Hughes, M. A., Martini, X., Kuhns, E., Colee, J., Mafra‐Neto, A., Stelinski, L. L., & Smith, J. A.

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Evaluation of repellents for the redbay
ambrosia beetle, Xyleborus glabratus , vector of
the laurel wilt pathogen
Article in Journal of Applied Entomology · February 2017
DOI: 10.1111/jen.12387
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J.Appl.Entomol.
ORIGINAL CONTRIBUTION
Evaluation of repellents for the redbay ambrosia beetle,
Xyleborus glabratus, vector of the laurel wilt pathogen
M.A.Hughes1,a,X.Martini2,3,a,E.Kuhns2,*,J.Colee4,A.Mafra-Neto5,L.L.Stelinski2&J.A.Smith1
1 SchoolofForestResourcesandConservation,UniversityofFlorida,Gainesville,FL,USA
2 DepartmentofEntomologyandNematology,CitrusResearchandEducationCenter,UniversityofFlorida,LakeAlfred,FL,USA
3 DepartmentofEntomologyandNematology,NorthFloridaResearchandEducationCenter,UniversityofFlorida,Quincy,FL,USA
4 IFASStatisticalConsultingUnit,UniversityofFlorida,Gainesville,FL,USA
5 ISCATechnologies,Inc.,Riverside,CA,USA
Keywords Abstract
forestpests,laurelwilt,methylsalicylate,
Theredbayambrosiabeetle,Xyleborusglabratus,isthevectorofthelaurel
redbayambrosiabeetle,repellents,
verbenone,Xyleborusglabratus wilt disease fungal pathogen, Raffaelea lauricola. Since the vector’s initial
detection in the USA in the early 2000s, laurel wilt has killed millions of
Correspondence redbay,Perseaborbonia,treesandothermembersoftheplantfamilyLau-
MarcA.Hughes(correspondingauthor), raceae. To protect host trees from beetle attack and laurel wilt infection,
SchoolofForestResourcesandConservation,
wetestedtheefficacyofhost-andnon-host-derivedandcommercialcom-
UniversityofFlorida,POBox110410,
poundsasX. glabratusrepellentsinfieldexperiments.Inourfirsttrial,the
Gainesville,FL32611,USA.
majorconstituentsofthenon-hosttree,longleafpine,Pinuspalustris,and
E-mail:Mhughes741@ufl.edu
SPLAT Verb (verbenone 10%) were paired with manuka oil attractants
*PresentAddress:ScottsMiracle-Gro and beetle captures were counted. Verbenone and a 1 : 1 blend of myr-
Company,Marysville,OH,USA cene and camphene were intermediate to both the manuka positive and
blank negative controls. Subsequently, we tested different blends of
Received:October14,2016;accepted:
methyl salicylate (MeSA), a host defence and signalling compound, and
December5,2016.
verbenoneinSPLATdispensersusingfreshlycutredbayboltsasanattrac-
tant.AlltreatmentsreducedX.glabratuscapturesandboringholesascom-
doi:10.1111/jen.12387
pared to the redbay (-) repellent positive control; however, SPLAT Verb
aTheseauthorscontributedequally. and SPLAT MeSA-Verb (5% each) achieved the highest repellency, with
resultscomparabletothatofthenon-host(laureloak).Thesetrialsestab-
lish that host-derived and commercially available repellent compounds
can reduce X. glabratus attacks and therefore have potential as part of an
integratedmanagementstrategyagainstlaurelwiltanditsvector.
trees (Romme et al. 1986; Herms and McCullough
Introduction
2014).Theterm‘ambrosiabeetle’isanecologicalclas-
Since establishment by European settlers, over 450 sificationthatcombinesdifferentphylogeneticgroups
invasive insects and several serious tree pathogens of minute wood-boring beetles belonging to the Cur-
havecolonizedtheurbanandruralforestsoftheUni- culionidae (subfamilies Scolytinae and Platypodinae).
ted States (Aukema et al. 2010). Of these accidental Unlike bark beetles, which feed on host phloem,
introductions, some species have become important ambrosia beetles are completely reliant on their sym-
forest pests, causing widespread tree mortality and bioticfungifornutrition(Batra1985).Ambrosiabee-
severeecologicaldisturbances(Wingfieldet al.2016). tles commonly attack and colonize stressed and
Woodborers,inparticular,can beanespeciallydam- wounded trees and are, therefore, usually considered
aging group, with introduced insects such as the benignorbeneficialtoecosystemhealthastheyaccel-
emerald ash borer (Agrilus planipennis Fairmaire) and erateforestnutrientrecycling(Rudinsky1962).How-
the native mountain pine beetle (Dendroctonus pon- ever, there are exceptions; non-native ambrosia
derosae Hopkins) killing billions of susceptible host beetle species may attack susceptible healthy trees
©2016BlackwellVerlagGmbH 1
EvaluationofX.glabratusrepellents M.A.Hughesetal.
and become detrimental pests in forestry, agriculture Hugheset al.2015a).AswiththeDutchelmandoak
and the nursery trade (Hulcr and Dunn 2011; Ploetz wilt diseases in America, proper sanitation consists of
et al.2013). treefellingandcoveringlogsorchipsinplasticsheets
Among the ambrosia beetles that attack live and to vector visitation, development and/or escape
apparently healthy trees, the redbay ambrosia beetle, (Bruhn and Heyd 1992; Haugen 2001; Spence et al.
