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