Directory UMM :Data Elmu:jurnal:B:Biochemical Systematics and Ecology:Vol28.Issue10.Dec2000:
Biochemical Systematics and Ecology 28 (2000) 933}947
Volatiles released from oak, a host tree for the
bark beetle Scolytus intricatus
PavlmH na Vrkoc\ ovaH *, Irena ValterovaH , Jan Vrkoc\ ,
BohummH r Koutek
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
Flemingovo na& me\ stn& 2, 166 10 Praha 6, Czech Republic
Received 15 November 1999; received in revised form 6 April 2000; accepted 10 April 2000
Abstract
Volatile compounds emitted in di!erent phases of oak (Quercus robur) development (bark,
unopened buds, young developing leaves, and blossoms) were analyzed with the aim of "nding
possible host-plant attractants for the European oak bark beetle, Scolytus intricatus. Complex
mixtures of aliphatic, aromatic, and terpenoid compounds were identi"ed in the samples.
(E)-2-Hexenal and hexanal dominated in samples of bark. In buds, (Z)-3-hexenyl acetate formed
a substantial part of the mixture. In both leaves and blossoms (E, E)-a-farnesene was the main
component.
Volatiles released from oak twigs and branches during both the maturation feeding and
construction of maternal galleries by Scolytus intricatus were also analyzed. Most compounds
found in the samples from females' and males' maturation feeding were identical. High contents
of anisole, (E)-b-ocimene, a-copaene, one unidenti"ed sesquiterpenic hydrocarbon C H and
15 24
b-caryophyllene were found in both samples of twigs attacked by beetles. During the construction of maternal galleries by bark beetles in oak logs, monoterpene hydrocarbons such as
p-cymene, (E)-b-ocimene, and c-terpinene, and sesquiterpenes a-copaene and b-caryophyllene
were released in large quantities. No new compound appeared when males were added to the
log with feeding females. ( 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Quercus; Monoterpenes; Sesquiterpenes; Oak wilt; Pathogenic fungi; Scolytus intricatus; Maturation feeding; Brood galleries
* Corresponding author. Tel.: #4202-201-83229; fax: #4202-243-10177.
E-mail address: vrkocova@uochb.cas.cz (P. Vrkoc\ ovaH ).
0305-1978/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 5 - 1 9 7 8 ( 0 0 ) 0 0 0 4 2 - 9
934
P. Vrkoc\ ova& et al. / Biochemical Systematics and Ecology 28 (2000) 933}947
1. Introduction
The oak wilt, caused by pathogens of the genera Ceratocystis, Ophiostoma and
others, is a disease leading to withering and death of oak trees in the eastern and
central states of the USA as well as in Europe (Doganlar and Schopf, 1984). Infected
trees die within a year, sometimes in less than a month. The disease is spread by
root-to-root grafts and the pathogenic fungi can be transmitted by insects.
The European oak bark beetle (Scolytus intricatus; Coleoptera: Scolytidae) is reported as a possible vector of the fungus (Doganlar and Schopf, 1984). The adult
beetles breed in cut or weakened oak branches by boring deep larval galleries in the
xylem. The cut branches are often infested by pathogens. S. intricatus overwinters in
the late larva stage or as a pupa (Schwenke, 1974). In the spring time, emerging adult
beetles visit healthy oaks and make twig cavities where they feed (maturation feeding).
Thus, beetles contaminated with spores or parts of mycelium can transmit the fungus
to the healthy trees. In the Czech Republic, the species usually has one generation per
year. In exceptionally warm years or localities, a partial second generation can occur.
Bark beetles feeding or breeding in trees must locate a suitable host from among the
relatively few scattered widely in the forest. Usually, chemicals are the most important
mediators of host-plant-searching behavior (Miller and Strickler, 1984; Visser, 1986;
StaK dler, 1992). It is believed that insects have evolved behavioral responses to volatile
host-plant chemicals that indicate the presence of a suitable host in which reproduction can occur. Depending on the species, bark beetles "nd their host either by
attraction to host volatiles from a distance or by random landing and probing (Byers,
1995). Chemical mediation of host "nding generally occurs via mixtures of chemicals,
some of them stimulants and others inhibitors.
Host volatiles are attractive to a number of forest scolytids including species in the
genus Scolytus. In case of S. intricatus, oak (Quercus spp.) is the main host tree, but
willow (Salix spp.) is also accepted by beetles for maturation feeding (Doganlar and
Schopf, 1984).
The main goal of our work is to search for chemical communication of the
European oak bark beetle by pheromones and for their potential primary attractants.
In the present study, we have investigated (a) volatiles emitted in di!erent phases of
the oak development (outer and inner bark, unopened buds, young developing leaves,
and blossoms), and (b) volatiles released from oak twigs and branches during both
maturation feeding and construction of maternal galleries. Biological activity was
observed by preliminary electrophysiological and behavioral tests.
2. Materials and methods
2.1. Collection of plant material and insects
Buds, leaves, bark and blossoms were obtained from healthy oak trees (Quercus
robur) in an area about 20 km from Prague in the spring of 1999. The bark was
collected twice, "rst when buds were developing and second when young leaves were
P. Vrkoc\ ova& et al. / Biochemical Systematics and Ecology 28 (2000) 933}947
935
developing. Logs, healthy as well as infested with Scolytus intricatus were collected in
the winter of 1998 in three di!erent localities within the Czech Republic. All logs were
kept at 53C until used. Infested logs were then placed into net cages at laboratory
temperature. Emerged beetles were collected daily, sexed and allowed to bore into
young oak twigs for maturation feeding and later for making maternal galleries and
eventual copulation. All plant materials studied were free of pathogenic fungi.
All samples were repeated "ve times and standard deviations are given in the
results.
2.2. Collection of volatiles
An air entrainment technique was used for trapping the volatile compounds
released from oak (Q. robur) buds, blossoms, young twig bark and young leaves, oak
twigs (diameter 2}6 mm), logs (diameter 4}5 cm) and beetle-infested twigs and logs (40
beetles in each). Modi"ed equipment earlier described by JursmH k et al. (1991) was used
for collection of volatiles. Buds, blossoms and bark were shredded and leaves were
crushed in liquid nitrogen before the entrainment. Biological material was then placed
in a glass vessel (3 l). Puri"ed air (silica gel, charcoal and molecular sieve) was drawn
through the vessel for 24 h at 40}50 ml/min and then through a glass tube "lter
containing Porapak Q (0.1 g; mesh size 100}120). An additional "lter was placed after
the "rst one for a breakthrough check. The captured volatiles were eluted from the
sorption material with hexane (150 ll).
2.3. Identixcation of volatile constituents
The components were identi"ed on a GC (Carlo Erba) coupled with a mass detector
Fisons MD 800, using a BPX5 capillary column (SGE, 30 m]0.22 mm, "lm thickness
0.25 mm) with helium #ow 0.94 and 0.55 ml/min (measured at 503C), respectively. The
temperature program was 503C for 2 min, then 43C/min up to 2603C and held for
20 min, resp. 703C for 2 min, then 43C/min up to 2603C and held for 20 min; inlet
temperature was 2003C in all cases, injector working in splitless mode. The mass
spectra (electron-impact) were compared to the Wiley Registry of Mass Spectral Data,
6th edition, the National Institute of Standards and Technology (NIST) Library, and
Identi"cation of Essential Oil Components by Gas Chromatography/Mass Spectrometry Library (R.P. Adams, Allured Publishing Corporation). Retention times of
analyzed compounds were compared with those of authentic samples wherever
possible.
The gas-phase infrared spectrum (GC-FTIR) of one of the isolated components,
a sesquiterpene hydrocarbon C H , was measured on an HP 5890 gas chromato15 24
graph coupled with an HP 5695A IRD equipped with a narrow-band
(4000}750 cm~1) infrared detector (mercury cadmium telluride).
Two-dimensional chromatography (2D-GC) was used for the determination of the
optical purity of chiral compounds, using available standards (a-pinene, b-pinene,
limonene, camphene). Two Varian 3400 gas chromatographs with two installed
Valco micro-valves were connected with a heated interface. The GC system was
936
P. Vrkoc\ ova& et al. / Biochemical Systematics and Ecology 28 (2000) 933}947
equipped with split/splitless injectors and #ame ionization detectors. Deactivated
fused silica capillaries were used as retention gaps and for the connection of the
di!erent valves with each other and with the detectors. A DB-WAX column was used
for pre-separation in the "rst chromatograph (GC-A). For chiral analysis the valve
was programmed according to the retention times determined in the "rst run so as to
let the chiral compounds pass to the second chromatograph (GC-B) with the Cyclodex B column. For further details see Borg-Karlson et al. (1993). For GC-A, helium
#ow 6.2 ml/min (403C), column DB-WAX (J&W, 30 m]0.25 mm, "lm thickness
0.25 mm) and temperature program 503C for 2 min, then 43C/min up to 2103C
and held for 20 min were used. For GC-B, helium #ow 9.2 ml/min (403C), column
Cyclodex B (J&W, 30 m]0.25 mm, "lm thickness 0.25 mm) and temperature program
553C for 13 min, then 13C/min up to 773C were used.
3. Results and discussion
Complex mixtures of aliphatic, aromatic, and terpenoid compounds were identi"ed
in the samples from oak (Table 1, Figs. 1 and 2). In the early and late bark samples,
aldehydes formed the main portion of the mixture, (E)-2-hexenal and hexanal 8.7 and
12.8% resp. 23.0% and 45.4% being the main components. n-Alkanes formed 13.7%
resp. 5.1% and aliphatic alcohols 6.2% resp. 4.3% of the compounds. Monoterpene
and sesquiterpene hydrocarbons were present as minor components. Esters were
almost absent. Proportion of compounds in early and late bark samples did not di!er
signi"cantly.
In the buds, esters dominated (36.5%) in the volatiles released. (Z)-3-hexenyl acetate
formed 21.5% of the mixture. Substantial proportions of alcohols (16.4%) and
aldehydes (13.0%) were found in the samples. Among the alcohols, 11.0% consisted of
(Z)-3-hexenol. In relatively high abundance were dioxaspiroalkanes (11.5%).
