farnesene were also released by the shoots of noninfested neighbouring conspecifics. These phenomena indicate the induction of specific VOCs
below- and above-ground upon M. melolontha larvae feeding on apple roots as well as plantplant
communication in apple plants.

Plants under insect herbivore attack are able to defend
themselves by releasing a range of chemicals that directly
protect them, including alkaloids, terpenes, and phenolic
compounds (Baldwin et al., 2006). They may also release
chemicals that could defend them indirectly by attracting
enemies of their enemies (i.e., predators and/or parasitoids), a phenomenon that is described as crying for help

*Correspondence and present address: John Abraham, Department

of Entomology and Wildlife, School of Biological Sciences, University
of Cape Coast, Ghana. E-mail:

(Whitfield, 2001; Gershenzon, 2007; Bruinsma & Dicke,

2008; Dicke, 2009; Dicke & Baldwin, 2010; Kaplan, 2012).
It has also been established that plants under attack from
herbivorous insects are able to warn their neighbours of
impending danger; additionally, neighbours of plants
under attack eavesdrop on chemicals released by these
plants and by so doing, prepare to defend themselves
before they are attacked (Baldwin & Schultz, 1983; Karban
et al., 2000; Baldwin et al., 2006; Heil & Silva Bueno,
2007). This occurrence is now popularly known as the
talking trees phenomenon.
It is clear from these phenomena that plants are able
to communicate and they do so through the language
of volatile organic compounds (VOCs). Furthermore,

2015 The Netherlands Entomological Society Entomologia Experimentalis et Applicata 156: 279289, 2015


280 Abraham et al.

predatory arthropods are known to follow odour plumes

of herbivore-induced plant volatiles (HIPVs) to navigate
their way towards target prey which may be feeding or ovipositing on host plants (Fatouros et al., 2012). For example, recent olfactometer and wind tunnel studies showed
that the parasitic wasps Trichogramma brassicae Westwood and Cotesia glomerata L. were attracted to volatile
compounds of a wild crucifer (Brassica nigra L.) induced
through contact of their leaves with eggs of Pieris brassicae
L. (Fatouros et al., 2012). In addition, neighbours of plants
under herbivore attack are primed for impending attack
through sensing HIPVs (Karban et al., 2000; Engelberth
et al., 2004; Baldwin et al., 2006; Kessler et al., 2006). For
example, (Z)-3-hexen-1-yl acetate was produced in
undamaged maize plants when exposed to woundinduced green leaf volatiles (GLVs), indicating plantplant
communication (Yan & Wang, 2006). This further triggered the release of the terpenoid (E)-4,8-dimethyl-1,3,7nonatriene, a typical defence compound likely to prime
them against attack (Yan & Wang, 2006). Moreover, herbivory on Lima bean plants (Phaseolus lunatus L.)
decreased when they were exposed to insect-induced volatiles released by conspecifics, another example of priming
among plants (Heil & Silva Bueno, 2007).
Besides plantplant communication in which receiving plants are primed, within-plant communication is
also possible. It has been found that herbivory by the
tobacco hornworm, Manduca sexta L., on lower leaves
of tomato plants (Solanum lycopersicum L.) elicited the
release of the monoterpenes a-pinene, -pinene, 2-carene, and -phellandrene, and the sesquiterpenes
-caryophyllene, a-humulene, and d-elemene, both at
the site of damage and systemically from the upper
undamaged leaves (Farag & Pare, 2002). Thus, HIPVs
may be released either at the site of herbivore attack or
systemically at other parts of the plant not under attack
(Heil & Ton, 2008) probably for priming. Indeed,
priming of plants against impending herbivore attack
helps them avoid possible heavy damage by herbivores
and increases resistance to the attack (Engelberth et al.,
2004; Baldwin et al., 2006; Turlings & Ton, 2006;
Rodriguez-Saona & Frost, 2010; Pastor et al., 2013).
Moreover, when undamaged leaves of P. lunatus were
exposed to volatiles released from damaged leaves of
the same plant, extra-floral nectar (EFN) secretions by
the undamaged leaves increased (Heil & Silva Bueno,
2007). On the other hand, when damaged leaves were
covered so that only internal signalling pathways were
possible, the release of EFN on undamaged leaves was
significantly lower, indicating that the VOCs were necessary to induce a full systemic response within the
plant (Heil & Silva Bueno, 2007). Thus, apparently the