Xyleborus glabratus Eichhoff, was first detected near 2013). Although effective in reducing vector popula-
Savannah, Georgia, in monitoring traps maintained tions, this treatment is only appropriate for diseased
bytheUSForestServicein2002(Haack2006).Native trees and may not be practical across a large area. A
to Asia (Rabaglia et al. 2006), X. glabratus was likely prophylactic application of the fungicide propicona-
introducedtotheUSAthroughuntreatedwoodpack- zolecanoffer protection(Mayfieldet al.2008);how-
ingmaterial(e.g.cratesandpallets).Soonafterinitial ever, the longevity ofthe treatment is variable, likely
detection, X. glabratus andits pathogenic fungal sym- requiring frequent re-applications. Propiconazole
biont, Raffaelea lauricola T.C. Harr., Fraedrich & applications, although cost prohibitive in forest set-
Aghayeva, were associated with attacks of healthy tings,isan optioncurrentlyusedbyavocadogrowers
redbay (Persea borbonia [L.] Spreng.) trees and the tolimitthespreadoflaurelwiltwithinagrove.Topi-
newly described, lethal vascular disease namedlaurel cal and systemic insecticides are relatively ineffective
wilt (Fraedrich et al. 2008; Harrington et al. 2008). in X. glabratus suppression due to their limited dura-
Laurel wilt disease affects most USA native and some tionofefficacyinhosttrees(Pen~aet al.2011;Carrillo
non-nativemembersoftheLauraceaefamily(Kendra et al. 2013). Entomopathogenic fungi have been
et al.2014;Hugheset al.2015a).Laurelmortalityhas describedthatinfectX. glabratus,buttheykillthebee-
been most severe in forests of the south-eastern tlesaftertheyboreintothewood,notpreventingthe
Atlantic and Gulf coastal plains where, in total, mil- inoculationwithR. lauricola(Carrilloet al.2015).For
lions of mature redbay, swamp bay (Persea palustris avocado, pre-symptomatic aerial detection of laurel
[Raf.] Sarg.) and silk bay (P. humilis Nash) trees have wiltdiseasedplantsusingspectralimageryisindevel-
beenattackedbythebeetle,infectedandkilled(Frae- opment(Sankaranet al.2012;deCastroet al.2015),
drich et al. 2008; Shields et al. 2011; Kendra et al. but not yet operational. Overall, there is no reliable
2012a;SpiegelandLeege2013;Cameronet al.2015). tool available to prevent R. lauricola infection; conse-
Xyleborus glabratus attacks apparently healthy Lau- quently,thereisacrucialneedformanagementtools
raceae, introducing the laurel wilt pathogen into the to prevent X. glabratus attack and to slow the spread
xylem, disrupting vascular function and causing the oflaurelwiltdisease.
leaves to wilt in a matter of weeks to a few months The host selection process of X. glabratus is largely
(Fraedrichet al.2008;Hugheset al.2015a,b). drivenbytheirchemicalecology,whichisalsoimpor-
Due to the wide distribution of Lauraceae in the tant to the epidemiology of laurel wilt. Xyleborus
Americas,laurel wiltthreatensa diversityofhabitats, glabratusdoesnotrespondtoethanol,achemicalindi-
including the central and western forests, where sas- cator of plant stress and a common lure component
safras (Sassafras albidum [Nutt.] Nees) and California for most ambrosia beetle species investigated to date
laurel (Umbellularia californica [Hook. & Arn.] Nutt.) (HanulaandSullivan2008;Johnsonet al.2014),but
are native, respectively (Fraedrich 2008; Fraedrich insteadrespondstootherhostvolatiles(Niogretet al.
et al.2015).In2012,laurelwiltwasfoundinanother 2011; Kendra et al. 2012a,b, 2014). Wood, leaf and
Lauraceae member, avocado (P. americana Mill.), fungalodours,inadditiontovisualcuessuchasstem
damaging commercial production (Mosquera et al. silhouette, can direct X. glabratus attack, suggesting
2015). However, in south Florida, other ambrosia that a complex combination of cues may be utilized
beetle species may also be involved in R. lauricola by the beetle for host selection (Hulcr et al. 2011;
transmission in avocado (Carrillo et al. 2012, 2014), Mayfield and Brownie 2013; Kuhns et al. 2014a,b;
requiring unique considerations for the management Martini et al. 2015). Additionally, lure development
ofthediseaseinthatsystem(Ploetzet al.2011;Crane has focused on the sourcing and enrichment of the
et al. 2015). Given its rapid spread, laurel wilt repre- attractanta-copaene(Kendraet al.2016a,b),aplant-
sents a threat to the commercial avocado growers of derived sesquiterpene common in laurel wilt hosts
California and, to the world’s largest producer, (Hanula and Sullivan 2008; Kendra et al. 2011;
Mexico(Pisaniet al.2015). Niogretet al.2011).
