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Attraction of Redbay Ambrosia Beetle, Xyleborus Glabratus,
To Leaf Volatiles of its Host Plants in North America
Xavier Martini
1
&Marc A. Hughes
2
&Jason A. Smith
2
&Lukasz L. Stelinski
1
Received: 14 January 2015 /Revised: 21 May 2015 / Accepted: 28 May 2015
#Springer Science+Business Media New York 2015
Abstract The redbay ambrosia beetle, Xyleborus glabratus,
is an important pest of redbay (Persea borbonia) and swamp
bay (P. palustris) trees in forests of the southeastern USA. It is
also a threat to commercially grown avocado. The beetle is
attracted to host wood volatiles, particularly sesquiterpenes.
Contrary to other ambrosia beetles that attack stressed, possi-
bly pathogen-infected, and dying trees, X. glabratus readily
attacks healthy trees. To date little is known about the role of
leaf volatiles in the host selection behavior and ecology of
X. glabratus. To address this question, an olfactometer bioas-
say was developed to test the behavioral response of
X. glabratus to plant leaf volatiles. We found that X. glabratus
was attracted to the leaf odors of their hosts, redbay and
swamp bay, with no attraction to a non-host tree tested (live
oak, Quercus virginiana), which served as a negative control.
Gas chromatography–mass spectrometry (GS/MS) analysis of
leaves revealed the absence of sesquiterpenes known to be
attractive to X. glabratus and present in host wood, suggesting
that additional leaf-derived semiochemicals may serve as at-
tractants for this beetle. An artificial blend of chemicals was
developed based on GC/MS analyses of leaf volatiles and
behavioral assays. This blend was attractive to X. glabratus
at a level that rivaled currently used lures for practical moni-
toring of this pest. This synthetic redbay leaf blend also was
tested in the field. Baited traps captured more X. glabratus
than unbaited controls and equivalently to manuka oil lures.
We hypothesize that leafvolatiles may beused by X. glabratus
as an additional cue for host location.
Keywords Host finding .Terpen es .Volatile organic
compounds .Laurel wilt .Lauraceae .Insect pest .Coleoptera
Introduction
Theexoticredbayambrosiabeetle,Xyleborus glabratus
Eichhoff (Coleoptera: Curculionidae: Scolytinae), is an inva-
sive species, native to Asia and established in the southeastern
United States (Fraedrich et al. 2008; Rabaglia et al. 2006).
Xyleborus glabratus is the vector of the fungal pathogen
Raffaelea lauricola T.C. Harr., Fraedrich and Aghayeva, that
causes laurel wilt, a highly lethal disease of the Lauraceae
(Fraedrich et al. 2008; Harrington et al. 2008). Boring by
X. glabratus inoculates host trees with R. lauricola,which,
in susceptible hosts, is followed by branch wilt that progresses
throughout the entire canopy, ultimately leading to tree death
(Fraedrich et al. 2008;Mayfieldetal.2008). Wild and urban
populations of redbay (Persea borbonia [L.] Spreng.) and
swamp bay (Persea palustris [Raf.] Sarg.) have been killed
by laurel wilt (Evans et al. 2013; Fraedrich et al. 2008; Shields
et al. 2011; Spiegel and Leege 2013), and practical concerns
are significant, given that the full impacts of this disease upon
the Florida Everglades and commercial avocado (Persea
americana Mill.) growing regions of south Florida have not
yet been realized (Ploetz et al. 2013; Rodgers et al. 2014).
Xyleborus glabaratus is restricted to lauraceous hosts with-
in the US (Hanula et al. 2008;Kendraetal.2014a;Mayfield
et al. 2013;Peñaetal.2012); these also are preferred in its
native host range (Hulcr and Lou 2013). Contrary to most
Xavier Martini and Marc A. Hughes contributed equally to this work.
*Xavier Martini
xavierp.martini@gmail.com
1
Entomology and Nematology Department, Citrus Research and
Education Center, University of Florida, 700 Experiment Station Rd,
Lake Alfred, FL 33850, USA
2
School of Forest Resources and Conservation, University of Florida,
136 Newins-Ziegler Hall, Gainesville, FL 32611-0410, USA
JChemEcol
DOI 10.1007/s10886-015-0595-5
ambrosia beetles that attack weakened, damaged or recently
dead trees (Hulcr et al. 2007;Lindgren1990), X. glabratus
can attack live and apparently healthy trees within its intro-
duced range in the US (Fraedrich et al. 2008; Mayfield et al.