same mechanism that primes neighbouring plants is

also responsible for intra-plant communication.
In apple [Malus 9 domestica Borkh. (Rosaceae)] orchards, Melolontha melolontha L. (Coleoptera: Scarabaeidae)
is one of the major insect pests (Zelger, 1996; Strasser,
2004). This pest spends about 98% of its 3- to 4-year life
cycle in the soil as egg, larva, and pupa (Fidler, 1936; Huiting et al., 2006; Ester et al., 2007). Melolontha melolontha
have three larval instars during their development, feeding
voraciously on the roots of many plant species including
annual and perennial plants (Huiting et al., 2006).
Although much is known about the ecology of M. melolontha adults above-ground (e.g., Reinecke et al., 2002a,b,
2005, 2006), very little information on the chemical ecology of the larvae is documented (e.g., Eilers et al., 2012;
Weissteiner et al., 2012). In below-ground interactions,
there is evidence that VOCs play a role in the search for
food resources (Wenke et al., 2010). Similarly, host plants
also utilise VOCs released below-ground to protect themselves from herbivory (Rasmann et al., 2012). For
instance, it was documented that feeding damage by larvae
of the maize pest, Diabrotica virgifera virgifera LeConte,
induces the release of (E)--caryophyllene, a sesquiterpene
that is attractive to entomopathogenic nematodes (Rasmann et al., 2005). A similar study was conducted on citrus where it was found that feeding damage by the root
weevil, Diaprepes abbreviatus L., induces the release of
terpenes which are attractive to the entomopathogenic
nematode Steinernema diaprepesi sp. nov. (Ali et al.,
2010). These two examples indicate that HIPVs released
below-ground have similar ecological functions as those
released above-ground in response to insect herbivore
attack. Unfortunately, the revelation that D. abbreviatesinduced terpenes from the roots of citrus are attractive to
enthomopathogenic nematodes is hampered by a new revelation that the same terpenes also attract phytopathogenic
nematodes (Ali et al., 2011). This means those terpenes
cannot be used in controlling the root weevils.
As the larvae of our study insect, M. melolontha, live in
soil and feed for a long time, we investigated whether the
feeding activity of M. melolontha larvae (1) alters the volatile profile of the roots of apple plants; (2) induces a systemic release of HIPVs above-ground in response to root
damage; and (3) induces chemical communication
between infested plants and their neighbouring noninfested conspecifics.

Materials and methods

Novel rhizobox for root volatile collection

To investigate the apple root volatiles without the need to

remove the root from the soil and the risk of causing

Cockchafer larvae induces HIPVs in Malus domestica 281

mechanical damage, a novel rhizobox was designed to

allow direct collection of VOCs released by the root into a
second empty compartment through a porous membrane
(Figure 1). The rhizobox was constructed with a white
foamed PVC Forex sheet (Tecnomag, Bolzano, Italy). It
consisted of two compartments (A: 520 ml, B: 430 ml)
separated by a diagonal window with a nylon monofilament gauze (30 lm mesh; Georg Becker Laboreinrichtungen, Vienna, Austria). We placed our plants, soil, and, if
applicable, insect larvae in compartment A. The bottom of
this compartment had four drainage holes, allowing extra
water to drain. Plant roots developed and branched in A
near the diagonal gauze. Compartment B was used to collect the volatile compounds released through the gauze
from the root-containing soil. The upper part of this compartment had two customised holes, to insert two Teflon
tubes for the circulation of air inside the compartment.


Malus 9 domestica rootstock M9 is one of the most popular rootstocks used in commercial apple production orchards. It is a dwarfing rootstock with a better water-use
efficiency than other rootstocks, making it popular among
apple growers (Soumelidou et al., 1994; Li et al., 2002).
Clones of overwintering M9 rootstocks without leaves
were obtained from a plant nursery (Fairplant, Luttelgeest,
The Netherlands). To enable the collection of constitutive
volatiles from the M9 roots, 10 rootstocks were cut to 12
nodes long and planted singly in rhizoboxes. Another
28 M9 rootstocks (also 12 nodes long) were planted in
800-ml plastic pots. The planting medium for both rootstocks in rhizoboxes and plastic pots was TerraBrill peat
moss (Agrochimica, Bolzano, Italy). The planted M9 rootstocks were placed in a growth chamber (Snijders Scientific, Tilburg, The Netherlands) with ca. 70  10% r.h.,
L16 (26 C):D8 (20 C) photoperiod, and ca. 10 000 lux
illuminance. The rootstocks were watered 39 per week
and they developed leaves after 3 weeks.

Third instars of M. melolontha with head capsule width

measuring 5.56.5 mm (Malysh & Frolov, 2009) were collected from an infested apple orchard in Trentino, Italy.
They were reared in pairs in plastic boxes filled with peat
moss in the laboratory at ca. L16(26 C):D8(20 C) photoperiod, ca. 2 500 lux illuminance, and ca. 70  10%
r.h. They were fed pieces of organically grown carrots
[Daucus carota L. (Apiaceae)] 29 per week. They were fed
only once during the week prior to the experiments.
Volatile samples collection

Figure 1 Rhizobox with two compartments. Compartment (A)
contains soil, plant, and insect larvae (520 ml) and in
compartment (B) root volatiles are sampled (430 ml). C is a
diagonal window made of nylon monofilament gauze (30 lm
mesh) to separate the two compartments. In the bottom of
compartment A, drainage holes are drilled (D) and through the
cover of compartment B two holes (E), to allow the passage of
Teflon tubes connected to the valves of a small graphite pump to
circulate air for volatile collection.

VOCs were collected from M. 9 domestica M9 by closedloop-stripping-analysis (CLSA) (Boland et al., 1984; Kunert et al., 2009). An absorbent trap loaded with 1.5 mg of
activated charcoal (CLSA-filter; Granicher Daniel, Daumazan sur Arize, France) was attached to a Teflon tube
fixed to the inlet valve of a small 12-V graphite vacuum
pump (F
urgut, Tannheim, Germany); a Teflon tube was
also attached to the outlet valve. These were passed
through the two holes at the top of the rhizobox (Figure 1), such that the pump circulated air at a rate of 1 l
per min within the rhizobox and trapped VOCs from the
root-containing soil onto the activated charcoal. In
the case of VOC sampling from leaf-bearing shoots of M9,
the shoots bearing ca. 2530 leaves were enclosed within a
Cuki oven bag (Cofresco, Volpiano, Italy) and the graphite vacuum pump with the CLSA filter attached was fixed
at the top of the enclosure. The pump was powered by
7.5 V of energy from a laboratory DC power supply (GW
Instek, Shanghai, China). VOCs were collected in three

