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Abstract
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.
Introduction
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
2015 The Netherlands Entomological Society Entomologia Experimentalis et Applicata 156: 279289, 2015
279
Plants
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.
Insects
D
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
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 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
Results
VOCs from roots 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.
No.
LRI
Reference
LRI
Compound
Non-infested
root
1
2
3
4
5
6
1030
1107
1149
1198
1208
1879
1031
1105
1151
1197
1208
1879
2-Ethyl hexan-1-ol
Nonanal
Camphor (root bark oil)
Methyl salicylate
Decanal
1-Hexadecanol
763
63
0
32
102
43
85
8
14
18
8
Infested
root
499
52
24
0
70
27
56
7
12
11
5
P
0.002
>0.05
0.01
<0.001
0.04
0.028
LRI, linear retention index; reference LRI, retention indices already published in peerreviewed literature and listed on NIST WebBook. P values are based on MannWhitney
U-tests.
900
Undamaged roots
800
Day 3
Day 7
ab
700
Day 14
600
500
bc
400
300
200
100
0
2-ethyl hexan-1-ol
140
B
a
120
ab
100
80
ab
60
a a
ab
40
20
0
Nonanal
Camphor
MeSA
Decanal
1-hexadecanol
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).
Discussion
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.,
Leaves of M9 with
Reference
LRI
No.
LRI
1008 1008
1030 1031
1059 1058
4
5
6
1102 1103
1107 1105
1188 1187
7
8
9
10
1198
1208
1428
1510
11
1563 1564
12
1570 1570
1197
1208
1428
1510
Compound
(Z)-3-hexen-1-yl
acetate1
2-Ethyl hexan-1ol1
3-Methylbutyl
butanoate1
Linalool1
Nonanal1
(Z)-3-hexen-1-yl
butanoate1
Methyl salicylate1
Decanal1
-Caryophyllene1
(E,E)-afarnesene1
2-Methyl
pentadecane2
3-Methyl
pentadecane2
Non-infested
root
Non-infested
root at a
different
location
Infested
root
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
0
79 6
378 70
0
94
80
106
82
0
34 4
114 38
0
41
13
28
35
+/ 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.
1
Identity confirmed by authentic standard compound.
2
Identity confirmed by published LRI.
14 000
Constitutive VOCs
Day 3
12 000
Day 7
10 000
Day 14
8000
6000
4000
2000
500
(Z ) -3-hexen-1-yl acetate
2-ethyl hexan-1-ol
450
400
350
300
a
250
a
200
a
a
150
aa
100
50
0
a
a
a
c
3-Mbb
Lin
aa
cc
a
a
b bc
c
bc
Non
(Z ) -3-Hb
MeSA
Dec
-Cary
-Far
300
A
Undamaged roots
a
250
Melolontha-damaged
200
150
a
100
50
b
160
Day 0
Day 3
Day 7
Day 14
B
a
140
a
120
100
80
60
40
20
0
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
could contribute to finding eco-friendly ways of controlling the Melolontha spp. damage in apple orchards and
forest ecosystems.
Acknowledgement
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.
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