Ambrosia Beetle, Xyleborus glabratus

13 Pages·2014·1.78 MB·English

North American Lauraceae: Terpenoid Emissions,
Relative Attraction and Boring Preferences of Redbay
Ambrosia Beetle, Xyleborusglabratus(Coleoptera:
Curculionidae: Scolytinae)
Paul E. Kendra1*, Wayne S. Montgomery1, Jerome Niogret1, Grechen E. Pruett2, Albert E. Mayfield III3,
Martin MacKenzie4, Mark A. Deyrup2, Gary R. Bauchan5, Randy C. Ploetz6, Nancy D. Epsky1
1UnitedStatesDepartmentofAgriculture,AgriculturalResearchService,SubtropicalHorticultureResearchStation,Miami,Florida,UnitedStatesofAmerica,2Archbold
BiologicalStation,LakePlacid,Florida,UnitedStatesofAmerica,3UnitedStatesDepartmentofAgriculture,ForestService,SouthernResearchStation,Asheville,North
Carolina,UnitedStatesofAmerica,4UnitedStatesDepartmentofAgriculture,ForestService,ForestHealthProtection,StanislausNationalForest,Sonora,California,
UnitedStatesofAmerica,5UnitedStatesDepartmentofAgriculture,AgriculturalResearchService,BeltsvilleAreaResearchCenter,ElectronandConfocalMicroscopyUnit,
Beltsville,Maryland,UnitedStatesofAmerica,6UniversityofFlorida,TropicalResearchandEducationCenter,Homestead,Florida,UnitedStatesofAmerica
Abstract
Theinvasiveredbayambrosiabeetle,Xyleborusglabratus,istheprimaryvectorofRaffaelealauricola,asymbioticfungusand
theetiologicagentoflaurelwilt.Thislethaldiseasehascausedseveremortalityofredbay(Perseaborbonia)andswampbay
(P. palustris) trees in the southeastern USA, threatens avocado (P. americana) production in Florida, and has potential to
impact additional New World species. To date, all North American hosts of X. glabratus and suscepts of laurel wilt are
members of the family Lauraceae. This comparative study combined field tests and laboratory bioassays to evaluate
attraction and boring preferences of female X. glabratus using freshly-cut bolts from nine species of Lauraceae: avocado
(one cultivar of each botanical race), redbay, swampbay, silkbay (Persea humilis), California bay laurel (Umbellularia
californica),sassafras(Sassafrasalbidum),northernspicebush(Linderabenzoin),camphortree(Cinnamomumcamphora),and
lancewood(Nectandracoriacea).Inaddition,volatilecollectionsandgaschromatography-massspectroscopy(GC-MS)were
conducted to quantify terpenoid emissions from test bolts, and electroantennography (EAG) was performed to measure
olfactory responses of X. glabratus to terpenoids identified by GC-MS. Significant differences were observed among
treatmentsinbothfieldandlaboratorytests.Silkbayandcamphortreeattractedthehighestnumbersofthebeetleinthe
field,andlancewoodandspicebushthelowest,whereasboringactivitywasgreatestonsilkbay,baylaurel,swampbay,and
redbay,andlowestonlancewood,spicebush,andcamphortree.TheGuatemalancultivarofavocadowasmoreattractive
thanthoseoftheotherraces,butboringresponseamongthethreewasequivalent.Theresultssuggestthatcamphortree
maycontainachemicaldeterrenttoboring,andthatdifferentcuesareassociatedwithhostlocationandhostacceptance.
Emissions of a-cubebene, a-copaene, a-humulene, and calamenene were positively correlated with attraction, and EAG
analysesconfirmedchemoreception ofterpenoidsby antennalreceptors ofX.glabratus.
Citation:KendraPE,MontgomeryWS,NiogretJ,PruettGE,MayfieldAEIII,etal.(2014)NorthAmericanLauraceae:TerpenoidEmissions,RelativeAttractionand
BoringPreferencesofRedbayAmbrosiaBeetle,Xyleborusglabratus(Coleoptera:Curculionidae:Scolytinae).PLoSONE9(7):e102086.doi:10.1371/journal.pone.
0102086
Editor:RobertGlinwood,SwedishUniversityofAgriculturalSciences,Sweden
ReceivedApril14,2014;AcceptedJune15,2014;PublishedJuly9,2014
Thisisanopen-accessarticle,freeofallcopyright,andmaybefreelyreproduced,distributed,transmitted,modified,builtupon,orotherwiseusedbyanyonefor
anylawfulpurpose.TheworkismadeavailableundertheCreativeCommonsCC0publicdomaindedication.
DataAvailability:Theauthorsconfirmthatalldataunderlyingthefindingsarefullyavailablewithoutrestriction.Allrelevantdataarewithinthepaperandits
SupportingInformationfiles.
Funding:FundingwasprovidedbytheUSDA-ARSNationalPlantDiseaseRecoverySystemandtheFloridaAvocadoAdministrativeCommittee.Thefundershad
noroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*Email:[email protected]
Introduction wood borer native to Southeast Asia [4]. Female beetles store
several fungal symbionts in cuticular pouches (mycangia) at the
LaurelwiltisadestructivevasculardiseaseofAmericantreesin baseofthemandibles,oneofwhich,RaffaelealauricolaT.C.Harr.,
the family Lauraceae, particularly members of the genus Persea. Fraedrich & Aghayeva (Ophiostamatales: Ophiostomataceae),
Over the last decade, large populations of native redbay and causeslaurelwilt[5–6].ThepresenceofR.lauricolainsusceptible
swampbay [P. borbonia (L.) Spreng. and P. palustris (Raf.) Sarg., hosts elicits a cascade of events, including secretion of resins and
respectively] have been decimated throughout the southeastern formation of extensive parenchymal tyloses that wall off conduc-
United States [1–2], and currently avocado (P. americana Mill.) is tive xylem vessels [7–8]. This defensive response results in
threatened in south Florida [3].The disease emerged subsequent diminished water transport, whichinitially impedes spread of the
to establishment of the redbay ambrosia beetle, Xyleborus glabratus mycopathogen,butultimatelyleadstosystemicwiltandhosttree
Eichhoff (Coleoptera: Curculionidae: Scolytinae), an invasive mortality.ArecentstudydocumentedlateraltransferofR.lauricola
PLOSONE | www.plosone.org 1 July2014 | Volume 9 | Issue 7 | e102086
HostPreferencesofRedbayAmbrosiaBeetle
to other species of ambrosia beetle (secondary colonizers that femaleslocatepotentialhosttreesbasedontheirvolatileemissions.
breed sympatrically with X. glabratus), and transmission of the Our current hypothesis is that X. glabratus detects (via antennal
pathogentohostsunderlaboratoryconditions[9],butitisnotyet olfactory receptors) a mixture of terpenoid compounds in which
knowniftheseadditionalspeciescontributetothespreadoflaurel the sesquiterpene a-copaene functions as a key long-range
wilt innatural ecosystems. attractant[29–35].Theseterpenoids–tantamounttoasignature
ThefirstredbayambrosiabeetledetectedinNorthAmericawas bouquetoftheLauraceae–havebeenfoundtobeconcentratedin
trappedinMay2002inamaritimeportnearSavannah,Georgia severalplant-derivedessentialoils,includingphoebe,manuka,and
[10].Sincethattime,itsgeographicrangehasexpandedatarate cubeboils,whichhavebeenutilizedasfieldluresfordetectionof
exceedingmodelpredictions[11].Southwardspreadthroughthe X. glabratus [21],[29],[31],[34–35].