2008). Additionally, X. glabratus is not attracted to ethanol
(Hanula and Sullivan 2008; Johnson et al. 2014), a semio-
chemical indicative of tree stress and decay (Kelsey et al.
2014; Kimmerer and Kozlowski 1982)thatisusedasanat-
tractant for monitoring of various ambrosia beetles and wood
borers of the Xyleborini tribe (Miller and Rabaglia 2009;
Montgomery and Wargo 1983; Ranger et al. 2010). Extensive
research has shown that X. glabratus is attracted to the
sequiterpenes found within the cambium of their lauraceous
hosts (Niogret et al. 2011); primarily α-copaene, but also to α-
cubebene, α-humulene, and calamenene (Hanula and Sullivan
2008; Kendra et al. 2012,2014a; Niogret et al. 2011). This led
to the widespread use of manuka oil, an essential oil contain-
ing high concentration of α-copaene as well as cubeb oil as the
primary attractants for trapping and surveying X. glabratus for
quarantine and management purposes (Hughes et al. 2015;
Johnson et al. 2014).
Thus far, the response of X. glabratus to chemical host cues
has been tested with the use of cut tree bolts, synthetic terpe-
noids, and essential oil lures (Hanula and Sullivan 2008l;
Kendra et al. 2014a,b; Kuhns et al. 2014a; Mayfield et al.
2013). Leaves represent a major tissue source of volatile emis-
sion from plants (Baldwin 2010), yet little has been done to
explore what role (if any) host leaves and their volatiles may
play in the ecology of X. glabratus. The purpose of this inves-
tigation was to determine the possible attractiveness of host and
non-host leaf volatiles to X. glabratus. Behavioral assays were
conducted by exposing X. glabratus to leaf odors in laboratory
olfactometers, which indicated beetle attraction to host leaf vol-
atiles. In addition, the volatile profile of a host plant (redbay)
leaf headspace was analyzed by GC/MS and compared to
redbay wood volatiles. The leaf chemical profile was
reconstituted in vitro, creating an artificial ‘redbay leaf blend’
that was later tested in laboratory and field experiments. This
synthetic blend, based on identified leaf volatiles, proved to be
attractive to X. glabratus in the laboratory and field.
Methods and Material
Insect and Plants Xyleborus glabratus beetles were reared
and emerged from infested swamp bay logs collected in
Wekiwa, FL, USA. The logs were stored at 23 ° C in large
plastic containers with humidified Kimwipe® papers
(Kimberly-Clark, Roswell, GA, USA) that were replaced ev-
ery 2 weeks. Beetles were collected, observed under a dissec-
tion microscope to ensure mobility (no missing legs and able
to walk) 1–2 hr prior to olfactometer assays. Plant material
consisted of clonally propagated redbays planted in 57 L
containers (Hughes and Smith 2014). Containerized (12.5 L)
nursery grown swamp bays and live oak branches (Quercus
virginiana Mill.) collected near Lake Alfred, FL, USA also
were investigated.
Chemicals Dichloromethane (99.8 % purity), nonyl acetate
(99 %), βCaryophyllene (80 %), β-pinene (99 %), camphor,
p-cymene (99 %), α-pinene (98 %), limonene (90 %),
sabinene (75 %), eucalyptol (99 %), borneol, terpinen-4-ol
(95 %), α-terpineol (96 %), bornyl acetate (98 %), and α-
terpinyl acetate (90 %) were purchased from Sigma-Aldrich
(St. Louis, MO, USA). Manuka oil was purchased from East
Cape Manuka oil (Meridian, ID, USA).
Olfactometer System A four-choice olfactometer (Vet et al.
1983) (Analytical Research System, Gainesville, FL, USA)
was used to evaluate the behavioral response of X. glabratus.
The olfactometer consisted of a four-armed star-shape of four
crescents within a 30 × 30 cm Teflon square (Vet et al. 1983).