282 Abraham et al.

experiments described below; each collection process

lasted 3 h.
All trapped VOC samples were eluted from the CLSA
filters with 100 ll of GC grade dichloromethane (SigmaAldrich, Milan, Italy). The elutes were stored in a freezer at
80 C pending gas chromatography-mass spectrometry
(GC-MS) analysis.

non-infested ones simultaneously after 3, 7, and 14 consecutive days after introduction of the larvae (n = 6 per sampling time). As control, another set of non-infested M9
rootstocks was placed in another growth chamber with the
same conditions and their VOCs were collected in the
same sequence as above (n = 6).
Standard compounds

Experiment 1

To investigate whether feeding damage on roots by

M. melolontha larvae alters the volatile profile of the roots,
we collected constitutive and M. melolontha damageinduced VOCs from the roots of M9 rootstocks planted in
rhizoboxes. Constitutive VOCs were collected from
undamaged roots in 10 replicates, as described above.
Immediately after the collection of constitutive VOCs
from the roots, three third instars of M. melolontha were
introduced into the peat moss and VOCs were collected
after 3, 7, and 14 consecutive days (10 replicates per sampling time). As control, the volatiles released by the rhizobox filled only with peat moss and three M. melolontha
larvae were collected using the same protocol (four replicates).
Experiment 2

To investigate whether systemic release of HIPVs aboveground occurs in response to M. melolontha larval damage
on the roots, we collected both constitutive and induced
VOCs from the leaf-bearing shoots of M9 rootstocks
planted in pots with intact and M. melolontha-damaged
roots, respectively (n = 10 each). After the collection of
constitutive VOCs from root-undamaged M9 shoots,
three third-instar M. melolontha were immediately introduced into the peat moss in which the M9 were planted
and the VOCs collection process was repeated 3, 7, and 14
consecutive days after the introduction of the larvae with
the same protocol. To detect potential contaminants
released by the oven bags, the VOCs within six empty oven
bags were collected.
Experiment 3

To investigate whether chemical communication occurs

between infested M9 plants and their neighbouring noninfested conspecifics, we collected VOCs from the leafbearing shoots of M9 rootstocks, which were under attack
by M. melolontha larvae and those of neighbouring conspecifics which were not under attack. For experimental
purposes, we placed a non-infested M9 rootstock 20 cm
away from each of their conspecifics which were under
M. melolontha attack. VOCs were collected from the leafbearing shoots of both M. melolontha-infested plants and

Reference standard compounds were purchased from

Sigma-Aldrich to aid identification and quantification
of volatile compounds collected from the M9 rootstock:
(Z)-3-hexen-1-yl acetate, 98%; 2-ethyl hexan-1-ol,
99.6%; 3-methylbutyl butanoate, 98%; linalool, 95%;
nonanal, 95%; camphor, 95.5%; (Z)-3-hexen-1-yl butanoate, >98%; methyl salicylate, 99%; decanal, 98%; caryophyllene, 80%; farnesene, mixture of isomers; and
1-hexadecanol, >90%.
GC-MS analysis

The VOCs were analysed in a networked GC system

(7890A) coupled with an MS (5975C Network) (Agilent
Technologies, Santa Clara, CA, USA). The GC had a nonpolar HP-5MS column (30 m 9 0.25 mm ID, 0.25 lm
film thickness; Agilent Technologies). Aliquots of 2 ll of
volatile sample were injected into the GC in the splitless
mode when the inlet valve was 280 C. Helium was used
as carrier gas at a flow rate of 1.2 ml per min and a velocity
of 39.92 cm s 1. Starting temperature was 50 C held for
1.5 min, followed by an increase of 7.5 C per min until a
temperature of 250 C was reached and held for 10 min.
The mass range of the mass spectra was 20400 amu. Data
acquisition was carried out using ChemStation software
(Agilent Technologies). All volatile compounds were initially identified by comparing their mass spectra with those
in the databases of NIST 11 (Gaithersburg, MD, USA) and
Wiley 7N (Wiley, Hoboken, NJ, USA). The identities of 12
of 14 compounds were confirmed by comparing their
mass spectra and retention times with those of the reference standard compounds. The linear retention indices of
the compounds were calculated using the retention times
of n-alkane series from C9 to C20 as reference compounds
(Van den Dool & Kratz, 1963) and were compared with
those already published.
Statistical analysis

To estimate the amount of volatiles emitted by the M9

rootstock, we purchased standards of the 12 available
compounds out of the total 14 identified. Five-point calibration curves were prepared for each corresponding
standard compound at 0, 0.01, 1, 100, and
10 000 lg g 1 in dichloromethane considering the total

Cockchafer larvae induces HIPVs in Malus domestica 283

ion current (TIC). We compared the amount of volatile

compounds emitted among experimental treatments
using the KruskalWallis test (SPSS version 20; IBM
SPSS, Armonk, NY, USA), followed by MannWhitney
U-test (IBM SPSS), where significant treatment effects
were observed.