Floridapeninsulawasparticularlyrapid,duetomildtemperatures, Thepresentstudywasinitiatedtoinvestigatein-flightattraction
ample host availability, and human transport of infested material and boring preferences of female X. glabratus for the dominant
(e.g.firewood[12]).InMarch2010,fiveyearsbeforethepredicted speciesofLauraceaeintheU.S.,andtoevaluatetherelationship
date [11], X. glabratus had reached southernmost Florida (Miami- between behavioral response and phytochemical emissions from
DadeCounty);thiswasfollowedbyconfirmationoflaurelwiltin wood substrates. Of the ,50 described genera within the
the county by 2011 [13] and in commercial avocado groves by Lauraceae, only nine occur in North America: Cassytha, Cinnamo-
2012[3].AsofFebruary2014,thevector-pathogencomplexhad mum, Licaria, Lindera, Litsea, Nectandra, Persea, Sassafras, and
been confirmed in portions of six southeastern states: North Umbellularia [36]. We compared responses of X. glabratus to cut
Carolina, South Carolina, Georgia, Florida, Alabama, and bolts from nine tree species, representative of six of these genera.
Mississippi [14], but further expansion is likely (see [15–16] for Cassytha was excluded from the study because it is a non-woody,
recent reviews of the epidemic). Avocado production provides an parasitic vine [36] not susceptible to attack by a wood boring
estimated $23.5 million in sales annually for the state of Florida beetle.LitseaandLicariawerealsoomittedsincetheyarerareplant
[17]. With continued spread, laurel wilt could pose a serious speciesofconservationconcernintheU.S.;however,bothgenera
economic threat to the avocado industries in California ($468 havebeenevaluatedpreviouslyforsusceptibilitytolaurelwilt[23],
million in annual sales [17]) and Mexico ($1.2 billion in export [27]. Specific components of our study included (i) field tests to
revenue alone [18]). Moreover, there is potential for severe determinerelativeattractionamongtheninespeciesofLauraceae,
ecological impact on forest ecosystems in Mexico, Central and (ii) laboratory bioassays to assess female boring behavior as an
South America, areas rich in species of Persea and other genera indicator of host recognition and acceptance, (iii) volatile
withintheLauraceae [16],[19]. collections followed by gas chromatography-mass spectroscopy
Development of effective semiochemical-based detection and (GC-MS)toquantifyterpenoidemissionsfromthebolttreatments,
control programs for X. glabratus willrequire an understanding of and (iv) electroantennography (EAG) to measure the beetle’s
its unique chemical ecology. Like other ambrosia beetles within peripheral olfactory response to the major constituents identified
the tribe Xyleborini, X. glabratus is known to have extensive by GC-MS. Chemicals of primary interest were volatile com-
inbreeding,haplo-diploidsexdetermination,andasexratiohighly poundspreviouslyreportedaspotentialattractantsforX.glabratus,
skewed toward females[10].Species-specific pheromones are not includinga-copaeneandseveralothersesquiterpenehydrocarbons
used,asfemalestypicallymatewithflightless,siblingmalespriorto [21], [29–35], [37–38], as well as the monoterpene ether
emergence from natal trees. However, females of X. glabratus are eucalyptol (1,8-cineole) [37],[39].
behaviorally atypical for this taxonomic group. While most
xyleborines are broad generalists that target stressed or dying Materials and Methods
trees (saprotrophic symbiosis) [20], X. glabratus functions ecolog-
icallyasaprimarycolonizer,capableofattackinglive,apparently- Ethics Statement
healthyhosts[1].Consequently, itisnotattractedtoethanol [2], Field studies were conducted at the Lake Wales Ridge
[21], a signature volatile of tree decay, which serves as the Environmental Management Area under special use permit
standard lure for detection of most ambrosia beetles [22]. In #SUO-33630 issued by the Florida Fish and Wildlife Conserva-
addition, X. glabratus is not a host generalist in the United States, tion Commission. Test bolts from Matheson Hammock were
butappearstoberestrictedtotheLauraceae.Todate,atleast12 collected under research permit #148 from the Miami-Dade
U.S.species–allintheLauraceae–havebeenreportedaseither County Parks and Recreation Department. Collection of bolts
hostsofX.glabratusand/orsusceptsoflaurelwilt[1–2],[23–27].It from other sites did not require specific permits; however, verbal
was thought that this host specificity represented a major permission was obtained from, and collection activities were
behavioral shift that accompanied establishment of the founder coordinatedwithappropriatecurators,landstewards,andrangers
populationinNorthAmerica[15],butarecentinspectionofhost (listed in the acknowledgments). Field studies did not involve any
recordsfromtheChineseNationalInsectCollectionsuggeststhat protected or endangered species.
X.glabratusshowsapreferenceforLauraceaeinAsiaaswell[28].
It is not a strict specialist, however, since reported hosts do Test Substrates
includerepresentatives fromotherfamilies,including Dipterocar- Plant material for field tests, bioassays, and chemical analyses
paceae, Fabaceae, Fagaceae, Theaceae, and Pinaceae [10], [28]. consisted of freshly-cut bolts (15–20cm long and 5–8cm in
InFlorida,itwasdiscoveredthatX.glabratusishighlyattractedto, diameter) obtained from multiple locations: avocado cultivars
andwillinitiateboringinto,freshly-cutbranches(bolts)oflychee, ‘Catalina’ (West Indian race; MIA# 17248, PI# 281923, WA2-
Litchi chinensis Sonn. (Sapindaceae) [29]. Subsequent evaluations 18-34),‘Duke’(Mexicanrace;MIA#17468,PI#277487,WA4-
indicatedthatL.chinensis,althoughattractivetoX.glabratusdueto 28-51),and‘Taylor’(Guatemalanrace;MIA#18262,PI#26710,
chemical similarities with the Lauraceae, is not a suitable WB3-13-02) from the National Germplasm Repository at the
reproductivehost.Thisisapparentlyduetotheinabilityoflychee USDA-ARS Subtropical Horticulture Research Station (SHRS;
wood to support growth of R. lauricola, the presumed primary Miami, FL); redbay, swampbay, silkbay (Persea humilis Nash), and
nutritional symbiont [30]. This latter work, in combination with live oak (Quercus virginiana L.) from Archbold Biological Station
otherfieldstudiesofX.glabratus,providesevidencethatdispersing (ABS;LakePlacid,FL);lancewood[Nectandracoriacea(Sw.)Griseb.]