Each arm of the star runs into a 15 mm (internal diam [ID])
Teflon tube (Fig. 1). Four odor fields were created in the
chamber by a constant airflow of 0.1 L/min pushed through
each arm of the olfactometer and by pulling air (0.4 L/min) out
through the floor’s central air evacuation hole (Fig. 1). The
olfactometer floor and arms were covered with filter paper
(25 cm diam laboratory filter paper, Curtin Matheson Scien-
tific, Houston, TX, USA) to improve beetle traction and
movement. The air evacuation hole was covered with Teflon
fabric to prevent beetles from entering.
Between each bioassay, the filter paper was changed, and
the olfactometer was washed with Sparkleen® detergent
(Fisherbrand, Pittsburgh, PA, USA) and acetone. Each arm
of the olfactometer was connected to the air delivery system
through a two-way opened 350 ml glass vial that served as a
collection trap for beetles choosing an arm/odor (Fig. 1). To
ensure a chemical-free ambient air supply, arms of the olfac-
tometer received charcoal purified air from a custom made air
delivery system (ARS, Gainesville, FL, USA). The airflow
was measured with a flowmeter (Varian, Walnut Creek, CA,
USA) to ensure equivalent velocity within each arm. The ol-
factometer was positioned under a 150 W high-pressure sodi-
um grow light (Hydrofarm, Petaluma, CA, USA). Twenty-
five X. glabratus adult females were released into the center
of the olfactometer, which was covered with a Plexiglas sheet
and black filter paper so that only the glass traps were illumi-
nated. Beetles were introduced into the olfactometer between
16:00 and 17:00 hr, and the number of beetles that entered the
arms and fell into each trap was counted 16 hr later. This
bioassay was performed overnight given that peak activity
for X. glabratus is between 17:00 and 19:00 hr (Brar et al.
2012;Kendraetal.2012). Beetles that did not leave the ol-
factometer arena were designated as non-responders (NR).
Preliminary negative control tests were conducted by running
JChemEcol
bioassays without odors (humidified air only), which support-
ed the assumption that beetles distributed randomly among the
four traps.
Xyleborus Glabratus Response to Leaf Volatiles For this
experiment, beetles were tested in the 4-choice olfactometer as
described above. Identical odor sources were randomly
assigned to two opposing arms of the olfactometer for each
treatment and, therefore, only two treatments were compared
simultaneously. Odor sources consisted of undamaged redbay
leaves that were enclosed within two-port glass domes (38 cm
height, 14.4 cm ID). Each plant was inserted into a 1 cm diam
hole within a 2.5 cm width polytetrafluoroethylene (PTFE)
board (thereafter referred as ‘guillotine’). The guillotine can
be opened so that the plant can be introduced within the hole
without damage. The guillotine was used to separate the upper
and lower portions of the tree canopy. With this procedure, a
known number of leaves were present within each glass dome.
Clean air was pushed through a water-filled bubbler, to hu-
midify it, into the glass dome (with leaves enclosed within),
and finally into the olfactometer at 0.1 L/min. Air was pulled
from the olfactometer’s central evacuation port by a vacuum
pump at 0.4 L/min to maintain a constant air stream. Labora-
tory conditions were maintained at 23±1 C°, 49 % RH and a
L14:D10 photoperiod. The response of X. glabratus to redbay,
swamp bay, and live oak leaf volatiles (plants described
above) was tested. Redbay and swamp bay are considered
optimal hosts for X. glabratus; whereas, live oak has already
been described as a non-host to X. glabratus (Kendra et al.
2014a; Mayfield et al. 2008) and, therefore, was used as a
negative control. Each leaf volatile treatment was tested with
three replicate trees and up to 25 beetles per assay (≈75 beetles
tested in total).
GC/MS Analysis of Leaf Volatiles A volatile collection sys-
tem was used to identify the profile of redbay leaf volatile
odors. It consisted of four parallel glass domes (38 cm height,
23 cm ID) each with two 3 cm outlets, one at the top,
connecting to the incoming airflow, and the other at the bot-
tom, connecting to the vacuum. Each glass dome was posi-
tioned onto a 5 cm PTFE guillotine so that each plant was
separated into two parts in terms of headspace collection. A
volatile collection trap (7.5 cm long) with 30 mg of HayeSep
Q adsorbent (Volatile Assay Systems, Rensselaer, NY, USA)
was connected to the bottom outlet with a PTFE fitting. Vol-
atiles emitted from the upper portion of each plant enclosed
within each glass chamber were swept downward by the in-
coming humidified and charcoa1 filter purified air at a rate of
1.0 L/min. The volatiles were forced to the bottom of the
chamber by pulling air at 0.6 L/min through volatile collection
traps with a controlled vacuum from the automated volatile
collection system.