VOCs from roots of M9 rootstocks with undamaged and Melolontha
melolontha-damaged roots

Analysis of volatile samples from intact and undamaged

M9 roots revealed five constitutive volatile compounds (2ethyl hexan-1-ol, nonanal, methyl salicylate, decanal, and
1-hexadecanol), which were not present in control rhizoboxes filled with only peat moss and M. melolontha larvae
(data not shown). Upon feeding by larvae of M. melolontha, camphor (root bark oil) was released (Table 1).
Methyl salicylate, that had been released constitutively,
was not detected in the volatile profile of roots fed on by
larvae of M. melolontha (Table 1; Figure 2B).
The amount of 2-ethyl hexan-1-ol released by undamaged roots of M9 decreased significantly after feeding by
larvae of M. melolontha for 7 and 14 days (v2 = 14.82,
d.f. = 3, P = 0.002; Figure 2A). The amount of decanal
released by undamaged roots was also significantly higher
than the amount released after 7 days of feeding by
M. melolontha larvae (U = 20.0, P = 0.023). Furthermore, the amount of 1-hexadecanol released by undamaged roots was significantly higher than the amount
released after 3 days (U = 23.5, P = 0.045) and 14 days
(U = 17.5, P = 0.014) of M. melolontha larvae feeding
damage. There was no difference in the amount of
non-anal released by undamaged and M. melolonthadamaged roots (v2 = 5.83, d.f. = 3, P = 0.12; Figure 2B).
Camphor was released only when the roots were fed on by
larvae of M. melolontha and the amount released after

Table 1 Mean ( SEM) volatile organic

compound emission rate (ng per 3 h)
from the roots of non-infested (n = 10)
and Melolontha melolontha-infested
(n = 30) M9 apple rootstocks

3 days of damage was significantly higher than that

released after 7 days (U = 21.0, P = 0.024) and 14 days
(U = 19.0, P = 0.012) (Figure 2B).
VOCs from leaves of M9 rootstocks with undamaged and Melolontha
melolontha-damaged roots

Ten constitutive VOCs were characterised from the leafbearing shoots of non-infested M9 rootstocks (Table 2).
When the roots of the M9 rootstocks were fed on by larvae
of M. melolontha, one of the constitutive VOCs (3-methylbutyl butanoate) was no longer detected in the volatile
profile; however, two HIPVs, [methyl salicylate and (E,E)a-farnesene] were released in addition to nine of the constitutive VOCs (Table 2).
(Z)-3-hexen-1-yl acetate and 2-ethyl hexan-1-ol were
released in higher amounts than virtually any other compound released by the leaf-bearing shoots of M9 rootstocks
(Figure 3A). The amount of (Z)-3-hexen-1-yl acetate
released constitutively was significantly higher than that
released by leaf-bearing shoots of M9 whose roots had been
damaged by M. melolontha larvae for 3, 7, and 14 consecutive days (v2 = 19.52, d.f. = 3, P<0.001). There was no difference in the amount of 2-ethyl hexan-1-ol released
constitutively and that released after M. melolontha larvae
damage (v2 = 3.63, d.f. = 3, P = 0.31) (Figure 3A).
There were differences in the amounts released of linalool, (Z)-3-hexen-1-yl butanoate, and -caryophyllene
(v2 = 24.93, 20.34, and 19.15, respectively; all d.f. = 3,
P<0.001); the amounts released constitutively were always
higher than those released after M. melolontha damage to
the roots (Figure 3B). 3-Methylbutyl butanoate was
released only constitutively. There were no statistical differences in the amounts of nonanal, methyl salicylate, decanal, and (E,E)-a-farnesene (Figure 3B). The amounts of
2-methyl pentadecane and 3-methyl pentadecane released
could not be quantified because the respective analytical
standards are currently not available on the market.









2-Ethyl hexan-1-ol
Camphor (root bark oil)
Methyl salicylate






LRI, linear retention index; reference LRI, retention indices already published in peerreviewed literature and listed on NIST WebBook. P values are based on MannWhitney

284 Abraham et al.


Undamaged roots


Day 3
Day 7



Day 14




Emission rate (ng per 3 h)

2-ethyl hexan-1-ol






a a








VOCs from leaves of Melolontha melolontha-damaged M9 and

neighbouring non-infested conspecics

The leaf-bearing shoots of M9 rootstocks whose roots were

infested with M. melolontha larvae released methyl salicylate and (E,E)-a-farnesene in addition to nine constitutive
volatiles (Table 2). The non-infested neighbouring M9
plants located 20 cm away also released methyl salicylate
and (E,E)-a-farnesene from their leaf-bearing shoots.
Moreover, methyl salicylate and (E,E)-a-farnesene were
not detected in the volatile profiles of a set of non-infested
M9 rootstocks used as control (Table 2). All volatile compounds that were released by the empty oven bags were
removed from the volatile profile of leaf-bearing M9
shoots (data not shown).
The amount of methyl salicylate released by the leafbearing shoots of M9 rootstocks whose roots had been
infested with M. melolontha larvae for 7 days was significantly higher than the amount released by the leaves of
their neighbouring non-infested conspecifics (U = 3.5,
P = 0.003). However, the amounts of methyl salicylate
released from the leaf-bearing shoots of both infested M9


Figure 2 Mean (+ SEM) emission rate (ng

per 3 h) of (A) 2-ethyl hexan-1-ol released
constitutively and (B) constitutive and
induced VOCs from roots of M9
rootstocks, after 3, 7, and 14 days of
Melolontha melolontha feeding on M9
apple roots. Means within a volatile
compound capped with different letters
are significantly different (MannWhitney
U-test: P<0.05). MeSA, methyl salicylate.

and neighbouring non-infested rootstocks were not significantly different after 3 days (U = 19.0, P = 0.21) and
14 days (U = 28.0, P = 0.82) (Figure 4A).
Similarly, the amount of (E,E)-a-farnesene released by
the leaf-bearing shoots of M9 rootstocks whose roots had
been infested with M. melolontha larvae for 7 days was significantly higher than the amount released by the leafbearing shoots of their non-infested neighbours (U = 2.0,
P = 0.002), whereas the amounts were not significantly
different 3 days (U = 18.0, P = 0.19) and 14 days
(U = 25.0, P = 0.59) after the roots of the M. melolonthainfested M9 were damaged (Figure 4B).