Currentlaurelwiltmanagementreliesonrapidsan- The study of insect repellents for tree protection,
itation and chemical treatments of susceptible host specifically against X. glabratus and laurel wilt, is just
trees (Mayfield et al. 2008; Spence et al. 2013; starting; however, trials with other ambrosia beetles
2 ©2016BlackwellVerlagGmbH
M.A.Hughesetal. EvaluationofX.glabratusrepellents
show the potential of the use of repellents as part of and is commonly deployed as spatial repellent
an integrated pest management programme (Borden (Amman et al.1989,1991; Gibson et al.1991;Miller
et al. 2001; Burbano et al. 2012; Ranger et al. 2013). et al. 1995; Mafra-Neto et al. 2014b; Fettig et al.
Toexpandlaurelwiltmanagementoptions,weinves- 2015; Perkins et al. 2015). Methyl salicylate is a
tigatedtheefficacyofacommercialrepellentformula- plant-derived volatile derivative of salicylic acid
tion and of additional host and non-host volatiles to (Loake and Grant 2007) and has been found to be
deter visitation and attack by X. glabratus and other repellent against X. glabratus in laboratory experi-
ambrosiabeetlespeciesontrapsandhostboltsinfield ments(X.MartiniandM.A.Hughes,unpublished).
experiments.
Non-hostplantvolatilecollectionandanalysis
MethodsandMaterials
Theneedlesofthenon-hosttree,longleafpine(Pinus
palustris Mill.), were found to be significantly repel-
Experimentallocations
lent to X. glabratus in laboratory olfactometer bioas-
Fieldsiteswereselectedbasedonthepresenceoflau- says(datanotshown).Therefore,themajorchemical
rel wilt and a mixture of diseased and asymptomatic constituents of longleaf pine were analysed by gas
redbays still onsite. Experiments were conducted at chromatography–mass spectrometry (GC-MS) as
Wekiwa Springs State Park (WSSP, Apopka, FL – describedbelow,andcombinationsofvolatilecompo-
28°4240.93″N, 81°2745.85″W) in 2012 and in the nentswereusedforrepellencyinfieldtrials.Longleaf
0 0
Historic Haile Homestead (HHH, Gainesville, FL – pine needle samples of approximately 1.0–1.5 g were
29°3540″N 82°267″W) and Ichetucknee Springs cutintosmallpiecesandwereplacedinaclean40-ml
0 0
State Park (ISSP, Fort White, FL – 29°582.47″N glass vial and sealed with a lid and septa for volatile
0
82°4633.82″W) in 2015. WSSP was a pine flatwoods equilibration.Afterequilibratingforatleast15 minat
0
forest dominated by redbay, slash pine (Pinus elliottii 21°C,atriphase50/30 lmDVB/Carboxen/PDMSSta-
Engelm.) and red maple (Acer rubrum L.). HHH con- bleFlexTM solid page microextraction (SPME) fibre
sistedofamixedpine–hardwoodforestdominatedby (Supelco, Bellefonte, PA) was inserted through the
live oak (Quercus virginiana Mill.), redbay, southern septum and exposed to the leaf odours for 30 s. The
magnolia (Magnolia grandiflora L.), cabbage palmetto SPME was desorbed onto a GC-MS wax column, and
(Sabal palmetto [Walter] Lodd. ex Schult. & Schult. f.) the odour constituents were separated over 40 min
andslashpine.ISSP’svegetationconsistedofanoak– on a Restek Stabilwax capillary column using a tem-
hickory overstory forest dominated by laurel oak peraturegradientfrom40to240°at 7°/min.Identifi-
(Quercus laurifolia Michx.), scrub hickory (Carya flori- cation of the compounds was performed using a
dana Sarg.), redbay, southern sugar maple (Acer flori- PerkinElmer Clarus 500 quadrupole mass spectrome-
danum[Chapm.]Pax)andcabbagepalmetto. ter and Turbo Mass software (GC-MS). Linear reten-
tiontimesofauthenticstandards,whenavailable,and
matchingmassspectratotheNISTdatabasewereused
Chemicalsandformulations
to identify components. The per cent abundance of
Manuka oil lures were purchased from Synergy majorconstituentsisprovidedintable 1.