Fig. 1 Graphic representation of
a 4-way olfactometer. aglass
capture trap, bvacuum air
evacuation port, codor fields,
with different colors/shades
representing different treatments.
Arrows indicate direction of
airflow
JChemEcol
Volatiles also were collected from the undamaged trunks of
redbay trees in a separate experiment. Ten cm of the main stem
(2.5 cm diam) was enclosed within an oven bag (Reynolds, Lake
Forest, IL, USA) and tied at the top and bottom with rubber
bands. Air was pulled at a rate of 1.0 L/min and sampled at
the bottom of the bag by pulling it at a rate of 0.6 L/min through
volatile collection traps during a 24 hr collection period.
Finally, redbay wood volatiles also were collected by
rasping 2 g of stem tissue (bark, cambium, and sapwood)
and placing this material within a 20 cm glass tube. Air was
pushed at a rate of 1.0 L/min and pulled at 0.6 L/min through
volatile collection traps for a 15 m collection period.
Volatiles were extracted from the collection traps by wash-
ing with 150 μl of dichloromethane. Nonyl acetate (1080 ng)
was added as an interna1 standard to the extracts. For each
collection sample, 1 μl was manually injected into a Clarus
500 GC/MS (PerkinElmer, Shelton, CT, USA). The gas chro-
matograph was equipped with a column capillary injector sys-
tem and flame ionization detector. Data collection, storage,
and subsequent analysis were performed on Perkin Elmer
chromatographic data system TurboMass™. Helium at a lin-
ear flow velocity of 2 ml/min was used as the carrier gas. All
samples were analyzed on a fused silica RTX-5 capillary col-
umn (Restek Corporation, Bellefonte, PA, USA), 60 min ×
0.25 mm ID. The temperature of the column oven was main-
tained at 40 °C for 1 min and then increased at a rate of
7 °C/min to a final temperature of 300 °C and maintained at
300 °C for 6 min. The injector temperature was set at 270 °C
with the detector set at 200 °C. Quantitations were based on
GC/MS profiles and were assigned by comparing peak areas
of known amounts of nonyl acetate (1080 ng) with the peak
areas of compounds extracted from the leaves. Constituents of
the plant volatile emissions were identified by comparison of
mass spectra with spectra in the National Institute of Standards
and Technology database, and the spectra obtained from au-
thentic reference compounds, when available. Additionally,
GC retention times of plant volatiles were compared with
those of authentic compounds on the RTX-5 column, when
available.
Xyleborus Glabratus Response to Synthetic Volatiles The
behavioral response of X. glabratus to synthetic volatiles was
tested based on the above GC/MS analyses. Test compounds
were dissolved in 100 μl of dichloromethane at a 0.1 μg/μl
dosage rate and pipetted onto 2 cm Richmond cotton wicks
(Petty John Packaging, Inc. Concord, NC, USA). These re-
lease devices were placed into two opposing glass olfactome-
ter traps, as described above. Manuka oil was chosen as a
positive control due to its known attractiveness to X. glabratus
(Hanula and Sullivan 2008). Five replicate bioassays of up to
25 beetles (total of=125 beetles) were performed for each
treatment. The response of X. glabratus was tested in the
four-choice olfactometer system to: (1) manuka oil vs. solvent
(dichloromethane), (2) redbay leaf blend (a mix of synthetic
volatiles representing 94.10 % of the redbay leaf volatiles
found after GC/MS analysis [Table 1]) vs. solvent, and (3)
the redbay leaf blend vs. manuka oil.
Test of Redbay Leaf Blend Under Field Conditions The
field trapping site consisted of a hardwood hammock border-
ing the Kanapaha Botanical Garden (29°36′41.0″N 82°24′
35.8″W) in Gainesville, Florida. This site was chosen because
X. glabratus was known to be abundant, and infested redbays
occurred in various stages of laurel wilt decline, as well as,
apparently healthy (asymptomatic) trees. Other abundant tree
species within the area included: live oak, bluff oak (Quercus
astrina Small), sweet gum (Liquidambar styraciflua L.), and
American holly (Ilex opaca Aiton). Laurel wilt was present on
the site for approximately 2 years (Adam Black, personal
communication) when this field experiment was initiated (Au-
gust 2014). Traps were constructed from a 1.5 m wooden post
with a 30 cm Plexiglas® square affixed to the upper portion.