Homoterpene hydrocarbons were present as minor components (2.0%). Monoterpene
and sesquiterpene hydrocarbons and n-alkanes were present in small quantities only.
After the buds opened and small leaves appeared, the spectrum of volatiles changed
again. Aldehydes (3.7%) and alcohols (9.6%) were less abundant, while n-alkanes were
moderately abundant (19.3%) constituents. Esters (31.7%) and sesquiterpene hydrocarbons (38.9%) dominated in the samples. (Z)-3-Hexenyl acetate and (Z)-3-hexenyl
butanoate were the major esters while (E,E)-a-farnesene (37.5%) was the most important sesquiterpene. Also, homoterpenes were present as minor components (4.8%).
Volatiles from opened oak blossoms were included in the analytical study although
the #owers open later than bark beetles start their maturation feeding. (E, E)-afarnesene dominated in the sample (41.7%). All other types of compounds were
present in moderate abundance (from 3 to 7%).
The period of maturation feeding is supposed to be critical for the transfer of fungal
pathogens. The attack of beetles on young twigs for maturation feeding coincides
approximately with the development of oak leaves. The knowledge of volatile pro"les
of four main types of tissue may lead to better understanding of the beetle host seeking
behavior.
Table 1
Compounds emitted from oak (Quercus robur) in di!erent phases of its development
Compound!
MS,
MS,
MS
MS,
MS,
MS,
MS,
MS
MS
MS,
MS
MS,
MS
MS,
MS,
MS
MS,
MS,
MS,
MS
MS,
MS,
MS,
MS,
MS,
MS,
MS,
MS,
standard
standard
standard
standard
standard
standard
standard
standard
standard
standard
standard
standard
standard
standard
standard
standard
standard
standard
standard
standard
standard
Relative % (standard deviation)
Bark I#
Bark II$
Buds
3.37
*
*
8.71
3.87
23.03
2.32
0.48
*
0.82
0.17
0.05
*
*
0.27
*
*
*
2.83
*
*
*
*
*
*
1.00
1.33
*
1.11
*
*
12.78
*
45.40
4.31
0.19
*
0.38
*
0.14
*
0.20
0.25
*
0.08
1.78
0.44
*
*
0.18
*
*
0.09
2.93
1.08
*
0.61
0.53
1.29
9.37
11.04
3.60
5.22
*
0.38
*
*
*
0.24
*
*
*
*
0.33
*
*
*
*
21.25
6.89
*
0.09
0.18
(3.83)
(5.78)
(3.32)
(21.29)
(1.91)
(0.45)
(0.84)
(0.15)
(0.04)
(0.28)
(2.97)
(0.87)
(0.70)
(0.25)
(6.17)
(4.32)
(2.17)
(0.16)
(0.11)
(0.09)
(0.18)
(0.14)
(0.08)
(0.49)
(0.21)
(0.05)
(0.11)
(2.47)
(0.76)
Leaves
(0.28)
(0.20)
(0.43)
(1.60)
(4.34)
(5.35)
(2.02)
(0.13)
(0.09)
(0.10)
(5.42)
(4.17)
(0.08)
(0.08)
0.08
*
0.46
3.50
8.94
0.22
0.69
0.03
*
0.02
*
0.13
*
0.02
0.01
*
0.08
0.03
*
0.40
*
15.22
1.01
*
0.02
0.07
0.03
(0.09)
(0.30)
(1.74)
(3.54)
(0.44)
(0.34)
(0.02)
(0.01)
(0.07)
(0.03)
(0.00)
(0.03)
(0.02)
(0.30)
(7.70)
(0.60)
(0.03)
(0.06)
(0.01)
Blossoms
1.59
*
*
5.84
6.26
1.04
0.80
0.68
*
0.15
*
*
*
*
0.37
0.15
*
0.68
0.22
0.32
*
*
3.99
0.34
*
*
0.43
*
(0.70)
(8.73)
(3.40)
(0.62)
(0.58)
(0.28)
(0.05)
(0.21)
(0.09)
(0.20)
(0.11)
(0.17)
(1.17)
(0.14)
(0.09)
937
3.284
3.501
3.600
3.667
4.517
4.400
4.617
4.717
4.718
4.984
5.101
5.201
5.367
5.601
5.851
6.584
6.768
7.117
7.251
7.501
7.534
7.669
7.717
7.801
7.985
8.317
8.384
8.467
Method of
identi"cation
P. Vrkoc\ ova& et al. / Biochemical Systematics and Ecology 28 (2000) 933}947
Toluene
Heptane
Ethyl butanoate
Hexanal
(Z)-3-Hexen-1-ol
(E)-2-Hexenal
1-Hexanol
Dimethylbenzene, isomer I
Isopentyl acetate
Nonane
Dimethylbenzene, isomer II
Heptanal
Pentyl acetate
a-Thujene
a-Pinene
Ethylmethylbenzene
Sabinene
Myrcene
Decane
Trimethylbenzene
(E)-3-Hexenyl acetate
Octanal
(Z)-3-Hexenyl acetate
n-Hexyl acetate
a-Terpinene
p-Cymene
Limonene
(Z)-b-Ocimene
RT (min)"
938
Table 1. Continued
Compound!
Method of
identi"cation
Relative % (standard deviation)
Bark I#
Bark II$
Buds
Leaves
0.03 (0.03)
0.25 (0.18)
10.82 (6.42)
0.02 (0.02)
0.70 (0.84)
0.04 (0.03)
0.33 (0.29)
0.61 (0.49)
0.60 (0.33)
*
*
0.03 (0.03)
*
0.16 (0.08)
*
0.04 (0.05)
*
8.534
8.801
9.184
MS, standard
MS, standard
MS, standard
*
*
*
*
*
*
9.269
9.715
9.717
10.203
MS, standard
MS, standard
MS, standard
MS
0.32 (0.42)
*
0.34 (0.24)
*
2.89 (2.84)
*
0.22 (0.15)
*
0.03 (0.07)
0.18 (0.10)
*
0.73 (0.25)
10.301
10.353
10.368
10.551
10.701
10.769
10.951
11.001
12.018
12.268
13.651
13.801
13.885
14.401
14.521
15.185
15.368
15.601
17.171
17.418
18.602
MS,
MS,
MS
MS
MS,
MS
MS,
MS
MS
MS
MS
MS
MS,
MS,
MS,
MS
MS
MS
MS,
MS,
MS
0.88
2.07
*
*
*
*
0.47
*
*
*
0.36
*
1.34
1.99
*
*
*
*
*
1.01
*
0.81
0.30
*
*
*
*
0.23
*
*
*
*
*
0.45
0.22
0.10
*
*
*
*
0.65
*
0.09
*
*
0.25
*
0.14
*
1.27
0.22
0.13
2.59
0.76
*
0.83
*
0.50
0.65
0.13
0.22
0.09
*
standard
standard
standard
standard
standard
standard
standard
standard
standard
(0.63)
(1.81)
(0.42)
(0.23)
(0.66)
(3.44)
(0.52)
(0.81)
(0.34)
(0.09)
(0.10)
(0.21)
(0.13)
(0.20)
(0.07)
(0.11)
(0.18)
(0.78)
(0.06)
(0.10)
(0.72)
(0.57)
(0.89)
(0.25)
(0.18)
(0.04)
(0.08)
(0.03)
0.04
*
0.15
0.69
0.01
*
*
0.91
0.89
0.03
10.96
0.15
*
*
*
0.83
0.32
0.03
(0.08)
(0.06)
(0.37)
(0.03)
(0.42)
(0.61)
(0.02)
(3.71)
(0.05)
(0.40)
(0.21)
(0.04)
0.06 (0.07)
0.04 (0.03)
Blossoms
0.04
0.33
*
*
*
*
*
0.83
*
*
1.55
*
0.39
*
*
*
*
*
*
0.79
*
(0.07)
(0.19)
(0.84)
(0.79)
(0.18)
(1.09)
P. Vrkoc\ ova& et al. / Biochemical Systematics and Ecology 28 (2000) 933}947
1,8-Cineole
(E)-b-Ocimene
(E)-7-Methyl-1,6-dioxaspiro [4.5]
decane
c-Terpinene
1-Octanol
Linalool oxide, furanic, cis
2-Ethyl-1,6-dioxaspiro [4.4]
nonan MchalcogranN
Linalool oxide, furanic, trans
Undecane
n-Pentyl butanoate
(Z)-3-Hexenyl propanoate
Linalool
n-Hexyl propanoate
Nonanal
(E)-4,8-Dimethyl-1,3,7-nonatriene
(Z)-3-Hexenyl isobutanoate
n-Hexyl isobutanoate
(Z)-3-Hexexyl butanoate
n-Hexyl butanoate
Dodecane
Methyl salicylate
Decanal
(Z)-3-Hexenyl 2-methylbutanoate
n-Hexyl 2-methylbutanoate
n-Hexyl pentanoate
Ethyl salicylate
Tridecane
(Z)-3-Hexenyl tiglate
RT (min)"
19.318
20.418
20.468
20.635
20.868
21.952
22.089
22.390
22.540
22.785
23.402
24.085
24.202
24.702
25.202
26.752
MS
MS,
MS
MS
MS,
MS,
MS,
MS
MS,
MS,
MS,
MS
MS,
MS,
MS
MS
27.152
27.319
30.302
33.153
33.970
MS
MS, standard
MS, standard
MS, standard
MS
standard
standard
standard
standard
standard
standard
standard
standard
standard
*
0.72
*
*
1.87
*
*
*
*
*
*
*
1.29
*
0.76
*
*
1.26
0.78
0.41
0.42
(0.55)
(1.03)
(0.56)
(1.07)
(1.11)
(0.48)
(0.28)
(0.37)
0.17 (0.10)
*
*
1.30
*
*
*
0.18
0.20
0.62
0.04
0.83
0.27
*
0.40
1.40
0.73
0.75
(0.25)
(1.01)
(0.89)
(0.78)
0.01
0.05
0.25
0.08
0.05
0.67
*
*
*
0.03
0.01
0.62
*
37.46
0.03
3.93
*
0.42
0.57
0.17
0.06
*
0.07 (0.02)
0.12 (0.03)
0.05 (0.01)
*
0.03
0.04
0.03
19.02
*
*
0.12 (0.07)
*
*
0.67 (0.26)
*
*
*
*
*
*
*
1.02 (0.24)
(0.10)
(0.16)
(0.03)
(0.02)
!The compounds are listed in elution order on a 30 m DB-5ms column (5% phenyl methyl silicone).