Semiochemicals Corp. (Burnaby, BC, Canada), and
the chemicals eucalyptol (C80601), myrcene
Treatmentrepellentsandexperimentaldesign
(M100005), limonene (62122), a-pinene (147524),
b-pinene (W290300) and ocimene (W353901) were Experiment1:Testofrepellentswithmanukaoilasattractant
purchased from Sigma-Aldrich (St. Louis, MO). The goal of this field trial was to provide an initial
SPLAT! (ISCA Technologies, Riverside, CA) is an screening of non-host volatiles and verbenone for
inert, wax-based matrix that allows for the infusion potential repellency to X. glabratus. At WSSP, Elm
and subsequent slow release of volatile compounds. BarkBeetleStickyTraps(GreatLakesIPM,Vestaburg,
ISCATechnologiessuppliedSPLATVerb(activeingre- MI) were prepared as two sticky cards (46 9 32 cm)
dient verbenone at 10%) (Mafra-Neto et al. 2014a; that were affixed to a wooden post at 1 m in height
Fettig et al. 2015) and formulated SPLAT MeSA (see Kuhns et al. 2014a). Treatments consisted of the
(methyl salicylate at 10%) and a SPLAT MeSA/Verb pairing of two separate components: a manuka oil
combination(5%each).Verbenoneisaketonic,anti- lure (attractant) (Hanula and Sullivan 2008) and
aggregation semiochemical produced by the mycan- either a non-host synthetic volatile blend or SPLAT
gial fungi of certain bark beetles (Brand et al. 1976) Verb (potential repellents). Manuka lures were
©2016BlackwellVerlagGmbH 3
EvaluationofX.glabratusrepellents M.A.Hughesetal.
Table1GC-MS analysis ofvolatiles emitted from longleaf pine (Pinus tests (X. Martini and M. A. Hughes, unpublished) as
palustris)needlesandthesyntheticvolatileblendsusedinfieldrepel- compared with natural tree host tissue. Field trials
lentexperiment1.Mixturesweremadebyvolume wereconductedatHHHandISSPin2015.Toincrease
the number of X. glabratus captured on controls, we
Syntheticblendsforfield
Linear Relative utilizedfreshlycutredbayboltsastheattractant(posi-
testing(%byvolume)
retention abundance(%) tive control) instead of manuka lures, and non-host,
time Identification LongleafPine Pine1 Pine2 Pine3 laurel oak bolts as a negative control. To obtain the
freshwoodtissue,asingleuninfectedredbayandlau-
1038 a-Pinene 23.1 67 – –
rel oak were cut down and the main stem sectioned
1097 Camphene 1.1 – 50 –
intosegments( 30 cmlength,10 cmdiam.).Alarge
1131 b-Pinene 51.8 33 – –
"
1135 Sabinene 2.2 – – – constructionnailwashammeredintothebottomofa
1189 Myrcene 1.8 – 50 – cutend,andtheboltwasthenplacedintothetopofa
1220 Limonene 1.6 – – 50 hollowmetalpole(3 cmdia.)thatwaspreviouslydri-
1248 Ocimene 2.0 – – 50 venintotheground 1.5 minheight,anappropriate
Total 83.6 100 100 100 "
location for capture of X. glabratus (Brar et al. 2012).
Two(28 9 23 cm)stickycards(Wingtrapstickyliner
– Scentry Biologicals Inc., Billings, MT) were stapled
constructed from an internal matrix soaked in man- tothelowerportionofthetreatmentboltonopposite
uka oil, enclosed in a plastic pouch (Synergy Semio- sides (Figure S1). Repellent treatments consisted of
chemicals Corp.). The putative X. glabratus repellents the redbay bolts plus SPLAT MeSA, SPLAT Verb or a
consisted of the following synthetic blends: ‘pine 1 MeSA/Verbmixture(1 : 1).Dollops(17.5 g)ofSPLAT
0
wasa2 : 1mixtureofa-pineneandb-pinenebyvol- compounds were applied directly to the bolts with a
ume, ‘Pine 2’ was a 1 : 1 mixture of myrcene and pre-calibrated caulking gun and allowed to cure for
camphenebyvolume, ‘Pine3’wasa1 : 1mixtureof 24 hbeforefielddeployment(FigureS1).Positiveand
limonene and ocimene by volume (table 1), and negativecontrolboltswereleftuntreated.
SPLAT Verb contained 10% verbenone by volume. Traps were arranged in a randomized complete
Synthetic liquid blends (5 ml) of each artificial plant block design. A block consisted of a linear transect
odour were injected directly into the manuka lure with treatments spaced at 10 m and blocks 30 m
using a 21-gauge hypodermic needle and glass syr- apart. Treatments were randomly positioned within
inge.Theholewascoveredwithasmallpieceofcello- blocks and were rotated to the next position every
phane tape. Control manuka lures were also trappingperiodtominimizepositionaleffects.Tofur-
puncturedandpatchedinasimilarmanner,butnoth- therreducethepotentialofapositionalbias,notraps
ing was injected into the lure. For the SPLAT Verb were placed within 3 m of symptomatic redbays.