An Elm Bark Beetle trap (46 × 64 cm) (Great Lakes IPM Inc.)
then was folded over the Plexiglas® square and held with
binder clips, resulting in a double-sided sticky-panel (46×
32 cm). Traps were deployed 10 m from the closest redbay
tree, at minimum, to avoid bias due to potential mass emer-
gence of X. glabratus. Lures consisted of 7 ml BEEM vials
(Thermo Fisher Scientific, Waltham, MA, USA) filled with
1 ml of the redbayleaf odor blend without solvent affixed with
twist-ties to the upper-center of the sticky, trapping panel.
Immediately prior to deployment, four holes were poked into
the vials with a pushpin to facilitate release of volatiles. Traps
were baited with one of three treatments: water (negative con-
trol), 1 ml of manuka oil without solvent (positive control),
and 1 ml of the redbay leaf blend lure. Three posts/traps,
baited with their respective treatment lures were spaced
10 m apart, representing a block. Each block was separated
by at least 50 m, and 3–5 blocks were deployed per trapping
period. The total number of X. glabratus captured was record-
ed during 7 days trapping periods. Three trials were conduct-
ed: August 25 through September 1, 2014 (N=4); September
8 through September 15, 2014 (N=5),andOctober6through
October 13, 2014 (N=3). Four blocks were similar during the
three trials, and the treatments were rotated within these
blocks for each trial. In one case, an entire block was removed
from the analysis to maintain a balanced design because a
single trap was found on the ground.
Statistical Analysis To analyze olfactometer data, a chi-
squared test on the pooled values of the different replicates
was performed. Beforehand, a heterogeneity chi-squared test
was conducted to ensure that data from each replicate were
homogenous (Zar 2009). Xyleborus glabratus response data
obtained per replicate were found to be homogenous if the
sum of the individual chi-squares for each replicate was not
JChemEcol
significantly different (α>0.05) from the overall chi-squared
of the pooled data (Zar 2009). For the field trapping experi-
ments, capture data were log transformed to account for a non-
normal distribution and were analyzed with a linear mixed
model with Gaussian distribution. The fixed variable was the
treatment lure and the random variable was the block number.
Pairwise paired t-test with Bonferonni correction was used to
determine differences among treatments.
Results
Xyleborus glabratus Response to Leaf Volatiles.Preliminary
experiments indicated no bias in the response of X. glabratus
among the four unbaited olfactometer arms receiving humid-
ified air (χ
2
=2.03, df=3, P>0.05). Xyleborus glabratus was
significantly attracted to the leaf volatiles from its redbay and
swamp bay host plants (Fig. 2). The heterogeneity test per-
formed on redbay was significant (χ
2
=17.94,df =2,P<0.001)
indicating that the three replicates for this treatment were not
homogeneous. Xyleborus glabratus were highly attracted to-
ward redbay leaf volatiles on two replicates (P<0.001), but
there was more variation on the third one (P>0.05). However,
the overall chi-squared tests performed on pooled data re-
vealed that redbay (χ
2
=12.65, df=1, P<0.001) and swamp
bay leaf volatiles (χ
2
=22.44, df=1, P<0.001)weremoreat-
tractive to X. glabratus than clean air. In contrast, leaf volatiles
from the non-host tree, live oak, were not attractive to
X. glabratus, as compared with the clean air control (Fig. 2)
(χ
2
=0.25, df=1, P>0.05).
GC/MS Analysis of Leaf Volatiles The leaf volatiles of eight
potted redbay trees were examined to identify compounds that
maybeattractivetoX. glabratus (Fig. 3a). Most notably, the
sesquiterpenes found in redbay wood and that are known to be
attractive to X. glabratus were absent from leaf volatile emis-
sion profiles (Table 1). To ensure that our collection and ex-
traction method allowed for the detection of those compounds,
a GC/MS analysis of rasped wood samples of the same redbay
individuals was performed using HayeSep Q according to
Niogret et al. (2011), but with dichloromethane as a solvent.