"Temperature program: 503C (2 min); 43/min to 2603 (20 min); helium #ow 0.94 ml/min at 503C.
#Collected at time when buds were developing.
$Collected at time when leaves were developing.
(0.80)
(0.09)
(0.24)
(0.76)
(0.09)
(1.56)
(0.19)
(0.02)
(0.04)
(0.13)
(0.11)
(0.02)
(0.72)
(0.01)
(0.01)
(0.26)
(10.55)
(0.02)
(3.16)
(0.02)
(0.02)
(0.02)
(0.01)
*
0.17
*
*
0.49
0.45
*
*
*
*
*
*
2.19
41.72
*
*
(0.09)
(0.26)
(0.16)
(0.49)
(21.40)
*
0.56 (0.35)
1.27 (0.31)
0.35 (0.21)
*
P. Vrkoc\ ova& et al. / Biochemical Systematics and Ecology 28 (2000) 933}947
a-Cubebene
a-Copaene
(Z)-3-Hexenyl hexanoate
n-Hexyl hexanoate
Tetradecane
b-Caryophyllene
trans-a-Bergamotene
b-Gurjunene
(Z)-b-Farnesene
(E)-b-Farnesene
Aromadendrene
(Z,E)-a-Farnesene
Pentadecane
(E,E)-a-Farnesene
d-Cadinene
(E,E)}4,8,12-Trimethyl-1,3,7,11Tridecatetraene
(Z)-3-Hexenyl benzoate
Hexadecane
Heptadecane
Octadecane
Isopropyl myristate
939
940
P. Vrkoc\ ova& et al. / Biochemical Systematics and Ecology 28 (2000) 933}947
Fig. 1. Sum of relative percent of di!erent compound classes emitted from plant tissues.
Samples obtained from oak twigs during the maturation feeding of Scolytus intricatus females and males had many components in common, though their proportions di!ered (Table 2, Figs. 3, 4a and b). Generally, the twigs and logs without beetles
gave sample of much lower concentration. After beetles started to feed and build
galleries, the amounts of emitted volatiles increased substantially. n-Alkanes formed
a substantial part (8%) of the twig sample without beetles. During the maturation
feeding of both females and males, more compounds were released as a consequence of
the beetles' activity and the proportions of alkanes dropped to the barely detectable
level. In twigs, monoterpenes formed 24.8% of the compounds. The most abundant
compound was (E)-b-ocimene (22.5%). Linalool oxides were present at 6.8% and
aldehydes at 6.5%. Only small amounts of sesquiterpenes (4.9%) were found.
Most of the compounds found in the samples from females' and males' feeding were
identical. Their proportions in the two samples were not very di!erent (see Fig. 4a).
High contents of anisole, (E)-b-ocimene, a-copaene, one unidenti"ed sesquiterpenic
hydrocarbon C H (unidentixed 3& in Table 2) and b-caryophyllene were found in
15 24
both samples of twigs with beetles.
The structure of this sesquiterpenic hydrocarbon could not be fully determined
from its mass and infrared spectra. The retention time and mass spectrum of this
compound was not identical with any known sesquiterpene present in our databases
(see Section 2). The character of its mass spectrum corresponds with a cyclic type of
compound (low intensities of fragments m/z 69, 81 exclude the farnesene-type). The
infrared spectrum is in agreement with the proposed cyclic structure. No band in the
P. Vrkoc\ ova& et al. / Biochemical Systematics and Ecology 28 (2000) 933}947
941
Fig. 2. Relative percent and standard deviation of selected compounds (over 5%) emitted from plant
tissues.
CH-stretching area 3070}3080 cm~1 and only a weak band around 1640}1660 cm~1
(C"C stretch) excludes the presence of an exomethylene terminal C"CH group in
2
the compound (Svatos\ and Attygalle, 1997). Bands at 3052 and 3024 cm~1 (C}H
stretch) suggest the presence of two di!erent types of carbon}carbon double bonds.
Double bonds are probably not conjugated, as the C"C stretch (1642 cm~1) is not
too intense. Comparison with IR spectra of terpenes from the literature, the CH
stretch at 3052 cm~1 and CH wagging 780 cm~1 imply a trisubstituted double bond in
a ring.
Oak logs prepared for S. intricatus females to feed in and build brood galleries
released high amounts of monoterpenes (37.8%), and also substantial amounts of
sesquiterpenes (10.1%). Anisole was present as a moderately abundant compound
(6.0%). When the females bored in, monoterpenic (48.6%) and sesquiterpenic (47.2%)
hydrocarbons made up most of the volatiles. When males were added
for copulation to the logs with females, the proportion of terpene hydrocarbons
changed in favor of sesquiterpenes (73.0%). p-Cymene (12.0 and 6.4%, respectively),
Compound!
Method of
identi"cation
Relative % (standard deviation)
Twigs
Maturation
Maturation
feeding}females feeding}males
Logs
Making
Copulation
galleries}females
6.134
6.317
MS
MS
*
0.77 (0.57)
*
0.08 (0.06)
*
0.06 (0.09)
*
1.22 (1.60)
0.01 (0.02)
0.10 (0.12)
*
0.01 (0.02)
6.634
6.900
MS, standard
MS
1.25 (1.01)
0.25 (0.49)
0.28 (0.24)
*
0.22 (0.21)
0.02 (0.03)
0.61 (0.46)
0.46 (0.67)
0.14 (0.14)
*
0.03 (0.02)
*
7.034
7.534
7.567
7.801
MS,
MS,
MS,
MS,
standard
standard
standard
standard
0.14 (0.28)
*
*
0.50 (0.52)
*
*
7.01 (6.83)
0.90 (0.39)
*
*
15.99 (12.74)
1.43 (0.68)
*
0.92 (0.95)
6.03 (7.39)
2.45 (2.48)
*
4.22 (3.74)
0.03 (0.06)
2.17 (1.85)
*
0.94 (1.23)
0.13 (0.14)
0.34 (0.27)
8.367
MS, standard
0.42 (0.85)
1.48 (0.90)
1.96 (1.40)
2.03 (2.38)
0.52 (0.38)
0.13 (0.08)
8.734
8.967
9.134
9.217
9.317
9.434
9.817
9.984
10.084
10.434
10.767
10.884
MS
MS,
MS,
MS,
MS,
MS,
MS
MS,
MS,
MS,
MS,
MS,
standard
standard
standard
standard
standard
0.78
*
0.20
*
*
*
1.26
1.83
*
*
0.38
0.37
*
0.17
*
0.23
0.47
0.39
*
*
*
*
0.54
0.71
(0.46)
(0.36)
*
0.37
*
0.30
0.61
0.21
*
*
*
*
1.34
0.52
(1.16)
(0.78)
0.40
1.88
*
0.65
2.43
*
*
*
*
0.25
9.20
4.78
10.934
11.034
11.317
11.751
MS,
MS,
MS,
MS,
standard
standard
standard
standard
0.65
0.06
22.49
0.89
(1.31)
*
(0.11)
0.05 (0.06)
(26.91) 20.34 (2.12)
(1.15)
0.37 (0.17)
0.49
0.34
19.45
0.38
(0.43)
(0.23)
(6.77)
(0.35)
*
3.56 (2.13)
5.38 (4.48)
*
11.867
12.384
MS, standard
MS, standard
0.63 (0.60)
*
7.80 (6.35)
*
standard
standard
standard
standard
standard
(1.41)
(0.39)
(2.51)
(0.68)
(0.77)
(0.45)
*
3.05 (2.71)
(0.05)
(0.11)
(0.14)
(0.36)
0.31 (0.31)
*
(0.14)
(0.24)
(0.34)
(0.25)
(0.40)
(1.31)
(0.81)
(1.36)
(0.31)
(4.87)
(2.91)
*
0.67
*
0.16
1.00
0.10
*
*
0.08
0.70
12.01
3.41
*
0.07
*
0.01
0.39
0.02
*
*
0.02
0.21
6.42
0.64
(0.03)
(0.28)
(9.07)
(1.29)
*
0.19 (0.20)
12.23 (7.43)
0.10 (0.15)
0.59
0.03
8.26
0.10
(0.77)
(0.03)
(3.04)
(0.13)
11.46 (10.25)
*
4.05 (5.53)
*
(0.59)
(0.13)
(0.49)
(0.11)
(0.07)
(0.62)
(10.01)
(2.19)
(0.09)
(0.02)
(0.17)
(0.03)
P. Vrkoc\ ova& et al. / Biochemical Systematics and Ecology 28 (2000) 933}947
Ethylbenzene
Dimethylbenzene,
Isomer I
Nonane
Dimethylbenzene,
Isomer II
Heptanal
a-Thujene
Anisole
a-Pinene (!/#:
96.2/3.8)#
Camphene (!/#:
100/0)#
Ethylmethylbenzene
Sabinene
Benzaldehyde
b-Pinene (!/#: 100/0)#
Myrcene
Decane
Trimethylbenzene
Octanal
a-Phellandrene
a-Terpinene
p-Cymene
Limonene (!/#:
83.1/16.9)#
(Z)-b-Ocimene
1,8-Cineole
(E)-b-Ocimene
(E)-7-Methyl-1,6dioxaspiro[4,5]decane
c-Terpinene
Linalool oxide, furanic,
cis
RT (min)"
942
Table 2
Compounds captured from oak twigs and logs during maturation feeding and making maternal galleries of Scolytus intricatus
12.834
12.951
12.984
MS, standard
MS, standard
MS, standard
*
*
3.78 (3.34)
0.09 (0.11)
0.40 (0.37)
*
0.02 (0.04)
0.16 (0.26)
*
0.66 (0.41)
*
*
0.71 (0.59)
0.06 (0.12)
*
0.29 (0.37)
*
*
13.634
13.668
MS, standard
MS
2.58 (3.28)
2.10 (3.18)
*
0.18 (0.06)
*
0.74 (0.55)
*
3.27 (2.70)
*
0.31 (0.30)
*
0.04 (0.05)
16.568
17.368
18.451
19.118
20.302
21.535
22.368
23.552
23.768
23.935
23.952
24.652
25.219
25.352
25.535
25.635