treatment,onedollop(17.5 gdeployedas1 cmdiam- Traps were inspected and rotated every 14 days for
eter sphere) of SPLAT was wrapped in medical gauze HHH and ISSP. At each trapping period, two circular
and attached with a paper clip to the sticky trap in wounds (2 cm dia.) were made with a cork borer to
closeproximitytothemanukalure.Trapsbaitedwith exposefreshredbayxylemandthusrefreshthebolts’
manukaoilonlyservedaspositivecontrols,andnon- attractiveness. At the end of every trapping period,
baited traps served as negative controls. Traps were sticky cards were replacedand brought to the labora-
arrangedinarandomizedcompleteblockdesign,con- torywherebeetleswereidentifiedandcountedunder
sisting of four blocks of six beetle traps (n = 24). adissectionmicroscope.Thetrialwasterminatedonce
Within-block traps were placed no less than 6 m each treatment completed a full positional rotation
apart, with blocks no less than 60 m apart. Sticky cycle along the linear transect (five rotations/
cardswerecollectedweekly,andX. glabratuscaptured 10 weeks). At the end of the trial, trap bolts were
were identified and counted. The mean temperature transported to the laboratory (University of Florida,
was 25.5 0.2°C and relative humidity Gainesville), where they were debarked and
!
78.0% 1.3%. X. glabratusentranceholeswerecountedasdescribed
!
in Hanula et al. (2008). A subsample of bolts with
Experiment2:Testofrepellentswithcutlogsasattractant beetle entrance holes was further dissected using a
Thegoalofthisfieldtrialwastotesttherepellencyof bandsawandexaminedforthepresenceofX. glabra-
verbenone and MeSA, the only volatiles that showed tus and the extent of gallery formation. The mean
repellency against X. glabratus in the initial field temperaturewas22.9 0.3°C,andrelativehumidity,
!
experiment (see Results section) and in laboratory 87.0% 0.7%.
!
4 ©2016BlackwellVerlagGmbH
M.A.Hughesetal. EvaluationofX.glabratusrepellents
catches (fig. 1). Captures decreased for all treatments
Statisticalanalysis
after 4 weeks (fig. 1). Mean weekly X. glabratus cap-
The reported means SE and sums were calculated tures were statistically higher (P = 0.007) for
!
inMicrosoftExcel(2010).Statisticalanalysesofbeetle manuka + pine 1 (4.4 1.0), manuka + pine 3
!
captures and X. glabratus entrance holes were calcu- (5.1 1.2) and manuka alone (5.6 1.0) than the
! !
lated using generalized linear mixed models with a negative control treatment (0.3 0.1) (table 2).
!
negative binomial distribution using the GLIMMIX Mean beetle captures with manuka + pine 2
procedure on SAS 9.4 (SAS Institute, Cary, NC). For (2.9 1.0), and manuka + SPLAT Verb (2.1 0.5)
! !
boring activity analysis, laurel oak data were omitted did not differ significantly to all other treatments
due to the lack of X. glabratus entrance holes on this (table 2). The manuka positive control captured the
host. Weekly capture data were analysed using a lin- mosttotalX. glabratusthroughoutthedurationofthe
earmixedmodelwhereWSSPwassquareroot-trans- experiment (90), and SPLAT Verb was the repellent
formed to meet the assumptions of the model, while with the fewest (34) total captures (table 2). Non-
counts at HHH and ISSP were not transformed. At X. glabratus ambrosia beetle captures were low and
WSSP, the violation of the model assumptions was statistically similar (P = 0.42) among all treatments,
probably due to low beetle counts. Block and site with means ranging from 0.9 to 1.9 weekly catches
locationsweretreatedasrandomeffectsinallmodels. and total sums 14–30 beetles (table 2). Non-target
Type III tests were used to determine significance of ambrosiabeetlescomprised17%–53%ofalltotalcap-
thefixedeffects.Multiplecomparisonsofleastsquares tures per treatment (table 2) and included members
means were analysed and adjusted for using Tukey’s of the genera Xyleborus and Xylosandrus (data not
multiplecomparisonprocedure.Standarderrorswere shown).
calculatedas[stddev/√n],wheren = numberofrepli-
cates.FigureswerepreparedusingGraphPadPrismv.
Experiment2:Testofrepellentswithcutlogsas
7.0 (GraphPad Software, La Jolla, CA) and edited in
attractant
Adobe Illustrator CS6 (Adobe System Inc., San Jose,
CA). All mean temperature and relative humidity The effects of block and site location (ISSP and HHH)
data were collected and analysed from the Florida werenotstatisticallysignificant(atP > 0.05,datanot
Automated Weather Network (FAWN) provided by shown); thus, data from both locations were com-
theUniversityofFlorida. bined. For X. glabratus, all repellent treatments and
the laurel oak negative control captured significantly
fewer beetles than the redbay positive control in our
Results
14-daytrappingperiods(P = 0.0001)(fig. 2,table 3).