The results we have obtained using our novel rhizobox
have demonstrated the validity of this technique for collecting volatile samples from roots in situ without touching the roots after earlier trials (e.g., Ali et al., 2010, 2011,
2012). This new technique adds to the very few available
for intact root volatile collection (Campos-Herrera et al.,

Cockchafer larvae induces HIPVs in Malus domestica 285

Table 2 Mean ( SEM) volatile organic

compound (VOC) emission rate (ng per
3 h) from the leaf-bearing shoots of noninfested M9 apple and from leaf-bearing
shoots of M9 whose roots have been
infested with larvae of Melolontha melolontha

Leaves of M9 with




1008 1008

1030 1031

1059 1058


1102 1103
1107 1105
1188 1187




1563 1564


1570 1570


2-Ethyl hexan-1ol1
Methyl salicylate1


root at a


9160  2839 1289  318

2444  631

1451  325

5868  1752
452  73

110  56

97  17

173  26
120  10
81  11

28  9
113  16
27  5

83  9
63  7
45  8

79  6
378  70


34  4
114  38


+/ indicates presence/absence of VOCs that could not be quantified; LRI, linear retention
index; reference LRI, retention indices already published in peer-reviewed literature and
listed on NIST WebBook.
Identity confirmed by authentic standard compound.
Identity confirmed by published LRI.

2013; Robert et al., 2013). This technique enabled us to

detect differences in the volatile profiles of undamaged
and M. melolontha-damaged roots of M9. The roots
released typical plant volatiles comprising five constitutive
volatile compounds, consisting of two alcohols (2-ethyl
hexan-1-ol and 1-hexadecanol), two aldehydes (nonanal
and decanal), and one sesquiterpene (methyl salicylate).
Upon feeding by M. melolontha larvae, camphor was
released by the M9 roots; proving that feeding activity of
M. melolontha larvae on roots alters the volatile profile of
M. 9 domestica. Interestingly, camphor was also detected
in a recent investigation as an induced compound from
the roots of Quercus petraea (Mattuschka) Lieblein and
Quercus robur L. damaged by Melolontha hippocastani
Fabricius, a closely related species of M. melolontha
(Weissteiner et al., 2012). Although in that study camphor
was detected in the volatile blends of undamaged, mechanically damaged, and M. hippocastani-damaged Quercus
spp., the concentration detected in M. hippocastani-damaged Quercus spp. was 69 higher than that detected in
mechanically damaged, and 209 higher than in undam-

aged Quercus spp. (Weissteiner et al., 2012). This together

with our finding suggests that the release of camphor from
roots may be induced generally by the feeding activity of
Melolontha spp. Moreover, it is worth noting that the
undamaged roots of Quercus spp. referred to in Weissteiner et al. (2012) were actually washed with water after the
soil on them have been removed. These processes may
have caused some degree of mechanical damage to the
roots, hence the emission of camphor.
In our study, methyl salicylate and (E,E)-a-farnesene
were released systemically in the leaf-bearing shoots, when
there was M. melolontha attack on the roots. This indicates
within-plant chemical communication and proves that
there is an induced systemic release of HIPVs aboveground in response to M. melolontha damage on the roots
of the host plant. Methyl salicylate and (E,E)-a-farnesene
have already been characterised as induced compounds
released by plants under attack to defend themselves
(Engelberth et al., 2004; Kessler et al., 2006). As HIPVs,
they could either be released at the site of attack, or systemically at other parts of the plant not under attack (Heil &

286 Abraham et al.

14 000

Constitutive VOCs

Day 3

12 000

Day 7

10 000

Day 14


Emission rate (ng per 3 h)



(Z ) -3-hexen-1-yl acetate

2-ethyl hexan-1-ol















b bc



(Z ) -3-Hb



Ton, 2008). To the best of our knowledge, our finding is

the first record of systemic release of HIPVs in apple
plants. Previous studies have shown that other plants are
able to release chemicals systemically to protect themselves
from insect herbivory. This has been demonstrated in
tomato plants (S. lycopersicum) (Farag & Pare, 2002),
Lima bean plants (P. lunatus) (Heil, 2004; Heil & Silva Bu
eno, 2007; Heil & Adame-Alvarez,
2010), and blueberry
(Vaccinium corymbosum L.) (Rodriguez-Saona et al.,
2009). Recently, Rodriguez-Saona & Frost (2010) further
suggested that the release of HIPVs within a plant is primarily for the protection of undamaged parts of that plant
against impending attack.
Earlier studies also demonstrated that plants under
attack are capable of communicating to their conspecific
neighbours that are not under attack through the release
of HIPVs (Baldwin et al., 2006; Heil & Silva Bueno,

2007; Heil & Adame-Alvarez,
2010; Heil & Karban,
2010). In fact, communication between plants has been
shown to be more efficient between plants that are genetically identical (clones) (Karban & Shiojiri, 2009). M9
rootstocks are clones and plantplant communication