26.452
26.602
27.035
27.085
27.219
27.285
27.635
27.752
27.819
28.352
MS,
MS,
MS
MS
MS,
MS
MS
MS,
MS
MS,
MS
MS
MS,
MS,
MS
MS,
MS
MS,
MS
MS
MS,
MS
MS
MS
MS
MS
1.74
1.90
*
*
2.87
*
*
*
*
0.83
*
*
0.27
0.66
*
*
*
1.02
*
0.04
1.96
*
*
1.59
*
1.35
standard
standard
standard
standard
standard
standard
standard
standard
standard
standard
(1.04)
(2.56)
(1.26)
(1.02)
(0.54)
(1.33)
(0.94)
(0.07)
(1.24)
(1.05)
(2.04)
0.25
*
0.01
0.11
0.09
*
0.15
16.02
16.46
*
0.48
0.09
9.72
1.48
1.06
1.02
1.87
0.46
0.00
*
*
0.57
2.29
1.99
*
1.98
(0.23)
(0.02)
(0.06)
(0.13)
(0.09)
(5.54)
(12.36)
(0.29)
(0.06)
(2.90)
(0.91)
(0.53)
(0.66)
(0.65)
(0.12)
(0.00)
(0.29)
(0.85)
(0.68)
(0.70)
0.09
*
0.07
*
*
*
0.12
15.25
16.86
*
0.66
0.31
5.98
1.51
0.98
0.96
1.56
0.64
0.67
*
*
1.03
1.13
2.06
*
3.23
(0.14)
(0.09)
(0.05)
(9.60)
(8.79)
(0.65)
(0.26)
(5.24)
(0.67)
(0.49)
(0.69)
(1.44)
(0.57)
(0.44)
(1.08)
(0.62)
(1.40)
(3.22)
*
*
*
*
*
*
*
2.59
5.37
*
*
*
0.33
0.73
*
0.38
*
0.37
*
*
*
*
*
0.14
*
0.10
(2.68)
(6.88)
(0.67)
(1.02)
(0.46)
(0.74)
(0.28)
(0.20)
(0.05)
(0.02)
(0.32)
(0.03)
(0.05)
(0.22)
(12.25)
(3.36)
(0.81)
(1.81)
(4.79)
(0.16)
(2.31)
(0.37)
(1.90)
(0.48)
(0.33)
(1.09)
(1.73)
(0.99)
(4.82)
(1.03)
*
*
0.03
0.37
*
0.21
0.33
26.73
7.57
*
1.58
1.37
8.87
0.55
2.96
0.36
3.84
0.76
1.07
*
*
2.63
2.67
0.97
6.87
3.90
(0.04)
(0.30)
(0.12)
(0.21)
(7.96)
(6.52)
(0.68)
(1.70)
(3.69)
(0.44)
(1.87)
(0.39)
(1.85)
(0.16)
(0.12)
(1.38)
(1.77)
(1.24)
(5.44)
(0.34)
943
!The compounds are listed in elution order on a 30 m BPX-5 column (5% phenyl methyl silicone).
"Temperature program: 703C (2 min); 43/min to 2603 (20 min); helium #ow 0.55 ml/min at 503C.
#Proportion of enantiomers determined on 2D-GC system.
$Mass spectrum * m/z%: 77(57), 79(56), 91(100), 105(52), 119(25), 133(14), 147(34), 162(29).
%Mass spectrum * m/z%: 41(79), 91(100), 105(53), 115(47), 117(44), 119(74), 131(56), 145(44), 159(86), 187(19), 202(16).
&Mass spectrum * m/z%: 41(100), 77(63), 91(79), 93(90), 105(25), 119(75), 161(4), 189(1), 204(0.6).
IR spectrum: 3052, 3024, 2969, 2925, 2876, 2741, 1642, 1452, 1381, 1349, 1159, 1111, 1027, 943, 845, 779 cm~1.
0.03
*
0.01
0.26
0.02
0.04
0.20
15.81
5.61
*
1.03
1.99
5.65
0.52
2.12
0.29
2.17
0.46
0.82
*
*
1.28
1.50
1.00
3.89
2.80
P. Vrkoc\ ova& et al. / Biochemical Systematics and Ecology 28 (2000) 933}947
Terpinolene
Undecane
Linalool oxide, furanic,
trans
Nonanal
(E)-4,8-Dimethyl1,3,7-nonatriene
Dodecane
Dodecanal
Thymol, methyl ether
Unidenti"ed 1$
Tridecane
Unidenti"ed 2%
a-Ylangene
a-Copaene
Unidenti"ed 3&
Tetradecane
b-Elemene
a-Gurjunene
b-Caryophyllene
trans-a-Bergamotene
a-Guaiene
(E)-b-Farnesene
a-Humulene
Aromadendrene
a-Amorphene
a-Curcumene
Pentadecane
Germacrene D
b-Selinene
a-Muurolene
b-Bisabolene
d-Cadinene
944
P. Vrkoc\ ova& et al. / Biochemical Systematics and Ecology 28 (2000) 933}947
Fig. 3. Sum of the relative percent of di!erent compound classes released from host branches and
beetle-attacked branches.
(E)-b-ocimene (12.2 and 8.3%, respectively), c-terpinene (11.5 and 4.0%, respectively),
a-copaene (15.8 and 26.7%, respectively), unidenti"ed sesquiterpene (5.6 and 7.4%,
respectively) and b-caryophyllene (5.7 and 8.9%, respectively) were the most abundant
compounds in logs attacked by beetles. Only relative proportion of anisole, a-copaene
and b-caryophyllene changed signi"cantly with the beetle attack (see Fig. 4b).
Terpenes and homoterpenes are known to be produced by plants in response to
herbivore feeding. (E)-b-Ocimene and (E)-4,8-dimethyl-1,3,7-nonatriene were released
in large amounts from infested cucumber plants (Takabayashi et al., 1994). It is not
clear whether the biosynthesis of the volatiles released from infested plants is induced
by herbivore feeding or if they are stored in the plant and released at the time of
infestation (PareH and Tumlinson, 1996). Infested cotton plants released high amounts
of a-pinene, b-caryophyllene, (E,E)-a-farnesene, (E)-b-farnesene, (E)-b-ocimene, and
(E)-4,8-dimethyl-1,3,7-nonatriene (Loughrin et al., 1994). Loughrin suggested from the
released timing and rate that a-pinene and b-caryophyllene come from the constitutive compounds (stored in the plant) while the other compounds were biosynthesized
de novo in response to insect feeding.
It can be seen from Table 2 that the types of compounds captured from oak twigs
and logs with feeding and breeding beetles correspond quite well with those described
for other herbivore-infested plants. No special compound was found for either
females' or males' feeding samples that would lead us to suspect the presence of
a pheromone. Although results of our behavioral tests indicate that secondary
P. Vrkoc\ ova& et al. / Biochemical Systematics and Ecology 28 (2000) 933}947
945
Fig. 4. (a) and (b). Relative percent and standard deviation of selected compounds (over 5%) released from
host branches and beetle-attacked branches.
chemical communication between sexes exists (Hovorka et al., 2000), a chemical basis
supporting this fact could not be found. Either the concentration of the active
compound(s) was too low or the signal is produced in a di!erent phase of the beetles'
946
P. Vrkoc\ ova& et al. / Biochemical Systematics and Ecology 28 (2000) 933}947
development. Electrophysiological testing of potential host-plant attractants indicated the activity of several components of our samples (dimethylbenzene, nonane,
a-thujene, sabinene, p-cymene, c-terpinene, terpinolene, dodecane, tridecane, and
a-gurjunene).
Many forest bark beetles are attracted by host volatiles. Some of them, possessing
a multicomponent aggregation pheromone, are attracted by host volatiles at the
beginning of their mass attack. Another smaller group of scolytids may not use an
aggregation pheromone, but generally they are strongly attracted either to the host
monoterpenes, ethanol, or a combination of both. The compounds released from
a host tree in the particular development state are not only important for primary
attraction to plants but they may play a role in enhancing the bark beetles' response to
the aggregation pheromone, if any.
The production of an aggregation or sex pheromone by S. intricatus is still uncertain
despite the evidence from behavioral trials that seems to indicate its presence. The
situation could be similar to that in Scolytus ventralis where, according to latest data,
the attack dynamics can be explained solely by its sensitive primary attraction
response to host volatiles (MarcmH as-SaH mano et al., 1998) or to Tomicus piniperda where
the pheromone exists, but it remains elusive due to a strong masking e!ect of tree
odors (Czokajlo, 1998).
Acknowledgements
The "nancial support of this work by the Grant Agency of the Czech Republic
(grants nos. 203/97/0037 and 203/00/0219) and by COST E16.10. is gratefully acknowledged. The authors also wish to thank Dr. A.-K. Borg-Karlson (KTH, Stockholm,
Sweden) for 2D-GC instrument time, Dr. A.B. Attygalle (Cornell University, Ithaca,
USA) for GC-FTIR instrument time, Dr. S. Vas\ mH c\ kovaH and Dr. A. Svatos\ (Institute of
Organic Chemistry and Biochemistry, Prague, Czech Republic) for the GC-IR consulting, and Prof. R.M. Coates (University of Illinois, Urbana, USA) for sending
a sample of trans-a-bergamotene.