Our tests were able to detect attractive sesquiterpenes (α-
copaene and calamenene) from the rasped wood, thus validat-
ing that they were undetectable or absent from the redbay leaf
emissions (Fig. 3c). The volatile profiles obtained from the
bagging of undamaged redbay stems was similar to that of
an empty oven bag (blank control), indicating that volatiles
emitted from the stem tissue were undetectable by our method
(data not shown) and that rasping/shaving of the tissue was
necessary to trap wood volatiles.
Xyleborus Glabratus Response to Synthetic Volatiles The
olfactometer bioassays revealed that diluted manuka oil was
attractive to X. glabratus when compared to the dichlorometh-
ane solvent (χ
2
=18.89, df=1, P<0.001) (Fig. 2), validating
the effectiveness of the bioassay. Xyleborus glabratus also
preferentially chose the redbay leaf blend (χ
2
=10.18, df=1,
P=0.001) as compared to the solvent negative control (Fig. 2),
with 65 % of beetles migrating to traps with the redbay leaf
blend. Finally, there was no statistical preference between the
redbay leaf blend and manuka oil (χ
2
=1.13, df=1,P>0.05) at
the dosage tested, although manuka oil captured slightly more
beetles (Fig. 2).
Test of Redbay Leaf Blend Under Field Conditions The
redbay leaf blend was similar to the leaf volatile odors
Tabl e 1 Compounds identified in redbay leaf volatiles and used to make the synthetic ‘redbay leaf blend’
a
Name of the compound KRI % in redbay leaf volatiles % in redbay blend
α- Thujene 936 0.37±0.18 0.00
α-Pinene 948 11.48±1.54 12.60
Camphene 965 0.27±0.15 0.00
Sabinene 987 41.28±7.78 37.13
b
β-Pinene 994 6.57±0.60 8.15
b
Myrcene 996 1.31±0.40 0.00
Cymene 1038 3.61±1.71 3.96
Limonene 1043 8.15±1.74 8.94
Eucalyptol 1048 7.31±2.29 8.02
Camphor 1172 19.31 ±5.74 21.19
α-Terpineol 1212 0.34±0.30 0.00
a
Average percentage (±SE) of chemicals present in the leaf volatile odor of redbay trees (N=8), and the percentage included in the blend used for
olfactometer bioassays and field tests. KRI: Kovats retention indexes calculated based on the injection of a standard mix of alkanes (Sigma-Alrdich.St.
Louis, MO, USA)
b
The purchased sabinene solution used in this blend contained 18 % of β-pinene (personal measurement)
JChemEcol
collected from redbay trees (Figs. 3a and b). There were dif-
ferences in X. glabratus captures between the three treatment
lures (χ
2
=19.98, df=2, P<0.001) (Fig. 4). Traps baited with
manuka oil or the redbay leaf blend captured more
X. glabratus than the negative controls (P<0.001 and P=
0.032, respectively). Although traps baited with manuka oil
caught the most beetles, captures were statistically similar to
those obtained with the redbay leaf blend (P>0.05) (Fig. 4).
Discussion
The olfactometer bioassays and field trapping experiments
demonstrated that X. glabratus is attracted to redbay and
swamp bay leaf volatiles, indicating that they may act as an
additional cue for locating hosts by X. glabratus. The lack of
attraction to live oak samples suggests that X. glabratus can
distinguish between host and non-host leaf volatiles within
100% 50% 0% 50% 100%
Percent response of X. glabratus
Humidified air
Manuka oil
Swamp bay
Redbay
Oak
Redbay blend
Redbay blend
Manuka oil (N=124, NR=13.70%)
Humidified air(N=48, NR=39.6%)
Solvent (N=123, NR=13.0%)
Humidified air (N=72, NR=15.3%)
Humidified air (N=72, NR=16.7%)
Humidified air (N=74, NR=13.5%)
Solvent (N=125, NR=10.40%)