Figure 3 Mean (+ SEM) emission rate (ng

per 3 h) of (A) (Z)-3-hexen-1-yl acetate
and 2-ethyl hexan-1-ol and (B) VOCs
from leaf-bearing shoots of M9 apple
rootstocks constitutively and after larvae of
Melolontha melolontha have fed on their
roots for 3, 7, and 14 consecutive days.
Means within a volatile compound capped
with different letters are significantly
different (MannWhitney U-test: P<0.05).
Emission of 2-methyl pentadecane and 3methyl pentadecane could not be
quantified. 3-Mbb, 3-methylbutyl
butanoate; Lin, linalool; Non, nonanal;
(Z)-3-Hb, (Z)-3-hexen-1-yl butanoate;
MeSA, methyl salicylate; Dec, decanal;
-Cary, -caryophyllene; a-Far = (E,E)-afarnesene.

has been demonstrated by our experiments, indicating

that the volatile profile of non-infested M9 plants was
altered after they were placed within 20 cm radius of
M. melolontha-infested plants. In particular, the noninfested M9 released relatively small amounts of methyl
salicylate and (E,E)-a-farnesene when their neighbours
were under M. melolontha attack in preparation of any
eventual insect herbivore attack. Through this plant
plant communication, the M9 rootstocks that were under
attack were likely warning their neighbouring clones of
impending attack.
The release of camphor at the site of M. melolontha
attack and induction of methyl salicylate and (E,E)-afarnesene systemically in the leaf-bearing shoots of both
M9 with M. melolontha-damaged roots and neighbouring non-infested conspecifics is likely a defence strategy
of the M9 rootstock, as these VOCs are among typical
HIPVs (Arimura et al., 2005; Dicke & Baldwin, 2010).
Camphor is known to have insecticidal and repellent
properties (Kumar & Ando, 2003; Chen et al., 2013),
achieving a repellence of 80100% in the beetles Sitophilus granarius (L.), Sitophilus zeamais Motschulsky,

Cockchafer larvae induces HIPVs in Malus domestica 287


Undamaged roots






Emission rate (ng per 3 h)



Day 0

Day 3

Day 7

Day 14




Figure 4 Mean (+ SEM) emission rate (ng

per 3 h) of (A) methyl salicylate and (B)
(E,E)-a-farnesene from the leaf-bearing
shoots of Melolontha melolontha-infested
M9 apple rootstocks and their
neighbouring conspecific after the infested
M9 had been fed on for 3, 7, and 14
consecutive days. Means within a day
capped with different letters are
significantly different (MannWhitney
U-test: P<0.05).




Day 0

Tribolium castaneum (Herbst), and Prostephanus truncatus Horn (Obeng-Ofori et al., 1998). Recently, camphor
has also been confirmed to have toxic effect on T. castaneum although at relatively high doses (10.0 ll per
adult) achieving 78.5% mortality (Liska et al., 2010).
Likely, M9 rootstock release camphor at the site of
attack in an attempt to defend itself against the feeding
M. melolontha. However, behaviour studies with pure
(1R)-camphor and (1S)-camphor on M. hippocastani
did not show a clear repellent effect, although some
M. hippocastani were repelled (Weissteiner et al., 2012).
The induction of methyl salicylate and (E,E)-a-farnesene in the leaf-bearing shoots of the M. melolonthainfested M9 and neighbouring non-infested M9 were
likely to prime them against any possible impending
attack above-ground (Rodriguez-Saona & Frost, 2010;
Pastor et al., 2013). For instance, it has been shown
recently that fields treated with HIPVs could recruit as
many as 225 predators m 2 compared to 125 predators m 2 where the fields have not been treated with
HIPVs (Kaplan & Lewis, 2015). By attracting predators

Day 3

Day 7

Day 14

or parasitoids, plants are able to prepare to defend

themselves even before an attack occurs; however, the
downside is that pollinators may be adversely affected
(Kaplan, 2012; Farre-Armengol et al., 2013). This also
amounts to deceit of predators as in the case of the
flowers of Epipactis helleborine (L.) Crantz and Epipactis
purpurata Sm., which release damage-induced GLVs
that attract the wasps Vespula germanica (Fabricius)
and Vespula vulgaris L. (Brodmann et al., 2008).
In conclusion, with this study we have demonstrated
that feeding damage by larvae of M. melolontha on M9
rootstocks causes a release of HIPVs at the site of attack
and systemically from the leaf-bearing shoots that are not
under attack. Moreover, neighbouring M9 that are not
under attack are able to eavesdrop on the volatiles released
by the shoots of those whose roots are under attack. This
shows that there is chemical communication within and
between M9 rootstocks through the induction of HIPVs.
Our study adds to knowledge on tritrophic interactions in
apple orchards and may lead to the use of natural plant
defence strategies in apple-growing regions. Our results

288 Abraham et al.

could contribute to finding eco-friendly ways of controlling the Melolontha spp. damage in apple orchards and
forest ecosystems.

We are grateful to Damiano Zanotelli, Daniela Campana,
and Irene Castellan for their help during the search and
rearing of Melolontha melolontha larvae. We are also grateful to Walther Turk for his help in the construction of our
rhizoboxes. We thank Clive Nuttman for comments on an
early draft of the manuscript. This project was funded by
the Internal Research Funds of Free University of BozenBolzano, Italy.