PLOSONE | www.plosone.org 2 July2014 | Volume 9 | Issue 7 | e102086
HostPreferencesofRedbayAmbrosiaBeetle
from Matheson Hammock (Coral Gables, FL); camphor tree eachrow(block).Thelatterstepensuredthateachtreatmentwas
[Cinnamomumcamphora(L.)J.Presl]fromBokTowerGardens(Lake rotatedthrougheachofthefieldpositionswithinablock,thereby
Wales, FL);sassafras [Sassafras albidum (Nutt.) Nees] andnorthern minimizingpositional effects on beetle capture.
spicebush [Lindera benzoin (L.) Blume] from Bent Creek Experi- Allsamplecollectionsweresortedunderadissectingmicroscope
mental Forest (Asheville, NC); and California bay laurel in the laboratory at SHRS. Species of Scolytinae were removed
[Umbellularia californica (Hook. & Arn.) Nutt.] from Stanislaus fromthestickypanels,soakedinhistologicalclearingagent(Histo-
National Forest (Tuolumne County, CA). Since California bay clear II; National Diagnostics, Atlanta, GA, USA) to remove
laurel is a foliar host of the oomycete plant pathogen Phytophthora adhesive,andthenstoredin70%ethanol.Beetleswereidentified
ramorumWerres,DeCock&Manin’tVeld(Pythiales:Pythiaceae), according to Rabaglia et al. [10] and voucher specimens were
the causal agent of sudden oak death [40], bolts from bay laurel deposited at SHRSandABS.
were collected outside the quarantine zone for P. ramorum; in
addition,boltsfromthissitewereassayedandfoundnegativefor Test Insects
thepathogenpriortoshipmenttoFlorida,asdescribedpreviously Insects used in laboratory bioassays and electrophysiology
for parallel tests conducted concurrently in South Carolina [25]. studies were host-seeking female X. glabratus collected in the field
At time of collection, the ends of each bolt were wrapped in (atABSandseveralsitesalongtheLakeWalesRidgeEcosystem)
ParafilmM(PachineyPlasticPackaging, Inc.,Chicago,IL,USA) using a published baiting method [43]. The procedure used
to minimize desiccation and loss of volatile phytochemicals. freshly-cut Persea wood and several manuka oil lures (Synergy
Samples from California and North Carolina were packed in Semiochemicals Corp., Burnaby, BC, Canada) as bait placed in
insulated coolers and shipped overnight to Florida. At test the center of a white cotton sheet. At 15–20min intervals, fresh
deployment, a thin section (,0.5cm) was cut from the ends of wood was added to the pile and the lures were fanned. This
each bolt using a battery operated reciprocating saw (Craftsman; generated a pulsed plume of attractive volatiles that effectively
Sears, Roebuck and Co., Chicago, IL, USA). All tests were ‘luredin’host-seekingfemales,whichhavepeakflightfrom17:30–
initiated within3 days ofboltcollection. 19:30 h(EDST)insouthFlorida[33].Asbeetleslanded,theywere
collected by hand with a soft brush and placed in plastic boxes
Field Tests containingmoisttissuepaper.Insectswerethenheldovernightin
Trappingexperimentswereconductedinsouth-centralFlorida the storage boxes until used in laboratory experiments early the
(Lake Placid, Highlands County) at two tracts within the Lake next morning.
Wales Ridge Environmental Management Area. Both sites had To visualize the fine morphological features of the X. glabratus
numerous swampbay trees exhibiting advanced stages of laurel antenna (the primary olfactory organ used by insects for
wilt, but there were also silkbay trees (mostly asymptomatic) in chemoreceptionandtransductionofenvironmentalodors),several
adjacent dry scrub habitats. Test 1 was conducted at the Royce females were examined by low-temperature scanning electron
RanchUnit(N27u3894100,W81u3493070)from24Septemberto microscopy (LT-SEM),usingmethods recently reported [44].
12 November 2010 (7-wk test), and evaluated attraction to five
species of Lauraceae: redbay, swampbay, silkbay, avocado, and Laboratory Bioassays
lancewood. In addition, the test included two controls, consisting Behavioral bioassays consisted of no-choice tests designed to
ofanunbaitedtrap(toassessrandombackgroundcaptures)anda document host recognition and boring behaviors, following
trap baited with live oak (a non-host bolt treatment, to assess published protocols [29–30]. Assays were conducted at ABS
potential captures resulting from visual cues [41]). Test 2 was under controlled laboratory conditions (25uC, 16:8h L:D). Test
conducted at the Highlands Park Estates (N 27u2190320, W arenas consisted of plastic buckets (4.4 liter) covered with cheese
81u1998370)from4Augustto15September2011(6-wktest),and clothmesh,securedwithrubberbands,andheldinscreenedinsect
compared captures of X. glabratus with bolts of camphor tree, cages (BioQuip, Rancho Dominguez, CA, USA). Into each
sassafras, northern spicebush, California bay laurel, silkbay (an bucket, lined with a filter paper disk (15cm diameter; Whatman
internal control for comparison with results from test 1), and an Intl.Ltd., Maidstone,England),were placed 10–15beetles anda
unbaited trap. single bolt of wood. Bolt treatments were identical to those
Trapdesignconsistedoftwoboltswiredtogether(side-by-side) evaluatedinthefieldtests.Abeetlewasscoredpositiveforboring
and hung vertically, to which were attached two white sticky whenitwasperpendiculartothewoodsubstrateandatleasthalf
panels (23628cm, Sentry wing trap bottoms; Great Lakes IPM, its body length (,1 mm) was inserted into the entrance hole.