Both the SPLAT MeSA-Verb (3.5 0.6) and SPLAT
Non-hostplantsyntheticblends !
Verb(2.2 0.6)repelledthemostbeetles,andmean
!
Volatileanalysisoflongleafpineneedlesrevealedthe trapcaptureswerestatisticallysimilartothenon-host
predominantconstituentsasaandbpinene;addition- laurel oak (5.8 0.8 beetles) (table 3). Initially,
!
ally, myrcene, camphene, limonene and ocimene SPLATMeSAhadarelativelyhighlevelofrepellency,
comprisedthelessabundantcomponentsofthevola- similar to the other best performing potential repel-
tile profile (table 1). All of these components except lents; however, after four weeks, the effectiveness of
ocimene are present in redbay wood odours (Kuhns this repellent treatment decreased, resulting in more
et al. 2014a; Martini et al. 2015); however, long leaf mean captures in SPLAT MeSA (14.6 2.4 beetles/
!
pine does not contain eucalyptol, a known attractant biweekly) than the treatments containing verbenone
ofredbayambrosiabeetle(Kuhnset al.2014a).Com- (fig. 2,table 3).TheredbayboltswithoutSPLATcap-
binationsofcomponentswereselectedforfieldtesting tured the most beetles per trapping period
based on their relative abundance in longleaf pine (39.8 3.9) (table 3). A total of 1992 X. glabratus
!
andareshownintable 1. beetles were captured with the redbay positive con-
trols during the experiment. SPLAT MeSA-Verb and
SPLAT Verb treatments captured fewer beetles than
Experiment1:Testofrepellentswithmanukaoilas
the laurel oak control (290 total X. glabratus), with a
attractant
totalof177and108,respectively(table 3).Compared
Manuka oil attracted significantly more X. glabratus to the positive control, SPLAT MeSA-Verb reduced
beetles than the non-baited control, with most of the total captures by 91% and SPLAT Verb by 94.5%,
treatment repellents showing intermediate trap indicating strong levels of repellency. Captures of
©2016BlackwellVerlagGmbH 5
EvaluationofX.glabratusrepellents M.A.Hughesetal.
Fig.1 TrappingexperimentatWekiwa
SpringsStatePark(WSSP)inFlorida.Mean
Xyleborusglabratuscaptures SEoverfour
!
weeks.Trapsconsistedofstickypanels
(46932cm)affixedtowoodenpostsbaited
withmanukaoil.Putativerepellentsynthetic
blendswerelabelledaspine1–3(seetable1)
andSPLATVerb.Trapsbaitedwithonlyman-
ukaoilwerepositivecontrols,andnegative
controlswereunbaitedtraps.Separationsof
meansarelistedatweeklyintervals.Columns
denotedwithdifferentlettersarestatistically
differentata=0.05accordingtoTukey’smul-
tiplecomparisonsprocedureoftreatment
leastsquaresmeans.n=24.
Table2SummaryresultsofX.glabratusrepel-
Xyleborusglabratus Non-targetambrosiabeetlespp.
lencyexperimentatWekiwaSpringsStatePark
(WSSP)
Sumof Sum Percent
Meanweekly trap Meanweekly oftrap oftotal
Treatment* trapcaptures† captures trapcaptures† captures captures
Manuka(pos.control) 5.6 1.0A 90 1.2 0.4A 19 17.4
! !
Manuka+pine1 4.4 1.0A 75 1.9 0.4A 30 28.6
! !
Manuka+pine2 2.9 1.0AB 46 0.9 0.3A 14 23.3
! !
Manuka+pine3 5.1 1.2A 81 0.9 0.2A 14 14.7
! !
Manuka+SPLATVerb 2.1 0.5AB 34 1.2 0.2A 19 35.8
! !
Blank(neg.control) 0.3 0.1B 16 1.2 0.4A 18 52.9
! !
Pvalue 0.007 0.42
Weeklymeans+SEandtotalsumsofX.glabratusandnon-targetambrosiabeetlescaptureddur-
ingfourweekexperiment(fourreplicatespertreatment,n=24).
*Treatmentsconsistedofmanukalures(attractant)pairedwithatestrepellent.Positiveandnega-
tive controls were manuka lure only and non-baited traps, respectively. See table1 for con-
stituentsofsyntheticblends.