References
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Volatiles released from oak, a host tree for the
bark beetle Scolytus intricatus
PavlmH na Vrkoc\ ovaH *, Irena ValterovaH , Jan Vrkoc\ ,
BohummH r Koutek
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
Flemingovo na& me\ stn& 2, 166 10 Praha 6, Czech Republic
Received 15 November 1999; received in revised form 6 April 2000; accepted 10 April 2000
Abstract
Volatile compounds emitted in di!erent phases of oak (Quercus robur) development (bark,
unopened buds, young developing leaves, and blossoms) were analyzed with the aim of "nding
possible host-plant attractants for the European oak bark beetle, Scolytus intricatus. Complex
mixtures of aliphatic, aromatic, and terpenoid compounds were identi"ed in the samples.
(E)-2-Hexenal and hexanal dominated in samples of bark. In buds, (Z)-3-hexenyl acetate formed
a substantial part of the mixture. In both leaves and blossoms (E, E)-a-farnesene was the main
component.
Volatiles released from oak twigs and branches during both the maturation feeding and
construction of maternal galleries by Scolytus intricatus were also analyzed. Most compounds
found in the samples from females' and males' maturation feeding were identical. High contents
of anisole, (E)-b-ocimene, a-copaene, one unidenti"ed sesquiterpenic hydrocarbon C H and
15 24
b-caryophyllene were found in both samples of twigs attacked by beetles. During the construction of maternal galleries by bark beetles in oak logs, monoterpene hydrocarbons such as
p-cymene, (E)-b-ocimene, and c-terpinene, and sesquiterpenes a-copaene and b-caryophyllene
were released in large quantities. No new compound appeared when males were added to the
log with feeding females. ( 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Quercus; Monoterpenes; Sesquiterpenes; Oak wilt; Pathogenic fungi; Scolytus intricatus; Maturation feeding; Brood galleries
* Corresponding author. Tel.: #4202-201-83229; fax: #4202-243-10177.
E-mail address: vrkocova@uochb.cas.cz (P. Vrkoc\ ovaH ).
0305-1978/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 5 - 1 9 7 8 ( 0 0 ) 0 0 0 4 2 - 9
934
P. Vrkoc\ ova& et al. / Biochemical Systematics and Ecology 28 (2000) 933}947
1. Introduction
The oak wilt, caused by pathogens of the genera Ceratocystis, Ophiostoma and
others, is a disease leading to withering and death of oak trees in the eastern and
central states of the USA as well as in Europe (Doganlar and Schopf, 1984). Infected
trees die within a year, sometimes in less than a month. The disease is spread by
root-to-root grafts and the pathogenic fungi can be transmitted by insects.
The European oak bark beetle (Scolytus intricatus; Coleoptera: Scolytidae) is reported as a possible vector of the fungus (Doganlar and Schopf, 1984). The adult
beetles breed in cut or weakened oak branches by boring deep larval galleries in the
xylem. The cut branches are often infested by pathogens. S. intricatus overwinters in
the late larva stage or as a pupa (Schwenke, 1974). In the spring time, emerging adult
beetles visit healthy oaks and make twig cavities where they feed (maturation feeding).
Thus, beetles contaminated with spores or parts of mycelium can transmit the fungus
to the healthy trees. In the Czech Republic, the species usually has one generation per
year. In exceptionally warm years or localities, a partial second generation can occur.
Bark beetles feeding or breeding in trees must locate a suitable host from among the
relatively few scattered widely in the forest. Usually, chemicals are the most important
mediators of host-plant-searching behavior (Miller and Strickler, 1984; Visser, 1986;
StaK dler, 1992). It is believed that insects have evolved behavioral responses to volatile
host-plant chemicals that indicate the presence of a suitable host in which reproduction can occur. Depending on the species, bark beetles "nd their host either by
attraction to host volatiles from a distance or by random landing and probing (Byers,
1995). Chemical mediation of host "nding generally occurs via mixtures of chemicals,
some of them stimulants and others inhibitors.
Host volatiles are attractive to a number of forest scolytids including species in the
genus Scolytus. In case of S. intricatus, oak (Quercus spp.) is the main host tree, but
willow (Salix spp.) is also accepted by beetles for maturation feeding (Doganlar and
Schopf, 1984).
The main goal of our work is to search for chemical communication of the
European oak bark beetle by pheromones and for their potential primary attractants.
In the present study, we have investigated (a) volatiles emitted in di!erent phases of
the oak development (outer and inner bark, unopened buds, young developing leaves,
and blossoms), and (b) volatiles released from oak twigs and branches during both
maturation feeding and construction of maternal galleries. Biological activity was
observed by preliminary electrophysiological and behavioral tests.
2. Materials and methods
2.1. Collection of plant material and insects
Buds, leaves, bark and blossoms were obtained from healthy oak trees (Quercus
robur) in an area about 20 km from Prague in the spring of 1999. The bark was
collected twice, "rst when buds were developing and second when young leaves were
P. Vrkoc\ ova& et al. / Biochemical Systematics and Ecology 28 (2000) 933}947
935
developing. Logs, healthy as well as infested with Scolytus intricatus were collected in
the winter of 1998 in three di!erent localities within the Czech Republic. All logs were
kept at 53C until used. Infested logs were then placed into net cages at laboratory
temperature. Emerged beetles were collected daily, sexed and allowed to bore into
young oak twigs for maturation feeding and later for making maternal galleries and
eventual copulation. All plant materials studied were free of pathogenic fungi.
All samples were repeated "ve times and standard deviations are given in the
results.
2.2. Collection of volatiles
An air entrainment technique was used for trapping the volatile compounds
released from oak (Q. robur) buds, blossoms, young twig bark and young leaves, oak
twigs (diameter 2}6 mm), logs (diameter 4}5 cm) and beetle-infested twigs and logs (40
beetles in each). Modi"ed equipment earlier described by JursmH k et al. (1991) was used
for collection of volatiles. Buds, blossoms and bark were shredded and leaves were
crushed in liquid nitrogen before the entrainment. Biological material was then placed
in a glass vessel (3 l). Puri"ed air (silica gel, charcoal and molecular sieve) was drawn
through the vessel for 24 h at 40}50 ml/min and then through a glass tube "lter
containing Porapak Q (0.1 g; mesh size 100}120). An additional "lter was placed after
the "rst one for a breakthrough check. The captured volatiles were eluted from the
sorption material with hexane (150 ll).
2.3. Identixcation of volatile constituents
The components were identi"ed on a GC (Carlo Erba) coupled with a mass detector
Fisons MD 800, using a BPX5 capillary column (SGE, 30 m]0.22 mm, "lm thickness
0.25 mm) with helium #ow 0.94 and 0.55 ml/min (measured at 503C), respectively. The
temperature program was 503C for 2 min, then 43C/min up to 2603C and held for
20 min, resp. 703C for 2 min, then 43C/min up to 2603C and held for 20 min; inlet
temperature was 2003C in all cases, injector working in splitless mode. The mass
spectra (electron-impact) were compared to the Wiley Registry of Mass Spectral Data,
6th edition, the National Institute of Standards and Technology (NIST) Library, and
Identi"cation of Essential Oil Components by Gas Chromatography/Mass Spectrometry Library (R.P. Adams, Allured Publishing Corporation). Retention times of
analyzed compounds were compared with those of authentic samples wherever
possible.
The gas-phase infrared spectrum (GC-FTIR) of one of the isolated components,
a sesquiterpene hydrocarbon C H , was measured on an HP 5890 gas chromato15 24
graph coupled with an HP 5695A IRD equipped with a narrow-band
(4000}750 cm~1) infrared detector (mercury cadmium telluride).
Two-dimensional chromatography (2D-GC) was used for the determination of the
optical purity of chiral compounds, using available standards (a-pinene, b-pinene,
limonene, camphene). Two Varian 3400 gas chromatographs with two installed
Valco micro-valves were connected with a heated interface. The GC system was
936
P. Vrkoc\ ova& et al. / Biochemical Systematics and Ecology 28 (2000) 933}947
equipped with split/splitless injectors and #ame ionization detectors. Deactivated
fused silica capillaries were used as retention gaps and for the connection of the
di!erent valves with each other and with the detectors. A DB-WAX column was used
for pre-separation in the "rst chromatograph (GC-A). For chiral analysis the valve
was programmed according to the retention times determined in the "rst run so as to
let the chiral compounds pass to the second chromatograph (GC-B) with the Cyclodex B column. For further details see Borg-Karlson et al. (1993). For GC-A, helium
#ow 6.2 ml/min (403C), column DB-WAX (J&W, 30 m]0.25 mm, "lm thickness
0.25 mm) and temperature program 503C for 2 min, then 43C/min up to 2103C
and held for 20 min were used. For GC-B, helium #ow 9.2 ml/min (403C), column
Cyclodex B (J&W, 30 m]0.25 mm, "lm thickness 0.25 mm) and temperature program
553C for 13 min, then 13C/min up to 773C were used.
3. Results and discussion
Complex mixtures of aliphatic, aromatic, and terpenoid compounds were identi"ed
in the samples from oak (Table 1, Figs. 1 and 2). In the early and late bark samples,
aldehydes formed the main portion of the mixture, (E)-2-hexenal and hexanal 8.7 and
12.8% resp. 23.0% and 45.4% being the main components. n-Alkanes formed 13.7%
resp. 5.1% and aliphatic alcohols 6.2% resp. 4.3% of the compounds. Monoterpene
and sesquiterpene hydrocarbons were present as minor components. Esters were
almost absent. Proportion of compounds in early and late bark samples did not di!er
signi"cantly.
In the buds, esters dominated (36.5%) in the volatiles released. (Z)-3-hexenyl acetate
formed 21.5% of the mixture. Substantial proportions of alcohols (16.4%) and
aldehydes (13.0%) were found in the samples. Among the alcohols, 11.0% consisted of
(Z)-3-hexenol. In relatively high abundance were dioxaspiroalkanes (11.5%).