Vestaburg, MI, USA) stapled back-to-back to the bottom of the (Previousobservationsindicatedthatfemalessometimes‘sampled’
bolts. The paired sticky panels were secured further with several the substrate, making shallow bore holes, but then aborted the
binder clips around the edges. The unbaited control traps attempt; however, once they inserted half theirbody length, they
consistedoftwostickypanelsstapledtogether.Fieldtestsfollowed typicallycontinuedtoborethroughthebarkandcambiumlayers
a randomized complete block design, with ten replicate blocks in into the sapwood.) To document behavioral response over time,
test 1, and five replicate blocks in test 2. Replicate bolts for each the number of beetles that were boring and their location on the
hostspecieswereobtainedfromdifferenttrees.Infieldtest1,the bolt were recorded at 1, 2, 4, 8, 12, and 24h. Tests were
ten replicate bolts of avocado consisted of four bolts of the replicated a minimum of five times for each substrate, and each
Mexican cultivar and three bolts each of the West Indian and replicatewasrunusingaseparatearena,anewboltofwood,anda
Mexican cultivars. Each block consisted of a row of traps hung newcohortof beetles.
from wire hooks ,1.5m above ground [42] in non-host trees,
withaminimumof10mspacingbetweenadjacenttrapsinarow, Chemical Collection and Analysis
and 30m spacing between rows. For both tests, traps were Samples for chemical analysis were prepared by manually
checked weekly. At each sampling date, the sticky panels were raspingtheouterlayersofbarkandunderlyingcambialtissuefrom
collected,athinlayerwassawedfromthebottomofeachboltto thebolttreatments,usingmethodsreportedpreviously[32],[45].
‘‘renew’’ release of wood volatiles, new sticky panels were Volatilechemicalswerecollectedfromfreshly-raspedshavings(6 g
attached, and the trap positions were rotated sequentially within samples, 3–10 replicates per species) by using Super Q traps
PLOSONE | www.plosone.org 3 July2014 | Volume 9 | Issue 7 | e102086
HostPreferencesofRedbayAmbrosiaBeetle
(AnalyticalResearchSystems,Gainesville,FL,USA)accordingto
published methods[46–47].Sampleswerespreadinacylindrical
glasschamber(4.5 cmdiameter625cmlength),purifiedairwas
introduced into the chamber (1 L/min), and headspace volatiles
werecollectedfor15min.SuperQtrapswerecleanedbysoxhlet
extraction with methylene chloride for 24h and dried in a fume
hood prior to each use. Volatile chemicals were eluted from the
SuperQadsorbentwith200mlofhighpuritymethylenechloride
(99.5%pure;ACROS,MorrisPlains,NJ,USA).AnaliquotofC
16
standard(5 mg)wasaddedtoeachsampleforquantitativeanalysis.
Chemicalextractswereanalyzedbyusinggaschromatography
(ThermoQuest Trace GC 2000, Austin, TX, USA). The column
wasfusedsilica,25mlong,0.25mmi.d.,DB-5MSphase(J&W
Scientific, Agilent Technologies, Santa Clara, CA, USA),
programmed from 50 to 130uC at 15.0uC/min, then from 130
to220uCat10.0uC/min,andthenheldat220uCfor4 min.The
column used in the gas chromatograph interface to the mass
spectrometer (Agilent Technologies 5975B) was 25m long,
0.25 mm i.d., DB-5MS phase (J & W Scientific, Agilent
Technologies), programmed at 40uC for 2 min, then from 40 to
130uC at 10.0uC/min, then from 130 to 220uC at 20.0uC/min,
and then held at 220uC for 4min. Chemicals were identified by
using the NIST mass spectral program (version 2.0 d) and the
NIST/EPA/NIH mass spectral library (NIST11) when Reverse
Matches and Matches were .950 and .900%, respectively.
Identifications werethenverifiedbycomparingthemeanKovats
Retention Index (RI) with the RI calculated from synthetic
chemicals, when commercially available [RI=1045, 1358, 1391,
1443, 1477, 1532 for eucalyptol ($99.0%; Fluka Analytical,
Steinheim, Germany), (2)-a-cubebene ($97.0%; Bedoukian
Research Inc., Danbury, CT, USA), (2)-a-copaene ($90.0%;
FlukaAnalytical),(2)-b-caryophyllene($98.5%;SigmaChemical Figure 1. Scanning electron micrographs of adult female
Co., St. Louis, MO, USA), a-humulene ($96.0%; Sigma Xyleborusglabratus.(A)Fullviewofheadshowingintactleftantenna;
the apical ventral surface of antennal club is flattened and bears
Chemical Co.), and (+)-d-cadinene ($97.0%; Fluka Chemie,
concentric arrays of sensilla. (B) Detail of antennal club reveals two
Buchs, Switzerland), respectively]. Alternatively, experimental RI types of sensilla: long tapered sensilla trichoidea (which bear minor
values were compared with previously published data (d-elemene branching morphology at the distal end), and more numerous short
[48],b-elemene[49–50], andcalamenene[48], [51]). bluntly-pointed sensilla basiconica. Antennal preparations for electro-
physiologicalrecordingsweremountedventralsurfacefacingupwards,
with electrode contact on the dorsum of the club to avoid coating
Electroantennography
olfactorysensillawithconductivegel.
EAG substrates consisted of: ethanol (5 ml ethyl alcohol 95%; doi:10.1371/journal.pone.0102086.g001
Pharmco-Aaper, Brookfield, CT, USA); a manuka oil lure (the
standard lure forX. glabratus [31],releaserate 50mgoilperday;
Fairfield,NJ,USA).LT-SEMrevealedthattheventralsurfaceat
Synergy Semiochemicals Corp.); silkbay shavings (15 g freshly-
theapexoftheantennalclubisflattenedandbearsadensearray
raspedbarkandcambium,collectedatABS,preparedasreported
ofconcentricallyarrangedolfactorysensilla(Fig.1);thus,carewas
previously [45]); and the synthetic terpenoids eucalyptol, a-
taken tonot coatthisregionwith conductive gel.
cubebene, a-copaene, b-caryophyllene, a-humulene, and d-
A stream of humidified air, purified with activated charcoal
cadinene (each 50ml neat oil; obtained from the suppliers
granules (grain size 1–2mm), was passed continuously over the
identified above). Each substrate was placed into a separate
antennalpreparationat400ml/min.Thetipofthedeliverytube
250ml hermetic glass bottle equipped with a lid that had been
was placed ,1 mm from theantenna, and theair controller was
fittedwithashortthru-hullport(Swagelok,Solon,OH,USA)and
configured to allow for pulse flow compensation during sample
siliconeseptum(Alltech,Deerfield,IL,USA).Samplebottleswere
delivery. Using gas tight syringes (SGE Analytical Science,
sealed and equilibrated for 2 h at 24uC to allow for headspace
Victoria, Australia), samples of saturated vapor were withdrawn
saturation withvolatiles.
fromthetestbottles,injectedintotheairstream,andpresentedto
Instrumentation consisted of a Syntech EAG system (Syntech
the antennae. In each recording session, the antenna was
Original Research Instruments, Hilversum, Netherlands), which
presented first with ethanol (2ml saturated vapor), which has
includedamicromanipulatorassembly(MP-15),adataacquisition
been shown previously to serve as an appropriate standard and
interfacebox(serialIDAC-232),astimulusaircontroller(CS-05),
positive control for Xyleborus species [33]. This was followed by
andEAG2000software.Olfactoryresponseswererecordedwitha
injection of test samples in random order, then with negative
newly developed technique [33] which utilized a gold-plated 2-
controls consisting of clean air injections equal in volume to the
pronged antennal holder (Syntech EAG Combi-Probe) modified
sample injections, and ended with a final injection of ethanol.
with thin gold wire to accommodate the minute antennae of X.