Ali JG, Alborn HT & Stelinski LL (2010) Subterranean herbivore-induced volatiles released by citrus roots attract entomopathogenic nematodes. Journal of Chemical Ecology 36:
Ali JG, Alborn HT & Stelinski LL (2011) Constitutive and
induced subterranean plant volatiles attract both entomopathogenic and plant parasitic nematodes. Journal of Ecology
99: 2635.
Ali JG, Alborn HT, Campos-Herrera R, Kaplan F, Duncan LW
et al. (2012) Subterranean, herbivore-induced plant volatile
increases biological control activity of multiple beneficial nematode species in distinct habitats. PLoS ONE 7: e38146.
Arimura G, Kost C & Boland W (2005) Herbivore-induced, indirect plant defences. Biochimica et Biophysica Acta 1734: 91
Baldwin IT & Schultz JC (1983) Rapid changes in tree leaf chemistry induced by damage: evidence for communication
between plants. Science 221: 277279.
Baldwin IT, Halitschke R, Paschold A, von Dah CC & Preston CA
(2006) Volatile signaling in plant-plant interactions: Talking
trees in the genomics era. Science 311: 812815.
Boland W, Ney P, Jaenicke L & Gassman G (1984) A closedloop-stripping technique as a versatile tool for metabolic studies of volatiles. Analysis of Volatiles: Methods and Applications
(ed. by P Schreier), pp. 371380. De Gruyter & Co, Berlin, Germany.
Brodmann J, Twele R, Francke W, H
olzler G, Zhang Q-H & Ayasse M (2008) Orchids mimic green-leaf volatiles to attract
prey-hunting wasps for pollination. Current Biology 18: 740
Bruinsma M & Dicke M (2008) Herbivore-induced indirect
defence: from induction mechanisms to community ecology.
Induced Plant Resistance to Herbivory (ed. by A Schaller), pp.
3160. Springer, Berlin, Germany.
Campos-Herrera R, Ali JG, Diaz BM & Duncan LW (2013) Analyzing spatial patterns linked to the ecology of herbivores and
their natural enemies in the soil. Frontiers in Plant Science 4:

Chen W, Vermaak I & Viljoen A (2013) Camphor a fumigant

during the black death and a coveted fragrant wood in ancient
Egypt and Babylon a review. Molecules 18: 54345454.
Dicke M (2009) Behavioural and community ecology of plants
that cry for help. Plant, Cell and Environment 32: 654665.
Dicke M & Baldwin IT (2010) The evolutionary context for herbivore-induced plant volatiles: beyond the cry for help. Trends
in Plant Science 15: 167175.
Eilers EJ, Talarico G, Hansson BS, Hilker M & Reinecke A (2012)
Sensing the underground-ultrastructure and function of sensory organs in root-feeding Melolontha melolontha (Coleoptera: Scarabaeinae) larvae. PLoS ONE 7: e41357.
Engelberth J, Alborn HT, Schmelz EA & Tumlinson JH (2004)
Airborne signals prime plants against insect herbivore attack.
Proceedings of the National Academy of Sciences of the USA
101: 17811785.
Ester A, Griepink FC, Huiting HF & Moraal LG (2007) Control
of Grubs and Monitoring of the Cockchafer, Melolontha melolontha: Laboratory and Greenhouse Experiments Concerning
Control and Field. Research Report. Applied Plant Research,
Wageningen, The Netherlands.
Farag MA & Pare PW (2002) C6-Green leaf volatiles trigger local
and systemic VOC emissions in tomato. Phytochemistry 61:
Farre-Armengol G, Filella I, Llusia J & Pe~
nuelas J (2013) Floral
volatile organic compounds: between attraction and deterrence
of visitors under global change. Perspectives in Plant Ecology,
Evolution and Systematics 15: 5667.
Fatouros NE, Lucas-Barbosa D, Weldegergis BT, Pashalidou FG,
van Loon JJA et al. (2012) Plant volatiles induced by herbivore
egg deposition affect insects of different trophic levels. PLoS
ONE 7: e43607.
Fidler JH (1936) An investigation into the relationship between
chafer larvae and the physical factors of their soil habitat. Journal of Animal Ecology 5: 333347.
Gershenzon J (2007) Plant volatiles carry both public and private
messages. Proceedings of the National Academy of Sciences of
the USA 104: 52575258.
Heil M (2004) Induction of two indirect defences benefits Lima
bean (Phaseolus lunatus, Fabaceae) in nature. Journal of Ecology 92: 527536.

Heil M & Adame-Alvarez
RM (2010) Short signalling distances
make plant communication a soliloquy. Biology Letters 6:
Heil M & Karban R (2010) Explaining evolution of plant communication by airborne signals. Trends in Ecology and Evolution 25: 137144.
Heil M & Silva Bueno JC (2007) Within-plant signaling by volatiles leads to induction and priming of an indirect plant defense
in nature. Proceedings of the National Academy of Sciences of
the USA 104: 54675472.
Heil M & Ton J (2008) Long-distance signalling in plant defence.
Trends in Plant Science 13: 264272.
Huiting HF, Moraal LG, Griepink FC & Ester A (2006) Biology,
Control and Luring of the Cockchafer, Melolontha melolontha: Literature Report on Biology, Life Cycle and Pest

Cockchafer larvae induces HIPVs in Malus domestica 289

Incidence, Current Control Possibilities and Pheromones.