***
***
***
***
NS
NS
NS
Fig. 2 Percentage of Xyleborus glabratus responding to natural or
synthetic odorants vs. humidified air or solvent (dichloromethane) nega-
tive controls, within a four-choice olfactometer. Ntotal number of
X. glabratus used during the experiments, NR Percent of non-responders.
Asterisks indicate significant differences between the two treatments
(***=P<0.001)
Fig. 3 GC/MS Profiles. aRepresentative GC/MS profile from redbay
leaves after 24 hr of collection. bredbay leaf blend that consists of a
reconstitution of the redbay leaf volatile profile with a mixture of synthetic
chemicals. credbay cambium/wood volatiles after 15 min of collection. a:
α-Pinene, b: Sabinene, c:β-Pinene, d: Myrcene, e:Cymene,f: Limonene,
g: Eucalyptol, h: Camphor, i: Borneol, j: Terpinen-4-ol, k;α-Terpineol, l:
Bornyl acetate, m:α-Terpinyl acetate, n:α-Copaene
*
,o:α-Bergamotene
*
,
p:βCaryophyllene, q: unidentified sesquiterpene, r: Calamenene*.
* Determined with NIST database only
JChemEcol
our olfactometer bioassays. Avoidance of non-host volatiles is
known to occur among some scolytid bark beetles, and syn-
thetic blends derived from these non-hosts also can inhibit
response to aggregation pheromones, resulting in possible
management options for the protection of forest trees (Byers
et al. 2000; Unelius et al. 2014; Zhang and Schlyter 2004). In
this case, we did not observe avoidance of the non-host tested,
but an absence of response (i.e., humidified air was not pre-
ferred over live oak).
Chemical analysis of attached and undamaged leaves re-
vealed the presence of eleven compounds, which were mostly
monoterpenes. The vast majority of the identified compounds
in leaf and wood redbay volatiles were chiral, however, the
enantiomeric composition of these volatiles was not deter-
mined in this study. Given that stereochemical properties of
enantiomers impacts odor and other biological activities, such
as insect behavior (Brenna et al. 2003;Mori2014), enantio-
meric compositions of wood and leaf volatiles of Laureacea
coupled with behavioral bioassays should be conducted to
determine if specific enantiomers are more effective attrac-
tants for X. glabratus. Nonetheless, our field trapping data
proved that the blend of available synthetic chemicals tested
here was attractive to X. glabratus. The volatile profile of
leaves differed from that derived from freshly rasped redbay
wood and lacked sesquiterpenes known to be attractive to
X. glabratus such as α- copaene and calamenene. The attrac-
tiveness of the leaf volatiles in the absence of wood-derived
terpenes was confirmed by in vitro reconstitution of the vola-
tile blend, which was found to be attractive both in laboratory
olfactometer and field tests. To assess the proximo-distal dis-
tributions of avocado (Persea americana Mill.) terpenes,
Niogret et al. (2013) sampled leaf, branch and trunk tissues.
In contrast to our results, the authors detected α-copaene and
several other sesquiterpenes among all tissue types sampled,
including leaves. They also noted a distinct gradient in the
abundances of these sesquiterpenes from trunk (highest) to
leaves (lowest), while the opposite relationship was seen for
monoterpenes. Similarly, in our work, redbay leaves
contained mostly monoterpenes but no sesquiterpenes, while
the wood tissue also had higher sesquiterpene content. The
chemical differences between our results and that of Niogret
et al. (2013) may be related to the phylogenetic separation
between the two plant species tested. Redbay is within the
subgenus Eriodaphne and avocado in Persea (Scora and Bergh
1992). Additionally, the size of the plants or solvents utilized
could have contributed to the differences found. Major differ-
ences also existed in the condition of the tissue used; while we
collected leaf volatiles from undamaged leaves, Niogret et al.
(2013) sectioned and cut the avocado leaves and rasped the
petioles, a procedure that may have released terpenes bound
within the plant’s cells, in a similar manner to herbivory
(Baldwin 2010). Interestingly, all the chemicals that we found
in redbay leaf volatiles also were found in the redbay wood
volatiles (Fig. 3), suggesting that the leaf volatile profile rep-
resents the monoterpene fraction of the wood volatile
headspace.
Some monoterpenes are known to be attractive to bark and
ambrosia beetles. α-Pinene, for instance, is widely used alone
or as part of blends to attract a wide range of bark beetles
feeding on gymnosperms (Duduman 2014; Miller et al.
2013; Miller and Rabaglia 2009). An increase of monoterpene
concentrations such as limonene, α-pinene, β-pinene, or
myrcene in the phloem and the sapwood of the Aleppo pine
(Pinus halepensis Mill.) has been correlated with higher infes-
tation by the Mediterranean pine shoot beetle (Tomicus
destruens Wollaston) (Kelsey et al. 2014). The attractiveness
of redbay leaves and the synthetic blend developed here may
be due to the presence of eucalyptol, which is a major com-
ponent of the wood volatile profile of redbay and California
bay laurel (Umbellularia californica [Hook. & Arn.] Nutt.).