There was a 2 min interval (clean air flush) between sample
glabratus (mean antennal length 0.3760.01 mm). Single excised
injections to prevent antennal adaptation (diminished EAG
antennae were mounted, ventral side facing up, between
electrodes using salt-free gel (Spectra 360, Parker Laboratories,
PLOSONE | www.plosone.org 4 July2014 | Volume 9 | Issue 7 | e102086
HostPreferencesofRedbayAmbrosiaBeetle
response as a result of repeated exposure to specific chemical
stimuli).
EAG responses to test substrates were measured initially in
millivolts (peak height of depolarization) and then normalized to
percentages relative to the EAG response obtained with ethanol.
Normalization with a standard reference chemical corrects for
time-dependent variability (gradual decline) in antennal perfor-
mance,andalsoallowsforcomparisonofrelativeEAGresponses
obtained with different substrates [45], [52] and with different
cohorts of insects [53]. Finally, any response recorded with the
negativecontrolwassubtractedfromthenormalizedtestresponses
to correct for ‘pressure shock’ caused by injection volume. All
statisticalanalyseswereperformedusingthecorrectednormalized
EAG values.
Two EAG experiments were conducted with host-based
attractants. The first experiment was designed to evaluate dose-
dependent EAG responses of female X. glabratus to various
terpenoidcompounds.Sixdoses,inatwo-foldseriesofheadspace
volumes ranging from 0.25 to 6.0 ml, were used to quantify
antennal response to volatiles emitted from three test substrates:
silkbayshavings,manukaoillure,andsynthetica-copaene.Based
on thedose-response results obtained in this initial experiment, a
second experiment was conducted using fixed 2 ml doses to
compareEAGresponsestosilkbaywood,manukalure,andallsix
synthetic terpenoids. To construct dose-response curves, EAG
responseswererecordedfromantennaeof10–15replicatefemales
for each substrate; for the comparative EAG experiment,
responses weremeasured from 15–20replicate females.
Statistical Analysis
Analysesofvariance(ANOVA)(ProcGLM,SASInstitute[54])
were conducted for results from the field tests and comparative
EAG experiments, followed bymean separations with Tukey test
(P,0.05).TheBox-Coxprocedure,whichisapowertransforma-
tion that regresses log-transformed standard deviations (y+1)
against log-transformed means (x+1), was used to determine the
type of transformation necessary to stabilize variance prior to
analysis [55]. Regression analysis (Systat Software [56]) was used
to describe the relationships between substrate dose and EAG
responses (with separate analyses for each substrate), and also to
document temporal patterns in boring behaviors observed in the
no-choice laboratory bioassays. Analysis by t-test [56] was
performed to measure differences between EAG responses to
equal doses of two different substrates, and differences between
responses to adjacent doses of the same substrate. For each
sesquiterpene and eucalyptol, the captures of X. glabratus in field
test 1 were compared to the quantity of chemical emitted per Figure2.Mean(±SE)capturesoffemaleXyleborusglabratusin
substrate (10 replicate bolts per tree species) by using Pearson fieldtestsconductedinFlorida,USA.(A)Test1evaluatedcaptures
product moment correlation [56]. instickytrapsbaitedwithboltsofsilkbayPerseahumilis,swampbayP.
palustris, redbay P. borbonia, avocado P. americana, lancewood
Nectandra coriaceae, and live oak Quercus virginiana. (B) Test 2
Results
evaluatedcaptureswithsilkbay(forcomparisonwithtest1),camphor
tree Cinnamomum camphora, California bay laurel Umbellularia
Field Tests
californica,sassafrasSassafrasalbidum,andnorthernspicebushLindera
Infieldtest1(Fig.2A,TableS1),thereweredifferencesinmean benzoin.Bothtestsincludedanunbaitedcontroltrap.(C)Toestimate
capture of X. glabratus among the seven treatments (F=14.58; relativeattractionamongallLauraceae,theresultsoftests1and2have
df=6, 63; P,0.0001). Traps baited with bolts of silkbay caught beennormalizedandcombined;normalizationconsistedofexpressing
captures as a percentage relative to silkbay (the most attractive
significantly morebeetles than anyothertreatment. Trapsbaited
treatment in both tests). Bars topped with the same letter are not
withswampbay,redbay,oravocado(allthreecultivarscombined)
significantly different (Tukey mean separation of square root [x+0.5]-
caught comparable numbers of beetles, which were significantly transformeddata,non-transformedmeanspresented,P,0.05).
higher than numbers caught with live oak or the unbaited trap. doi:10.1371/journal.pone.0102086.g002
Captures with lancewood were the lowest observed among the
Lauraceae treatmentsintest1,with resultsintermediatebetween were analyzed separately (Fig. 3), there were differences in mean
those obtained with known hosts (swampbay, redbay, avocado) capturesamongthethreevarieties(F=5.49;df=2,7;P=0.037).
and the non-host control (oak). When the results with avocado CaptureswiththeGuatemalancultivar‘Taylor’weresignificantly
PLOSONE | www.plosone.org 5 July2014 | Volume 9 | Issue 7 | e102086
HostPreferencesofRedbayAmbrosiaBeetle
before selecting a site and committing to boring activity. This
resultedinaconsiderablelagtime(relativetoPerseaandbaylaurel)
in boring response, and maximum percentages were not reached
until 12 to 15h. On bolts of sassafras, rates of boring were
intermediate between those for the two former groups (Fig. 4B).
Fewbeetlesrespondedtotheboltsofliveoak(Fig.4A).Although
most continued to wander throughout the test arena, several
females settled into natural crevices or under the bark at the cut
ends of the oak bolts. This behavior was interpreted as a
thigmotacticresponse andnot boring.