†Columnsdenotedwithdifferentlettersarestatisticallydifferentata=0.05accordingtoTukey’s
multiplecomparisonsprocedureoftreatmentleastsquaresmeans.
other bark and ambrosia beetles were low (0.4–1.6 with SPLAT MeSA (4.3 1.0) as compared with the
!
beetles/biweekly,22–79totalsum)andseemednotto positive control. SPLAT Verb was the most effective
beaffectedbytreatment(P = 0.48)(table 3).Non-tar- repellentwith1.2boringholesperboltatexperimen-
get ambrosia beetles comprised 3%–27% of all total tal conclusion. SPLAT MeSA-Verb (3.3 1.1 boring
!
captures per treatment (table 3) and included mem- holes) was statistically similar to the treatments con-
bers of the genera Xyleborus, Xylosandrus, Hypothene- tainingmethylsalicylateandverbenonealone.SPLAT
musandAmbrosiodmus(datanotshown). MeSA-Verb and SPLAT Verb suppressed X. glabratus
Xyleborus glabratus entrance-hole counts were boltboringbyover80%and92%,respectively,com-
higherfor theredbay positive controlwith amean of pared to the control with untreated redbay bolts
16.6 3.7 per bolt (table 3). Significantly fewer (table 3). No beetle boring was detected on the non-
!
mean boring holes occurred on redbay bolts treated host laurel oak. To explore the extent of X. glabratus
6 ©2016BlackwellVerlagGmbH
M.A.Hughesetal. EvaluationofX.glabratusrepellents
Fig.2 Trappingofcombinedexperimentsat
HistoricHaileHomestead(HHH)andIchetuck-
neeSpringsStatePark(ISSP)inFlorida.Mean
Xyleborusglabratuscaptures SEover
!
10weeks.Trapsconsistedofaredbaybolt
withtwostickycardsattached.Repellentscon-
sistedofSPLAT+methylsalicylate(MeSA),
SPLATVerbandablendofSPLAT
Verb+MeSA.Trapsbaitedonlywithredbay
boltswerepositivecontrolsandwithlaurel
oak(non-host)boltswerenegativecontrols.
Separationsofmeansarelistedat14-day
intervals.Columnsdenotedwithdifferentlet-
tersarestatisticallydifferentata=0.05
accordingtoTukey’smultiplecomparisons
procedureoftreatmentleastsquaresmeans.
n=50.
colonization, a subset of bolts were further dissected protectinglivehosttrees.Weselectedverbenonedue
by sectioning them into thin slices with a band saw. totherelativelylownumberofbeetlescapturedwhen
Most boring activity consisted of shallow tunnels it was included in the initial experiment and methyl
extending only 1 cm into the sapwood, lacking salicylate (MeSA) for its repellency to X. glabratus
"
signs of significant gallery formation or beetle under laboratory conditions (X. Martini and M. A.
reproduction(e.g.eggsorcallowbeetles). Hughes, unpublished). Methyl salicylate is a plant-
derived volatile ester released following stress events,
such as herbivory, pathogen infection and associated
Discussion
disease-resistance pathways (Shulaev et al. 1997).
Here, we evaluated volatile compounds for their Thecompoundisnormallyabsentinhealthyplanttis-
potential repellency against X. glabratus, the primary sue; however, upon herbivore and pathogen attack,
vector of the laurel wilt pathogen (Raffaelea lauricola) MeSA acts as a volatile signalling molecule that
that has been causing high levels of mortality in induces host systemic acquired resistance (SAR) and
native Lauraceae in the eastern USA. The volatile salicylic acid defence response pathways to other
blends chosen were based on chemical analyses of areas of the plant and neighbouring plants (Shulaev
non-host volatiles, and damage-induced volatiles et al. 1997; Loake and Grant 2007). Increased MeSA
known to repel this species, as well as known repel- levels induced by insect feeding have been linked to
lents for other bark beetle species.In our first experi- repellency in certain herbivores (Hardie et al. 1994;
ment, we paired manuka lures with three synthetic Losel et al. 1996) and the attraction of natural ene-
volatile blends and SPLAT Verb (verbenone 10%). A mies (Shimoda et al. 2002; James 2003; Mallinger
50 : 50blendofcampheneandmyrcene,aswellasa et al. 2011), suggesting that this volatile has a com-
commercially available formulation of verbenone plex and variable role in host signalling and defence.
(SPLAT Verb), was intermediate to all treatments. Bordenet al.(2001)foundthatMeSAreducedstriped
Furthertestingwithfreshhostboltsorimprovedlures ambrosia beetle (Trypodendron lineatum Oliver) densi-
wouldlikelyresultsinthehighercaptureratesneeded tiesintrapsbaitedwithanaggregationpheromonein
totrulydiscriminatebetweenthesetreatments. British Columbia. MeSA (10%) alone reduced cap-
In our subsequent experiments, we abandoned the turesandboringholesofX. glabratusfortheduration
useofmanukaoilluresasanattractanttoinvestigate oftheexperiment;however,longevityofeffectiveness
repellency in the field and instead used fresh redbay was lower as compared with verbenone from the
wood due to its superiority as an attractant (Kendra releasedevicestestedhere.Itispossiblethatoptimiz-
et al. 2011) and relevance to our overall goal of ingthereleaserateanddurationofeffectivereleaseof
©2016BlackwellVerlagGmbH 7
EvaluationofX.glabratusrepellents M.A.Hughesetal.
Table3Summary results of X.glabratus repellency experiment at Historic Haile Homestead (HHH) and Ichetucknee Springs State Park (ISSP)
combined.