Homoterpene hydrocarbons were present as minor components (2.0%). Monoterpene
and sesquiterpene hydrocarbons and n-alkanes were present in small quantities only.
After the buds opened and small leaves appeared, the spectrum of volatiles changed
again. Aldehydes (3.7%) and alcohols (9.6%) were less abundant, while n-alkanes were
moderately abundant (19.3%) constituents. Esters (31.7%) and sesquiterpene hydrocarbons (38.9%) dominated in the samples. (Z)-3-Hexenyl acetate and (Z)-3-hexenyl
butanoate were the major esters while (E,E)-a-farnesene (37.5%) was the most important sesquiterpene. Also, homoterpenes were present as minor components (4.8%).
Volatiles from opened oak blossoms were included in the analytical study although
the #owers open later than bark beetles start their maturation feeding. (E, E)-afarnesene dominated in the sample (41.7%). All other types of compounds were
present in moderate abundance (from 3 to 7%).
The period of maturation feeding is supposed to be critical for the transfer of fungal
pathogens. The attack of beetles on young twigs for maturation feeding coincides
approximately with the development of oak leaves. The knowledge of volatile pro"les
of four main types of tissue may lead to better understanding of the beetle host seeking
behavior.
Table 1
Compounds emitted from oak (Quercus robur) in di!erent phases of its development
Compound!
MS,
MS,
MS
MS,
MS,
MS,
MS,
MS
MS
MS,
MS
MS,
MS
MS,
MS,
MS
MS,
MS,
MS,
MS
MS,
MS,
MS,
MS,
MS,
MS,
MS,
MS,
standard
standard
standard
standard
standard
standard
standard
standard
standard
standard
standard
standard
standard
standard
standard
standard
standard
standard
standard
standard
standard
Relative % (standard deviation)
Bark I#
Bark II$
Buds
3.37
*
*
8.71
3.87
23.03
2.32
0.48
*
0.82
0.17
0.05
*
*
0.27
*
*
*
2.83
*
*
*
*
*
*
1.00
1.33
*
1.11
*
*
12.78
*
45.40
4.31
0.19
*
0.38
*
0.14
*
0.20
0.25
*
0.08
1.78
0.44
*
*
0.18
*
*
0.09
2.93
1.08
*
0.61
0.53
1.29
9.37
11.04
3.60
5.22
*
0.38
*
*
*
0.24
*
*
*
*
0.33
*
*
*
*
21.25
6.89
*
0.09
0.18
(3.83)
(5.78)
(3.32)
(21.29)
(1.91)
(0.45)
(0.84)
(0.15)
(0.04)
(0.28)
(2.97)
(0.87)
(0.70)
(0.25)
(6.17)
(4.32)
(2.17)
(0.16)
(0.11)
(0.09)
(0.18)
(0.14)
(0.08)
(0.49)
(0.21)
(0.05)
(0.11)
(2.47)
(0.76)
Leaves
(0.28)
(0.20)
(0.43)
(1.60)
(4.34)
(5.35)
(2.02)
(0.13)
(0.09)
(0.10)
(5.42)
(4.17)
(0.08)
(0.08)
0.08
*
0.46
3.50
8.94
0.22
0.69
0.03
*
0.02
*
0.13
*
0.02
0.01
*
0.08
0.03
*
0.40
*
15.22
1.01
*
0.02
0.07
0.03
(0.09)
(0.30)
(1.74)
(3.54)
(0.44)
(0.34)
(0.02)
(0.01)
(0.07)
(0.03)
(0.00)
(0.03)
(0.02)
(0.30)
(7.70)
(0.60)
(0.03)
(0.06)
(0.01)
Blossoms
1.59
*
*
5.84
6.26
1.04
0.80
0.68
*
0.15
*
*
*
*
0.37
0.15
*
0.68
0.22
0.32
*
*
3.99
0.34
*
*
0.43
*
(0.70)
(8.73)
(3.40)
(0.62)
(0.58)
(0.28)
(0.05)
(0.21)
(0.09)
(0.20)
(0.11)
(0.17)
(1.17)
(0.14)
(0.09)
937
3.284
3.501
3.600
3.667
4.517
4.400
4.617
4.717
4.718
4.984
5.101
5.201
5.367
5.601
5.851
6.584
6.768
7.117
7.251
7.501
7.534
7.669
7.717
7.801
7.985
8.317
8.384
8.467
Method of
identi"cation
P. Vrkoc\ ova& et al. / Biochemical Systematics and Ecology 28 (2000) 933}947
Toluene
Heptane
Ethyl butanoate
Hexanal
(Z)-3-Hexen-1-ol
(E)-2-Hexenal
1-Hexanol
Dimethylbenzene, isomer I
Isopentyl acetate
Nonane
Dimethylbenzene, isomer II
Heptanal
Pentyl acetate
a-Thujene
a-Pinene
Ethylmethylbenzene
Sabinene
Myrcene
Decane
Trimethylbenzene
(E)-3-Hexenyl acetate
Octanal
(Z)-3-Hexenyl acetate
n-Hexyl acetate
a-Terpinene
p-Cymene
Limonene
(Z)-b-Ocimene
RT (min)"
938
Table 1. Continued
Compound!
Method of
identi"cation
Relative % (standard deviation)
Bark I#
Bark II$
Buds
Leaves
0.03 (0.03)
0.25 (0.18)
10.82 (6.42)
0.02 (0.02)
0.70 (0.84)
0.04 (0.03)
0.33 (0.29)
0.61 (0.49)
0.60 (0.33)
*
*
0.03 (0.03)
*
0.16 (0.08)
*
0.04 (0.05)
*
8.534
8.801
9.184
MS, standard
MS, standard
MS, standard
*
*
*
*
*
*
9.269
9.715
9.717
10.203
MS, standard
MS, standard
MS, standard
MS
0.32 (0.42)
*
0.34 (0.24)
*
2.89 (2.84)
*
0.22 (0.15)
*
0.03 (0.07)
0.18 (0.10)
*
0.73 (0.25)
10.301
10.353
10.368
10.551
10.701
10.769
10.951
11.001
12.018
12.268
13.651
13.801
13.885
14.401
14.521
15.185
15.368
15.601
17.171
17.418
18.602
MS,
MS,
MS
MS
MS,
MS
MS,
MS
MS
MS
MS
MS
MS,
MS,
MS,
MS
MS
MS
MS,
MS,
MS
0.88
2.07
*
*
*
*
0.47
*
*
*
0.36
*
1.34
1.99
*
*
*
*
*
1.01
*
0.81
0.30
*
*
*
*
0.23
*
*
*
*
*
0.45
0.22
0.10
*
*
*
*
0.65
*
0.09
*
*
0.25
*
0.14
*
1.27
0.22
0.13
2.59
0.76
*
0.83
*
0.50
0.65
0.13
0.22
0.09
*
standard
standard
standard
standard
standard
standard
standard
standard
standard
(0.63)
(1.81)
(0.42)
(0.23)
(0.66)
(3.44)
(0.52)
(0.81)
(0.34)
(0.09)
(0.10)
(0.21)
(0.13)
(0.20)
(0.07)
(0.11)
(0.18)
(0.78)
(0.06)
(0.10)
(0.72)
(0.57)
(0.89)
(0.25)
(0.18)
(0.04)
(0.08)
(0.03)
0.04
*
0.15
0.69
0.01
*
*
0.91
0.89
0.03
10.96
0.15
*
*
*
0.83
0.32
0.03
(0.08)
(0.06)
(0.37)
(0.03)
(0.42)
(0.61)
(0.02)
(3.71)
(0.05)
(0.40)
(0.21)
(0.04)
0.06 (0.07)
0.04 (0.03)
Blossoms
0.04
0.33
*
*
*
*
*
0.83
*
*
1.55
*
0.39
*
*
*
*
*
*
0.79
*
(0.07)
(0.19)
(0.84)
(0.79)
(0.18)
(1.09)
P. Vrkoc\ ova& et al. / Biochemical Systematics and Ecology 28 (2000) 933}947
1,8-Cineole
(E)-b-Ocimene
(E)-7-Methyl-1,6-dioxaspiro [4.5]
decane
c-Terpinene
1-Octanol
Linalool oxide, furanic, cis
2-Ethyl-1,6-dioxaspiro [4.4]
nonan MchalcogranN
Linalool oxide, furanic, trans
Undecane
n-Pentyl butanoate
(Z)-3-Hexenyl propanoate
Linalool
n-Hexyl propanoate
Nonanal
(E)-4,8-Dimethyl-1,3,7-nonatriene
(Z)-3-Hexenyl isobutanoate
n-Hexyl isobutanoate
(Z)-3-Hexexyl butanoate
n-Hexyl butanoate
Dodecane
Methyl salicylate
Decanal
(Z)-3-Hexenyl 2-methylbutanoate
n-Hexyl 2-methylbutanoate
n-Hexyl pentanoate
Ethyl salicylate
Tridecane
(Z)-3-Hexenyl tiglate
RT (min)"
19.318
20.418
20.468
20.635
20.868
21.952
22.089
22.390
22.540
22.785
23.402
24.085
24.202
24.702
25.202
26.752
MS
MS,
MS
MS
MS,
MS,
MS,
MS
MS,
MS,
MS,
MS
MS,
MS,
MS
MS
27.152
27.319
30.302
33.153
33.970
MS
MS, standard
MS, standard
MS, standard
MS
standard
standard
standard
standard
standard
standard
standard
standard
standard
*
0.72
*
*
1.87
*
*
*
*
*
*
*
1.29
*
0.76
*
*
1.26
0.78
0.41
0.42
(0.55)
(1.03)
(0.56)
(1.07)
(1.11)
(0.48)
(0.28)
(0.37)
0.17 (0.10)
*
*
1.30
*
*
*
0.18
0.20
0.62
0.04
0.83
0.27
*
0.40
1.40
0.73
0.75
(0.25)
(1.01)
(0.89)
(0.78)
0.01
0.05
0.25
0.08
0.05
0.67
*
*
*
0.03
0.01
0.62
*
37.46
0.03
3.93
*
0.42
0.57
0.17
0.06
*
0.07 (0.02)
0.12 (0.03)
0.05 (0.01)
*
0.03
0.04
0.03
19.02
*
*
0.12 (0.07)
*
*
0.67 (0.26)
*
*
*
*
*
*
*
1.02 (0.24)
(0.10)
(0.16)
(0.03)
(0.02)
!The compounds are listed in elution order on a 30 m DB-5ms column (5% phenyl methyl silicone).