After 24h, there were significant differences among the ten
treatments (F=79.76; df=9, 49; P,0.001). Mean separation
analysis distinguished four groupings within the Lauraceae, with
the highest final percentages on 1) silkbay, California bay laurel,
swampbay, and redbay, followed by 2) avocado, 3) sassafras and
spicebush, and 4) spicebush, camphor tree, and lancewood
(Table 1). Most of the boring on these species occurred on the
cutendsoftheboltsratherthanthroughthebark,buttherewere
Figure 3. Mean (± SE) captures of femaleXyleborusglabratus
differences among treatments (F=3.99; df=8, 48; P=0.001).
with avocado cultivars in field test 1. Varieties tested included
‘Taylor’ (Guatemalan race), ‘Duke’ (Mexican race), and ‘Catalina’ (West When data for avocado were analyzed separately, there were no
Indian race). Bars topped with the same letter are not significantly differences among the three cultivars (F=0.46; df=2, 12;
different(Tukeymeanseparation,P,0.05). P=0.639); thus, results for avocado were pooled in Fig. 4 and
doi:10.1371/journal.pone.0102086.g003 Table 1.
higher than those obtained with the West Indian cultivar Chemical Analysis
‘Catalina’. Captures with the Mexican cultivar ‘Duke’ were Atotalof72volatilechemicals(detectedatquantities$0.5mg
intermediate.
in at least one sample) were isolated by Super Q collections and
Overall,numbersofX.glabratuswerehigherduringthesecond GC-MS analysis. Of these, there were eight sesquiterpenes
field test (Fig. 2B, Table S2), and there were differences in mean commonamongtheLauraceae(Table2,TableS4).Sesquiterpene
captures among the six treatments (F=32.32; df=5, 24; P, content varied both qualitatively and quantitatively among the
0.0001). As observed in test 1, highest captures were obtained in species(Fig.5),butonlyfourchemicalswerepositivelycorrelated
traps baited with silkbay bolts; however, captures obtained with with captures of X. glabratus in field tests: a-cubebene (Pearson
camphor tree were not significantly different. Traps baited with correlation coefficient=0.243, P=0.042; Fig. 5 peak 2), a-
either California bay laurel or Sassafras had the next highest copaene (coefficient=0.553, P,0.0001; Fig. 5 peak 3), a-
captures. Captures with spicebush were very low, and not humulene (coefficient=0.299, P=0.015; Fig. 5 peak 6), and
statistically different from those obtained with the unbaited calamenene (coefficient=0.465,P,0.0001;Fig.5 peak 8).
control. The volatile profile from camphor tree, one of the most
Since the population levels were different at the two sites used attractive species in the field (Fig. 2B) but with one of the lowest
for field testing, captures of X. glabratus were normalized to boringpercentagesinbioassays(Fig.4B),containedlargeamounts
facilitate comparison. Normalization consisted of converting raw of a-copaene and a-cubebene, but also contained a large
numericalcapturestopercentagesrelativetothecapturesobtained sesquiterpene peak (RI=1437) not detected in other Lauraceae
with silkbay, the most attractive treatment in each field test. The (tentative NIST library identification as b-santalene). The
combinednormalizeddataarepresentedinFig.2C,toprovidean Guatemalan avocado ‘Taylor’, the most attractive cultivar tested
estimate of relative attraction for all ten tree species evaluated in (Fig. 3), contained significantly higher quantities of many
thestudy. sesquiterpenes, including d-elemene, a-cubebene, b-elemene, b-
caryophyllene, and a-humulene (Table 2). ‘Taylor’ also had
Laboratory Bioassays detectablelevelsofeucalyptol, notseeninthetwootheravocado
Composite results of the no-choice bioassays are presented in cultivars.However,eucalyptolcontentwashighlyvariableamong
Fig. 4 (and Table S3); for comparative purposes, results are speciesofLauraceae.ItwasfoundatveryhighlevelsinCalifornia
groupedaccordingtotreatmentdeploymentinfieldtest1(Fig.4A) baylaurelandredbay,atrelativelylowlevelsinattractivespecies
orfieldtest2(Fig.4B).Boringwasobservedonboltsfromallnine like camphor tree and sassafras, and at moderate levels in
species of Lauraceae, and regression analysis with sigmoidal unattractive species like spicebush (Table 2, Table S4); conse-
models (sigmoid, three parameter models) best described the quently,eucalyptolwasnotcorrelatedwithcapturesofX.glabratus
relationships between the time after bolt presentation and the inthefield (coefficient=0.078,P=0.520).
percentage of females actively boring (Table 1). The sigmoidal
equation is expressed in the form: y=a/(1+e2[(x-b)/c]), where x Electroantennography
representstime(h),yrepresentsboringresponse(%),coefficient‘a’ The relationships between doses of volatile chemicals and
representsthemaximumboringresponse,andcoefficients‘b’and amplitudes of EAG responses (Fig. 6, Table S5) were best fit by
‘c’ reflect the rate at which maximum response is attained [29]. regression with hyperbolic models (single rectangular, two
BoringwasinitiatedmostquicklyonboltsofthefourPerseaspecies parameter models). The general equation is expressed in the
(Fig. 4A) and California bay laurel (Fig. 4B), and maximum form: y=ax/(b+x), where x represents the substrate dose (ml), y
percentageswereachievedwithin4to8 h.Incontrast,onboltsof representsthenormalizedEAGresponse(%),andthecoefficients
lancewood (Fig. 4A), camphor tree, and spicebush (Fig. 4B), ‘a’ and ‘b’ represent maximum EAG response and receptor
females spent considerably more time walking over the substrate binding affinity, respectively. Hyperbolic equations are used
PLOSONE | www.plosone.org 6 July2014 | Volume 9 | Issue 7 | e102086
HostPreferencesofRedbayAmbrosiaBeetle
Figure4.Mean(±SE)percentageoffemaleXyleborusglabratusboringintoboltsin24hrbioassay.Eachtreespecieswasevaluated
separatelyinno-choicetests,buttofacilitatecomparison,resultsaregroupedaccordingtotreatmentdeploymentinfieldtest1(A)orfieldtest2(B).
RateofboringwithallspeciesofLauraceaewasbestfitbyregressionanalysiswithsigmoidalmodels(seeTable1).
doi:10.1371/journal.pone.0102086.g004
frequently for ligand-binding studies, and have been shown df=28, P=0.073). When dosages were increased to 2 ml, there
previouslytoservewellforcharacterizationofEAGdose-response were significant differences in EAG response among the
relationships [45], [52]. The EAG regression equations were as treatments(F=14.186;df=2,42;P,0.001);responseelicitedwith
follows:silkbaywood:y=99.75x/(0.53+x),R2=0.996;manukaoil silkbaywoodwashigherthanthatwithmanukaoil,andresponse
lure: y=77.44x/(0.74+x), R2=0.988; and a-copaene: y=36.76x/ elicited with manukaoilwas higherthan that with a-copaene.