(Kendra et al. 2014a; Kuhns et al. 2014a). Eucalyptol is at-
tractive to X. glabratus and in electroantennographic tests
elicited a strong response from X. glabratus (Kendra et al.
2014a). In large quantities, this single chemical attracted
X. glabratus in the field (Kuhns et al. 2014a). Another major
peak that we found in undamaged redbay leaf volatiles was
camphor. Camphor is a significant volatile constituent of
camphortree (Cinnamomum camphora L.) wood (Li et al.
2014), which has been found to be more attractive to
X. glabratus than swamp bay, redbay, and avocado bolts in
field tests (Kendra et al. 2014a;Mayfieldetal.2013). The
positive control used in this study was manuka oil. Manuka
lures have been used since 2008 as a tool for X. glabratus
detection (Hughes et al. 2015). However, commercially avail-
able manuka lures are short-lived in activity as attractants for
X. glabratus compared to newly developed cubeb oil lures
(the efficiency of Manuka lures declines after 3 weeks of ac-
tivity) (Hanula et al. 2013. Kendra et al. 2015). Therefore,
further investigation of cubeb oil as a positive control on a
longer experimental period should be conducted to evaluate
Treatment
fo rebmuN
goL sutarbalg .
Xdetarutpac
0.0
0.5
1.0
1.5
2.0
2.5
Blank Manuka Redbay Blend
ab
b
Fig. 4 Log number of Xyleborus glabratus captured on sticky traps
baited with various lures during field trapping trials. Different letters
indicate significant differences between treatments at α<0.05
JChemEcol
the potential of our redbay leaf volatile blend for monitoring
of X. glabratus.
Xyleborus Glabratus potentially uses leaf volatile cues after
emergence, when the adult females attempt to locate host trees
during their dispersal flights (Hughes et al. 2015; Maner et al.
2013). Leaf volatiles potentially could be used by X. glabratus
as a long-range cue, or a cue indicating the presence of redbay
in the foraging area. Following this long-range attraction
through leaf volatiles, tree selection might be mediated
through visual rather than olfactory cues, as X. glabratus are
known to be attracted by artificial stems of larger diameter
(Mayfield and Brownie 2013). Additionally, bark requires
damage for volatile release, and once on the tree, the beetles
will subsequently determine if the host is suitable for coloni-
zation and reproduction, guiding the decision to bore into the
wood (Kendra et al. 2014a; Kuhns et al. 2014a). Additional
experiments are needed to investigate the potential interac-
tions between leaf volatiles and other known attractant cues
for X. glabratus such as the odors from its symbiotic fungus
(Hulcr et al. 2011; Kuhns et al. 2014b), as well as, other
possible microorganisms.
It is well established that leaf volatiles change qualitatively
and quantitatively in response to pathogen infection or herbiv-
ory (McLeod et al. 2005; Ponzio et al. 2013; Turlings and
Wäckers 2004), and observations suggest that more attacks
occur on moribund laurel wilt affected trees by X. glabratus
than on uninfected counterparts (Hughes et al. 2015). An in-
triguing question is whether X. glabratus responds to changes
in host volatile composition as a result of pathogen infection in
the same manner as has been observed for the Dutch elm
disease pathosystem (McLeod et al. 2005). In this case, native
elm bark beetles (Hylurgopinus rufipes Eichhoff) are signifi-
cantly more attracted to infected than uninfected trees. It is
possible that leaf volatile profiles may be modified by beetle
infestation or R. lauricola infection and that these changes
may affect host plant selection preferences by mobile
X. glabratus females.
Acknowledgments We thank Adam Black for acquiring infested
swamp bay logs for emergence; Kelsey Olson for help with Fig. 1;
Angelique Hoyte, Laurie Martini, and Barry Fleming for help with be-
havioral assays. Funding was provided by the USDA Forest Service
(Region 8) [Marc Hughes and Jason Smith] and USDA-APHIS grant
(Cooperative Agreement 13-8212-0990-CA) [Lukas L. Stelinski].
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