"Temperature program: 503C (2 min); 43/min to 2603 (20 min); helium #ow 0.94 ml/min at 503C.
#Collected at time when buds were developing.
$Collected at time when leaves were developing.
(0.80)
(0.09)
(0.24)
(0.76)
(0.09)
(1.56)
(0.19)
(0.02)
(0.04)
(0.13)
(0.11)
(0.02)
(0.72)
(0.01)
(0.01)
(0.26)
(10.55)
(0.02)
(3.16)
(0.02)
(0.02)
(0.02)
(0.01)
*
0.17
*
*
0.49
0.45
*
*
*
*
*
*
2.19
41.72
*
*
(0.09)
(0.26)
(0.16)
(0.49)
(21.40)
*
0.56 (0.35)
1.27 (0.31)
0.35 (0.21)
*
P. Vrkoc\ ova& et al. / Biochemical Systematics and Ecology 28 (2000) 933}947
a-Cubebene
a-Copaene
(Z)-3-Hexenyl hexanoate
n-Hexyl hexanoate
Tetradecane
b-Caryophyllene
trans-a-Bergamotene
b-Gurjunene
(Z)-b-Farnesene
(E)-b-Farnesene
Aromadendrene
(Z,E)-a-Farnesene
Pentadecane
(E,E)-a-Farnesene
d-Cadinene
(E,E)}4,8,12-Trimethyl-1,3,7,11Tridecatetraene
(Z)-3-Hexenyl benzoate
Hexadecane
Heptadecane
Octadecane
Isopropyl myristate
939
940
P. Vrkoc\ ova& et al. / Biochemical Systematics and Ecology 28 (2000) 933}947
Fig. 1. Sum of relative percent of di!erent compound classes emitted from plant tissues.
Samples obtained from oak twigs during the maturation feeding of Scolytus intricatus females and males had many components in common, though their proportions di!ered (Table 2, Figs. 3, 4a and b). Generally, the twigs and logs without beetles
gave sample of much lower concentration. After beetles started to feed and build
galleries, the amounts of emitted volatiles increased substantially. n-Alkanes formed
a substantial part (8%) of the twig sample without beetles. During the maturation
feeding of both females and males, more compounds were released as a consequence of
the beetles' activity and the proportions of alkanes dropped to the barely detectable
level. In twigs, monoterpenes formed 24.8% of the compounds. The most abundant
compound was (E)-b-ocimene (22.5%). Linalool oxides were present at 6.8% and
aldehydes at 6.5%. Only small amounts of sesquiterpenes (4.9%) were found.
Most of the compounds found in the samples from females' and males' feeding were
identical. Their proportions in the two samples were not very di!erent (see Fig. 4a).
High contents of anisole, (E)-b-ocimene, a-copaene, one unidenti"ed sesquiterpenic
hydrocarbon C H (unidentixed 3& in Table 2) and b-caryophyllene were found in
15 24
both samples of twigs with beetles.
The structure of this sesquiterpenic hydrocarbon could not be fully determined
from its mass and infrared spectra. The retention time and mass spectrum of this
compound was not identical with any known sesquiterpene present in our databases
(see Section 2). The character of its mass spectrum corresponds with a cyclic type of
compound (low intensities of fragments m/z 69, 81 exclude the farnesene-type). The
infrared spectrum is in agreement with the proposed cyclic structure. No band in the
P. Vrkoc\ ova& et al. / Biochemical Systematics and Ecology 28 (2000) 933}947
941
Fig. 2. Relative percent and standard deviation of selected compounds (over 5%) emitted from plant
tissues.
CH-stretching area 3070}3080 cm~1 and only a weak band around 1640}1660 cm~1
(C"C stretch) excludes the presence of an exomethylene terminal C"CH group in
2
the compound (Svatos\ and Attygalle, 1997). Bands at 3052 and 3024 cm~1 (C}H
stretch) suggest the presence of two di!erent types of carbon}carbon double bonds.
Double bonds are probably not conjugated, as the C"C stretch (1642 cm~1) is not
too intense. Comparison with IR spectra of terpenes from the literature, the CH
stretch at 3052 cm~1 and CH wagging 780 cm~1 imply a trisubstituted double bond in
a ring.
Oak logs prepared for S. intricatus females to feed in and build brood galleries
released high amounts of monoterpenes (37.8%), and also substantial amounts of
sesquiterpenes (10.1%). Anisole was present as a moderately abundant compound
(6.0%). When the females bored in, monoterpenic (48.6%) and sesquiterpenic (47.2%)
hydrocarbons made up most of the volatiles. When males were added
for copulation to the logs with females, the proportion of terpene hydrocarbons
changed in favor of sesquiterpenes (73.0%). p-Cymene (12.0 and 6.4%, respectively),
Compound!
Method of
identi"cation
Relative % (standard deviation)
Twigs
Maturation
Maturation
feeding}females feeding}males
Logs
Making
Copulation
galleries}females
6.134
6.317
MS
MS
*
0.77 (0.57)
*
0.08 (0.06)
*
0.06 (0.09)
*
1.22 (1.60)
0.01 (0.02)
0.10 (0.12)
*
0.01 (0.02)
6.634
6.900
MS, standard
MS
1.25 (1.01)
0.25 (0.49)
0.28 (0.24)
*
0.22 (0.21)
0.02 (0.03)
0.61 (0.46)
0.46 (0.67)
0.14 (0.14)
*
0.03 (0.02)
*
7.034
7.534
7.567
7.801
MS,
MS,
MS,
MS,
standard
standard
standard
standard
0.14 (0.28)
*
*
0.50 (0.52)
*
*
7.01 (6.83)
0.90 (0.39)
*
*
15.99 (12.74)
1.43 (0.68)
*
0.92 (0.95)
6.03 (7.39)
2.45 (2.48)
*
4.22 (3.74)
0.03 (0.06)
2.17 (1.85)
*
0.94 (1.23)
0.13 (0.14)
0.34 (0.27)
8.367
MS, standard
0.42 (0.85)
1.48 (0.90)
1.96 (1.40)
2.03 (2.38)
0.52 (0.38)
0.13 (0.08)
8.734
8.967
9.134
9.217
9.317
9.434
9.817
9.984
10.084
10.434
10.767
10.884
MS
MS,
MS,
MS,
MS,
MS,
MS
MS,
MS,
MS,
MS,
MS,
standard
standard
standard
standard
standard
0.78
*
0.20
*
*
*
1.26
1.83
*
*
0.38
0.37
*
0.17
*
0.23
0.47
0.39
*
*
*
*
0.54
0.71
(0.46)
(0.36)
*
0.37
*
0.30
0.61
0.21
*
*
*
*
1.34
0.52
(1.16)
(0.78)
0.40
1.88
*
0.65
2.43
*
*
*
*
0.25
9.20
4.78
10.934
11.034
11.317
11.751
MS,
MS,
MS,
MS,
standard
standard
standard
standard
0.65
0.06
22.49
0.89
(1.31)
*
(0.11)
0.05 (0.06)
(26.91) 20.34 (2.12)
(1.15)
0.37 (0.17)
0.49
0.34
19.45
0.38
(0.43)
(0.23)
(6.77)
(0.35)
*
3.56 (2.13)
5.38 (4.48)
*
11.867
12.384
MS, standard
MS, standard
0.63 (0.60)
*
7.80 (6.35)
*
standard
standard
standard
standard
standard
(1.41)
(0.39)
(2.51)
(0.68)
(0.77)
(0.45)
*
3.05 (2.71)
(0.05)
(0.11)
(0.14)
(0.36)
0.31 (0.31)
*
(0.14)
(0.24)
(0.34)
(0.25)
(0.40)
(1.31)
(0.81)
(1.36)
(0.31)
(4.87)
(2.91)
*
0.67
*
0.16
1.00
0.10
*
*
0.08
0.70
12.01
3.41
*
0.07
*
0.01
0.39
0.02
*
*
0.02
0.21
6.42
0.64
(0.03)
(0.28)
(9.07)
(1.29)
*
0.19 (0.20)
12.23 (7.43)
0.10 (0.15)
0.59
0.03
8.26
0.10
(0.77)
(0.03)
(3.04)
(0.13)
11.46 (10.25)
*
4.05 (5.53)
*
(0.59)
(0.13)
(0.49)
(0.11)
(0.07)
(0.62)
(10.01)
(2.19)
(0.09)
(0.02)
(0.17)
(0.03)
P. Vrkoc\ ova& et al. / Biochemical Systematics and Ecology 28 (2000) 933}947
Ethylbenzene
Dimethylbenzene,
Isomer I
Nonane
Dimethylbenzene,
Isomer II
Heptanal
a-Thujene
Anisole
a-Pinene (!/#:
96.2/3.8)#
Camphene (!/#:
100/0)#
Ethylmethylbenzene
Sabinene
Benzaldehyde
b-Pinene (!/#: 100/0)#
Myrcene
Decane
Trimethylbenzene
Octanal
a-Phellandrene
a-Terpinene
p-Cymene
Limonene (!/#:
83.1/16.9)#
(Z)-b-Ocimene
1,8-Cineole
(E)-b-Ocimene
(E)-7-Methyl-1,6dioxaspiro[4,5]decane
c-Terpinene
Linalool oxide, furanic,
cis
RT (min)"
942
Table 2
Compounds captured from oak twigs and logs during maturation feeding and making maternal galleries of Scolytus intricatus
12.834
12.951
12.984
MS, standard
MS, standard
MS, standard
*
*
3.78 (3.34)
0.09 (0.11)
0.40 (0.37)
*
0.02 (0.04)
0.16 (0.26)
*
0.66 (0.41)
*
*
0.71 (0.59)
0.06 (0.12)
*
0.29 (0.37)
*
*
13.634
13.668
MS, standard
MS
2.58 (3.28)
2.10 (3.18)
*
0.18 (0.06)
*
0.74 (0.55)
*
3.27 (2.70)
*
0.31 (0.30)
*
0.04 (0.05)
16.568
17.368
18.451
19.118
20.302
21.535
22.368
23.552
23.768
23.935
23.952
24.652
25.219
25.352
25.535
25.635
26.452
26.602
27.035
27.085