(0.40+x),R2=0.995.Withallsubstrates,EAGamplitudeincreased TherewasnosignificantincreaseinEAGresponsewhendoses
with dosage up through approximately 2 ml,and then reached a ofthetestsubstrateswereincreasedto4 mlor6 ml(Fig.6).Thus,
plateau. At doses #1ml, there was not consistent separation 2 mldoseswereassumedtosaturatetheolfactoryreceptorsofthe
among mean responses recorded with the three substrates. For antennae, and fixed 2 ml doses were used for the comparative
example, at 0.5ml, there was no difference between responses EAG experiment (Fig. 7, Table S6). There were significant
obtained with manuka oil and a-copaene (t=21.164, df=28, differences in antennal response elicited with the eight test
P=0.254), and at 1.0ml, there was no difference between substrates(F=58.153;df=7,133;P,0.001),andmeanseparation
responses obtained with manuka oil and silkbay wood (t=1.861, analysisidentifiedfourgroupings.Thehighestamplituderesponse
Table1. Analysisofboring responseoffemale Xyleborusglabratus presented with woodboltsfrom nine speciesof North
American Lauraceaeina 24hour no-choicebioassay (N$5per species).
Regression Finalboringpercentage Percentageboringoncutsurface
Species Equation R2 (Mean±SE)1 (Mean±SE)1
Silkbay y=98.28/(1+e-[(x-4.11)/1.44]) 0.991 97.862.2a 97.562.5a
Californiabaylaurel y=93.70/(1+e-[(x-3.09)/1.06]) 0.974 97.562.5a 86.066.0ab
Swampbay y=90.94/(1+e-[(x-2.82)/0.88]) 0.976 95.762.9a 83.665.5ab
Redbay y=90.70/(1+e-[(x-1.89)/0.24]) 0.991 95.063.1a 90.562.5ab
Avocado y=78.72/(1+e-[(x-1.93)/0.64]) 0.996 79.665.6b 79.264.1b
Sassafras y=65.92/(1+e-[(x-4.69)/1.22]) 0.997 66.764.8c 84.366.7ab
Spicebush y=52.62/(1+e-[(x-6.28)/1.67]) 0.997 52.062.0cd 59.767.5c
Camphortree y=50.05/(1+e-[(x-6.95)/2.60]) 0.976 50.063.2d 85.069.6ab
Lancewood y=44.12/(1+e-[(x-6.25)/1.68]) 0.988 43.863.6d 10060.0a
1Meansfollowedbythesameletterwithinacolumnarenotsignificantlydifferent(Tukeymeanseparation,P,0.05).
doi:10.1371/journal.pone.0102086.t001
PLOSONE | www.plosone.org 7 July2014 | Volume 9 | Issue 7 | e102086
HostPreferencesofRedbayAmbrosiaBeetle
d
n
a
e),
c
a
r
3 n
e a
n c
e xi
n e
e 7 2 0 0 0 0 0 0 0 0 0 0 M
calam 1536 63.20. 60.80. 60.00. 60.10. 61.00. 61.00. 60.00. 60.30. 60.50. 60.30. 60.10. 60.30. Duke’(
‘
e),
c
a
2d-cadinene 1530 60.70.1 62.40.3 61.30.1 60.60.1 60.50.1 60.80.3 61.60.7 62.50.1 62.40.2 61.30.3 64.20.7 60.90.1 (Guatemalanr
or’
yl
a
T
‘
2 d
e e
n d
e u
a-humul 1478 61.60.3 60.60.2 60.50.0 60.20.1 60.40.1 60.50.1 68.22.9 60.10.0 60.10.0 60.30.0 60.40.1 60.60.1 estedincl
t
ars
v
ulti
c
e. 2e do
oltsofNorthAmericanLauracea 3bb-elemene-caryophyllen 14001443 661.80.516.64.3 663.30.710.42.1 661.30.10.70.1 662.00.50.60.2 660.30.14.51.1 660.70.35.41.3 6619.29.028.512.6 660.90.07.90.6 663.50.35.50.5 662.70.414.63.8 660.60.50.13.5 661.300.24.20.6 alyzedbyGC-MS(DB-5MScolumn).Avoca msyntheticchemicals(seetext).mpublishedreports(seetext).
odb 2ne henan RIfroRIfro
wo pae 1.7 65.5 0.1 0.3 1.3 3.0 4.3 0.3 1.0 0.6 0.0 0.3 ndt withwith
edfrom a-co 1390 69.1 23.0 60.9 61.3 65.1 65.0 69.3 69.6 68.1 63.6 60.2 61.0 ollection,a mparisonmparison
penoidsemitt 2a-cubebene 1357 63.50.9 69.61.9 61.60.1 66.11.7 60.80.2 60.31.0 635.117.6 61.10.1 611.51.4 60.80.1 60.10.0 60.10.0 biumbysuperQc oltsperspecies.henverifiedbycohenverifiedbyco
6Mean(SE)quantity(g)ofvolatileterTable2.m 23deucalyptol-elemene 1RI10451343 66Silkbay47.711.60.30.1 66Camphortree7.63.54.31.9 66Cal.baylaurel396.536.90.00.0 66Sassafras2.00.60.00.0 66Swampbay60.110.10.10.0 66Redbay145.131.60.00.0 66Avocado-Guat.6.43.520.09.5 66Avocado-Mex.0.00.00.20.0 66Avocado-W.Ind.0.00.01.20.2 66Lancewood0.00.01.60.4 66Spicebush72.114.90.00.0 66Liveoak0.10.00.00.0 Volatileswereisolatedfrom6gsamplesofraspedbarkandcam‘Catalina’(WestIndianrace).1MeanKovatsRetentionIndexcalculatedfrom3–10replicateb2IdentificationbyNIST/EPA/NIHmassspectrallibrary(NIST11),t3IdentificationbyNIST/EPA/NIHmassspectrallibrary(NIST11),tdoi:10.1371/journal.pone.0102086.t002
PLOSONE | www.plosone.org 8 July2014 | Volume 9 | Issue 7 | e102086
HostPreferencesofRedbayAmbrosiaBeetle
Figure5.RepresentativechromatographicanalysesofsesquiterpenesfromNorthAmericanLauraceae.Volatileswereisolatedfrom
6gsamplesofraspedbarkandcambiumbysuperQcollection,andthenanalyzedbyGC-MS(DB-5MScolumn).Peakidentificationsareasfollows:
1=d-elemene,2=a-cubebene,3=a-copaene,4=b-elemene,5=b-caryophyllene,6=a-humulene,7=d-cadinene,8=calamenene.
doi:10.1371/journal.pone.0102086.g005
was obtained with 1) eucalyptol, and the next highest response host tree (New World Lauraceae) that is attractive to the vector,
with 2) silkbay shavings, followed by 3) manuka oil lure and a- supports growth of the symbiont, and recognizes the fungus as
cubebene, and 4) the other four sesquiterpenes: b-caryophyllene, foreign(bymechanismsyetunknown)toinducesystemicdefensive
d-cadinene, a-copaene, and a-humulene. responses.Thepresentinvestigationfocusedontheinitialstepsof
this process – host location and recognition by a foundress X.