Research Report. Applied Plant Research, Wageningen, The
Kaplan I (2012) Attracting carnivorous arthropods with plant
volatiles: the future of biocontrol or playing with fire? Biological Control 60: 7789.
Kaplan I & Lewis D (2015) What happens when crops are turned
on? Simulating constitutive volatiles for tritrophic pest suppression across an agricultural landscape. Pest Management
Science 71: 139150.
Karban R & Shiojiri K (2009) Self-recognition affects plant communication and defense. Ecology Letters 12: 502506.
Karban R, Baldwin IT, Baxter KJ, Laue G & Felton GW (2000)
Communication between plants: induced resistance in wild
tobacco plants following clipping of neighboring sagebrush.
Oecologia 125: 6671.
Kessler A, Halitschke R, Diezel C & Baldwin IT (2006) Priming of
plant defense responses in nature by airborne signaling
between Artemisia tridentata and Nicotiana attenuata. Oecologia 148: 280292.
Kumar M & Ando Y (2003) Single-wall and multi-wall carbon
nanotubes from camphor-a botanical hydrocarbon. Diamond
Related Materials 12: 18451850.
Kunert M, David A, Becher J & Boland W (2009) Volatile sampling from biological sources by the closed-loop-stripping
technique. Cold Spring Harbor Protocols 6: pdb.prot5233.
Li F, Cohen S, Naor A, Shaozong K & Ereze A (2002) Studies of
canopy structure and water use of apple trees on three rootstocks. Agricultural Water Management 55: 114.
Liska A, Rozman V, Kalinovic I, Ivezic M & Balicevic R (2010)
Contact and fumigant activity of 1,8-cineole, eugenol and camphor against Tribolium castaneum (Herbst). Julius-K
uhn-Archiv 425: 716720.
Malysh JM & Frolov AN (2009) Melolontha melolontha L. Common Cockchafer, May-bug, May Beetle. 2003-2009 Project
(Interactive Agricultural Ecological Atlas of Russia and Neighboring Countries. Economic Plants and Their Diseases, Pests
and Weeds). Available at:
pests/Melolontha_melolontha/ (accessed 10 August 2013)
Obeng-Ofori D, Reichmuth CH, Bekele AJ & Hassanali A (1998)
Toxicity and protectant potential of camphor, a major component of essential oil of Ocimum kilimandscharicum, against
four stored product beetles. International Journal of Pest Management 44: 203209.
Pastor V, Luna E, Mauch-Mani B, Ton J & Flors V (2013) Primed
plants do not forget. Environmental and Experimental Botany
94: 4656.
Rasmann S, K
ollner TG, Degenhardt J, Hiltpold I, Toepfer S et al.
(2005) Recruitment of entomopathogenic nematodes by
insect-damaged maize roots. Nature 434: 732737.
Rasmann S, Hiltpold I & Ali J (2012) The role of root-produced
volatile secondary metabolites in mediating soil interactions.
Advances in Selected Plant Physiology Aspects (ed. by G
Montanaro & B Dichio), pp. 269290. InTech, Rijeka, Croatia.

Reinecke A, Ruther J & Hilker M (2002a) The scent of food and

defence: green leaf volatiles and toluquinone as sex attractant
mediate mate finding in the European cockchafer Melolontha
melolontha. Ecology Letters 5: 257263.
Reinecke A, Ruther J, Tolasch T, Francke W & Hilker M (2002b)
Alcoholism in cockchafers: orientation of male Melolontha
melolontha towards green leaf alcohols. Naturwissenschaften
89: 265269.
Reinecke A, Ruther J & Hilker M (2005) Electrophysiological and
behavioural responses of Melolontha melolontha to saturated
and unsaturated aliphatic alcohols. Entomologia Experimentalis et Applicata 115: 3340.
Reinecke A, Ruther J, Mayer CJ & Hilker M (2006) Optimized
trap lure for male Melolontha cockchafers. Journal of Applied
Entomology 130: 171176.
Robert CAM, Erb M, Hiltpold I, Hibbard BE, Gaillard MDP et al.
(2013) Genetically engineered maize plants reveal distinct costs
and benefits of constitutive volatile emissions in the field. Plant
Biotechnology Journal 11: 628639.
Rodriguez-Saona CR & Frost CJ (2010) New evidence for a
multi-functional role of herbivore-induced plant volatiles in
defense against herbivores. Plant Signaling & Behavior 5: 58
Rodriguez-Saona CR, Rodriguez-Saona LE & Frost CJ (2009)
Herbivore-induced volatiles in the perennial shrub, Vaccinium
corymbosum, and their role in interbranch signaling. Journal of
Chemical Ecology 35: 163175.
Soumelidou K, Battey NH, John P & Barnett JR (1994) The
anatomy of the developing bud union and its relationship to
dwarfing in apple. Annals of Botany 74: 605611.
Strasser H (2004) Biocontrol of important soil-dwelling pests by
improving the efficacy of insect pathogenic fungi. Laimburg
Journal 1: 236241.
Turlings TCJ & Ton J (2006) Exploiting scents of distress: the
prospect of manipulating herbivore-induced plant odours to
enhance the control of agricultural pests. Current Opinion in
Plant Biology 9: 421427.
Van den Dool H & Kratz PD (1963) A generalization of the retention index system including linear temperature programmed
gas-liquid partition chromatography. Journal of Chromatography A 11: 463471.
Weissteiner S, Huetteroth W, Kollmann M, Weibecker B,
Romani R et al. (2012) Cockchafer larvae smell host root
scents in soil. PLoS ONE 7: e45827.
Wenke K, Kai M & Piechulla B (2010) Belowground volatiles
facilitate interactions between plant roots and soil organisms.
Planta 231: 499506.
Whitfield J (2001) Making crops cry for help. Nature 410: 736
Yan ZG & Wang CZ (2006) Wound-induced green leaf volatiles
cause the release of acetylated derivatives and a terpenoid in
maize. Phytochemistry 67: 3442.
Zelger R (1996) The population dynamics of the cockchafer in
South Tyrol since 1980 and the measures applied for control.
Bulletin OILB/SROP 19: 109113.