27.219
27.285
27.635
27.752
27.819
28.352
MS,
MS,
MS
MS
MS,
MS
MS
MS,
MS
MS,
MS
MS
MS,
MS,
MS
MS,
MS
MS,
MS
MS
MS,
MS
MS
MS
MS
MS
1.74
1.90
*
*
2.87
*
*
*
*
0.83
*
*
0.27
0.66
*
*
*
1.02
*
0.04
1.96
*
*
1.59
*
1.35
standard
standard
standard
standard
standard
standard
standard
standard
standard
standard
(1.04)
(2.56)
(1.26)
(1.02)
(0.54)
(1.33)
(0.94)
(0.07)
(1.24)
(1.05)
(2.04)
0.25
*
0.01
0.11
0.09
*
0.15
16.02
16.46
*
0.48
0.09
9.72
1.48
1.06
1.02
1.87
0.46
0.00
*
*
0.57
2.29
1.99
*
1.98
(0.23)
(0.02)
(0.06)
(0.13)
(0.09)
(5.54)
(12.36)
(0.29)
(0.06)
(2.90)
(0.91)
(0.53)
(0.66)
(0.65)
(0.12)
(0.00)
(0.29)
(0.85)
(0.68)
(0.70)
0.09
*
0.07
*
*
*
0.12
15.25
16.86
*
0.66
0.31
5.98
1.51
0.98
0.96
1.56
0.64
0.67
*
*
1.03
1.13
2.06
*
3.23
(0.14)
(0.09)
(0.05)
(9.60)
(8.79)
(0.65)
(0.26)
(5.24)
(0.67)
(0.49)
(0.69)
(1.44)
(0.57)
(0.44)
(1.08)
(0.62)
(1.40)
(3.22)
*
*
*
*
*
*
*
2.59
5.37
*
*
*
0.33
0.73
*
0.38
*
0.37
*
*
*
*
*
0.14
*
0.10
(2.68)
(6.88)
(0.67)
(1.02)
(0.46)
(0.74)
(0.28)
(0.20)
(0.05)
(0.02)
(0.32)
(0.03)
(0.05)
(0.22)
(12.25)
(3.36)
(0.81)
(1.81)
(4.79)
(0.16)
(2.31)
(0.37)
(1.90)
(0.48)
(0.33)
(1.09)
(1.73)
(0.99)
(4.82)
(1.03)
*
*
0.03
0.37
*
0.21
0.33
26.73
7.57
*
1.58
1.37
8.87
0.55
2.96
0.36
3.84
0.76
1.07
*
*
2.63
2.67
0.97
6.87
3.90
(0.04)
(0.30)
(0.12)
(0.21)
(7.96)
(6.52)
(0.68)
(1.70)
(3.69)
(0.44)
(1.87)
(0.39)
(1.85)
(0.16)
(0.12)
(1.38)
(1.77)
(1.24)
(5.44)
(0.34)
943
!The compounds are listed in elution order on a 30 m BPX-5 column (5% phenyl methyl silicone).
"Temperature program: 703C (2 min); 43/min to 2603 (20 min); helium #ow 0.55 ml/min at 503C.
#Proportion of enantiomers determined on 2D-GC system.
$Mass spectrum * m/z%: 77(57), 79(56), 91(100), 105(52), 119(25), 133(14), 147(34), 162(29).
%Mass spectrum * m/z%: 41(79), 91(100), 105(53), 115(47), 117(44), 119(74), 131(56), 145(44), 159(86), 187(19), 202(16).
&Mass spectrum * m/z%: 41(100), 77(63), 91(79), 93(90), 105(25), 119(75), 161(4), 189(1), 204(0.6).
IR spectrum: 3052, 3024, 2969, 2925, 2876, 2741, 1642, 1452, 1381, 1349, 1159, 1111, 1027, 943, 845, 779 cm~1.
0.03
*
0.01
0.26
0.02
0.04
0.20
15.81
5.61
*
1.03
1.99
5.65
0.52
2.12
0.29
2.17
0.46
0.82
*
*
1.28
1.50
1.00
3.89
2.80
P. Vrkoc\ ova& et al. / Biochemical Systematics and Ecology 28 (2000) 933}947
Terpinolene
Undecane
Linalool oxide, furanic,
trans
Nonanal
(E)-4,8-Dimethyl1,3,7-nonatriene
Dodecane
Dodecanal
Thymol, methyl ether
Unidenti"ed 1$
Tridecane
Unidenti"ed 2%
a-Ylangene
a-Copaene
Unidenti"ed 3&
Tetradecane
b-Elemene
a-Gurjunene
b-Caryophyllene
trans-a-Bergamotene
a-Guaiene
(E)-b-Farnesene
a-Humulene
Aromadendrene
a-Amorphene
a-Curcumene
Pentadecane
Germacrene D
b-Selinene
a-Muurolene
b-Bisabolene
d-Cadinene
944
P. Vrkoc\ ova& et al. / Biochemical Systematics and Ecology 28 (2000) 933}947
Fig. 3. Sum of the relative percent of di!erent compound classes released from host branches and
beetle-attacked branches.
(E)-b-ocimene (12.2 and 8.3%, respectively), c-terpinene (11.5 and 4.0%, respectively),
a-copaene (15.8 and 26.7%, respectively), unidenti"ed sesquiterpene (5.6 and 7.4%,
respectively) and b-caryophyllene (5.7 and 8.9%, respectively) were the most abundant
compounds in logs attacked by beetles. Only relative proportion of anisole, a-copaene
and b-caryophyllene changed signi"cantly with the beetle attack (see Fig. 4b).
Terpenes and homoterpenes are known to be produced by plants in response to
herbivore feeding. (E)-b-Ocimene and (E)-4,8-dimethyl-1,3,7-nonatriene were released
in large amounts from infested cucumber plants (Takabayashi et al., 1994). It is not
clear whether the biosynthesis of the volatiles released from infested plants is induced
by herbivore feeding or if they are stored in the plant and released at the time of
infestation (PareH and Tumlinson, 1996). Infested cotton plants released high amounts
of a-pinene, b-caryophyllene, (E,E)-a-farnesene, (E)-b-farnesene, (E)-b-ocimene, and
(E)-4,8-dimethyl-1,3,7-nonatriene (Loughrin et al., 1994). Loughrin suggested from the
released timing and rate that a-pinene and b-caryophyllene come from the constitutive compounds (stored in the plant) while the other compounds were biosynthesized
de novo in response to insect feeding.
It can be seen from Table 2 that the types of compounds captured from oak twigs
and logs with feeding and breeding beetles correspond quite well with those described
for other herbivore-infested plants. No special compound was found for either
females' or males' feeding samples that would lead us to suspect the presence of
a pheromone. Although results of our behavioral tests indicate that secondary
P. Vrkoc\ ova& et al. / Biochemical Systematics and Ecology 28 (2000) 933}947
945
Fig. 4. (a) and (b). Relative percent and standard deviation of selected compounds (over 5%) released from
host branches and beetle-attacked branches.
chemical communication between sexes exists (Hovorka et al., 2000), a chemical basis
supporting this fact could not be found. Either the concentration of the active
compound(s) was too low or the signal is produced in a di!erent phase of the beetles'
946
P. Vrkoc\ ova& et al. / Biochemical Systematics and Ecology 28 (2000) 933}947
development. Electrophysiological testing of potential host-plant attractants indicated the activity of several components of our samples (dimethylbenzene, nonane,
a-thujene, sabinene, p-cymene, c-terpinene, terpinolene, dodecane, tridecane, and
a-gurjunene).
Many forest bark beetles are attracted by host volatiles. Some of them, possessing
a multicomponent aggregation pheromone, are attracted by host volatiles at the
beginning of their mass attack. Another smaller group of scolytids may not use an
aggregation pheromone, but generally they are strongly attracted either to the host
monoterpenes, ethanol, or a combination of both. The compounds released from
a host tree in the particular development state are not only important for primary
attraction to plants but they may play a role in enhancing the bark beetles' response to
the aggregation pheromone, if any.
The production of an aggregation or sex pheromone by S. intricatus is still uncertain
despite the evidence from behavioral trials that seems to indicate its presence. The
situation could be similar to that in Scolytus ventralis where, according to latest data,
the attack dynamics can be explained solely by its sensitive primary attraction
response to host volatiles (MarcmH as-SaH mano et al., 1998) or to Tomicus piniperda where
the pheromone exists, but it remains elusive due to a strong masking e!ect of tree
odors (Czokajlo, 1998).
Acknowledgements
The "nancial support of this work by the Grant Agency of the Czech Republic
(grants nos. 203/97/0037 and 203/00/0219) and by COST E16.10. is gratefully acknowledged. The authors also wish to thank Dr. A.-K. Borg-Karlson (KTH, Stockholm,
Sweden) for 2D-GC instrument time, Dr. A.B. Attygalle (Cornell University, Ithaca,
USA) for GC-FTIR instrument time, Dr. S. Vas\ mH c\ kovaH and Dr. A. Svatos\ (Institute of
Organic Chemistry and Biochemistry, Prague, Czech Republic) for the GC-IR consulting, and Prof. R.M. Coates (University of Illinois, Urbana, USA) for sending
a sample of trans-a-bergamotene.
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