Discussion glabratus. There were two main objectives of the comparative
study.First,byassessingrelativeattractionandboringpreferences
Complexinteractionsunderlietheepidemiologyoflaurelwiltin within theLauraceae, wesought to identify thespecies that were
forestandagriculturalecosystems.Althoughrootgraftingbetween most susceptible to attack by X. glabratus. Second, by relating
adjacenttreesacceleratesspreadofthepathogeninaffectedareas, behavioral responses with volatile emissions from test substrates,
especially where there are high densities of host trees (e.g. wesoughttogainanenhancedunderstandingofthesemiochem-
commercialavocadoplantings),initialpathogentransmissionand icalsusedbyX.glabratusforhost-location.Thesetwoobjectiveswill
disease expression require an intimate association among three bediscussed separately.
species–aninsectvector(femaleX.glabratus,andpotentiallyother
species), a pathogenic fungal symbiont (R. lauricola), and a woody
PLOSONE | www.plosone.org 9 July2014 | Volume 9 | Issue 7 | e102086
HostPreferencesofRedbayAmbrosiaBeetle
Figure 6. Electroantennogram dose-response profiles con-
structed from mean (± SE) antennal responses of female
Xyleborusglabratus.Testsubstratesincludedfreshly-raspedwoodof
silkbayPerseahumilis,acommercialmanukaoillure,andsynthetica-
copaene.Responsesareexpressedasnormalizedpercentagesrelative
to a standard reference compound (ethanol, 2ml saturated vapor).
Dose-response curves generated with hyperbolic regression models
(seetext). Figure 7. Mean (± SE) electroantennogram responses of
doi:10.1371/journal.pone.0102086.g006 femaleXyleborusglabratustohost-basedvolatiles(2mldoses).
Testsubstratesincludedfreshly-raspedwoodofsilkbayPerseahumilis,a
commercial manuka oil lure, and six synthetic terpenoids. Responses
Susceptibility to Attack by X. glabratus are expressed as normalized percentages relative to a standard
Based on the high relative attraction in the field and the high referencecompound(ethanol,2mlsaturatedvapor).Barstoppedwith
thesameletterarenotsignificantlydifferent(Tukeymeanseparationof
percentages of individuals that exhibited boring behavior in
squareroot[x+0.5]-transformeddata,non-transformedmeanspresent-
bioassays($95%),thespeciesthatweremostvulnerabletoattack
ed,P,0.05).
weresilkbay,swampbay,redbay,andCaliforniabaylaurel.These doi:10.1371/journal.pone.0102086.g007
results are consistent with observations of native Persea species in
the southeastern U.S. to which this highly efficient vector has There is variation among the cultivated avocado varieties that
transmittedR.lauricola.IntheAtlanticCoastalPlaincommunities have been examined. There was no difference in attraction
of Georgia and South Carolina, redbay and swampbay popula- betweenavocadoandfourothertreespecieswhenresultsfromall
tionsfrequentlyexperiencemortalityinexcessof90percentwithin avocadotreatmentswerecombinedinthepresentstudy.However,
two years of the onset of laurel wilt [1]. With the more recent individualassessmentsindicatedthattheGuatemalancv.‘Taylor’
spread of laurel wilt into south-central Florida, stands of silkbay wassignificantlymoreattractivethantheothertwocultivars,and
are beginning to die off in the dry scrub habitats along the Lake this difference was associated with much higher terpenoid
WalesRidgeecosystem[33].AlthoughbreedingpopulationsofX. emissions in cv. ‘Taylor’ (discussed below). Previously, more X.
glabratus have not been detected west of Mississippi, future glabratus were caught with a Guatemalan cv., ‘Brooks Late’, than
expansion of the pest range could have a severe negative effect with West Indian (cv. ‘Simmonds’) or Mexican (cv. ‘Seedless
on California bay laurel, which is a significant component of Mexican’) genotypes [29]. Although these numerical differences
Pacific Coastal forests in California and Oregon. The strong were not statistically significant, GC-MS analysis indicated that
attraction and boring behaviors that were observed with this ‘BrooksLate’hadsignificantlyhighersesquiterpeneemissionsthan
species inthepresentstudy corroborate resultsfromparallel tests theother twocultivars [29].
conducted in South Carolina [25]. Notably, that latter study Additional evaluations are needed to determine if trees of the
demonstratedthatCaliforniabaylaurelisnotonlyattractivetoX. Guatemalanraceare,ingeneral,moreattractivetodispersingX.
glabratus,butisalsoasuitablereproductivehost;andpreviouswork glabratus than trees from the other two lineages. Clearly, these
indicatedthatthespeciesissusceptibletolaurelwiltafterartificial results have implications for breeding programs that would
inoculations of R.lauricola [57]. develop laurel wilt tolerant cultivars of this important crop.
Although avocado appears to be less suitable as a reproductive
Thepresentresultsindicatedthatavocadoandsassafrasareless
hostforX.glabratusthanU.S.nativePerseaspecies[58–59],beetle
vulnerable to attack by X. glabratus than the above species. Both
reproduction is not required for transmission of R. lauricola, only
specieswereasattractiveasswampbay,redbay,andCaliforniabay
hostrecognitionandboring.Inlaboratorybioassays,percentageof
laurelinthefieldtrial,butexhibitedlowerratesofboringactivity
boringwasequivalentamongthecultivarscomparedinthisstudy
inthelaboratorybioassay(80%and67%,respectively).Atafield
andamong thosecompared previously[29].Moreinformationis
sitein SouthCarolina, thenumbersof X.glabratus entranceholes
neededonthetransmissionofthispathogentoavocadoandother
were significantly lower on sassafras than on swampbay bolts in
host species by X. glabratus and other potential vector species [9],
2010[26],butweresignificantlyhigheronsassafrasboltsthanon
[16].
swampbay in 2011 [25]. A possible explanation for these
The remaining species in the Lauraceae that were tested –
seeminglyconflictingresultsisthat severedepletionofswampbay
camphor tree, lancewood, and northern spicebush – are appar-
trees between 2010 and 2011 may have led to the selection of
ently less vulnerable to attack by X. glabratus. Despite attracting
beetles that could successfully colonize a ‘less preferred’ host.
highnumbersofX.glabratusinthefieldtest,relativelylowboring
Alternatively, there may begeneticvariation intheattractiveness
activitywasobservedoncamphortreeinthelaboratorybioassay.
of these nativetrees toX. glabratus.
Low boring incidences were also observed on lancewood and
PLOSONE | www.plosone.org 10 July2014 | Volume 9 | Issue 7 | e102086