Xyleborusglabratus Non-targetambrosiabeetlespp.
Sum Sum Percent
Meanbiweekly oftrap Meanboring Meanbiweekly oftrap oftotal
Treatment* trapcaptures† captures holesperbolt† trapcaptures† captures captures
Redbay(pos.control) 39.8 3.9A 1992 16.6 3.7A 1.4 0.2A 70 3.4
! ! !
Redbay+SPLATMeSA 14.6 2.4B 724 4.3 1.0B 0.9 0.2A 44 5.7
! ! !
Redbay+SPLATMeSA-Verb 3.5 0.6CD 177 3.3 1.1BC 0.4 0.1A 22 11.1
! ! !
Redbay+SPLATVerb 2.2 0.6D 108 1.2 0.6C 0.8 0.2A 39 26.5
! ! !
Laureloak(neg.control)‡ 5.8 0.8CD 290 0 0.0 1.6 0.3A 79 21.4
! ! !
Pvalue <0.0001 <0.0001 0.48
Biweekly(14days)means+SEandtotalsumsofX.glabratusandnon-targetbarkandambrosiabeetlespeciescapturedduring10-weekexperiment.
n=50.
*Treatmentsconsistedofacutredbayboltattractantwitha17.5gdollopofSPLATtestrepellent.Positiveandnegativecontrolswereuntreatedred-
bayandalaureloakbolts,respectively.
†Columnsdenotedwithdifferentlettersarestatisticallydifferentata=0.05accordingtoTukey’smultiplecomparisonsprocedureoftreatmentleast
squaresmeans.
‡Duetothelackofboringholesinthenon-hostlaureloak,thesedatawereomittedfromstatisticalanalysis.
MeSAfromSPLATorothersemiochemicaldispensers saxesenii Ratzeburg in Hawaii (Burbano et al. 2012)
may further improve the effectiveness of MeSA as a and Euwallacea validus Eichoff, Xylosandrus germanus
repellentagainstX. glabratus. BlandfordandtwoothertaxainOhioinethanol-bai-
In our experiments, X. glabratus was repelled most tedtraps(Rangeret al.2013).Collectively,thesedata
effectively by the commercial SPLAT Verb formula- suggest that verbenone-based compounds should
tionofverbenone(10%)aloneandSPLATformulated have potential as an effective X. glabratus repellent
with a 1 : 1 mixture of verbenone and MeSA (5% andmanagement tool,especially in conjunction with
each). These two repellent treatments reduced cap- additional components of integrated management
ture of X. glabratus to levels observed with the laurel (i.e.sanitationandfungicides).
oak negative control. Boring by X. glabratus was Although these results are promising, further test-
reduced most effectively by the SPLAT MeSA-Verb ingisrequiredtoassesssomeoftheuniquedifficulties
and SPLAT Verb treatments over 10 weeks, decreas- ofprotectinghost treesagainst X. glabratus andlaurel
ing entrance holes by fivefold and over 10-fold, wilt.Mostnotableistheextremevirulenceofthefun-
respectively, compared to the untreated redbay bolts. gal pathogen, R. lauricola, to host Lauraceae. Unlike
Interestingly, beyond the initial entrance cavities other pestiferous bark and ambrosia beetles that
(Brar et al. 2013), no galleries, eggs or larva were require a high density of attacks in a very short time
observedinanyredbaytransversesections,regardless to overwhelm and kill their hosts (Lee et al. 2011;
oftreatment.ThisobservationiscongruentwithFrae- Tarno et al. 2011; Gitau et al. 2013), even very few
drichet al.(2008)whosuggestthatredbayhosttrees attacksofX. glabratusandalowloadoffungalpropag-
are not suitable for X. glabratus colonization and ulesaresufficienttoinoculateandkillhealthyredbay
reproduction until the advanced stages of laurel wilt trees (Fraedrich et al. 2008; Hughes et al. 2013,
andmortalityinducedbyR. lauricola. 2015b). As our tests utilized cut bolts as attractants,
Verbenonehasbeenwidelytestedasananti-aggre- which likely differed in terms of volatile release pro-
gant to protect pine trees in the USA against several file from non-wounded intact stems, further tests on
Dendroctonus barkbeetlespecies,includingthemoun- healthy, standing forest and landscape trees are
tain (D. ponderosae Hopkins) (Lindgren et al. 1989; requiredtofullyevaluatethepracticalutilityofthese
Shea et al. 1992; Fettig et al. 2015), southern compounds. Bolts treated with verbenone or the
(D. frontalis Zimmerman) (Payne and Billings 1989) MeSA-verbenone mixture had very few successful
and western (D. brevicomis) pine beetles (Fettig et al. boring attacks, indicating that larger-scale testing is
2008). When examined with ambrosia beetles, ver- warranted.Theverbenonealoneand1 : 1MeSAand
benone reduced captures of Xylosandrus compactus verbenoneblendtreatmentsweresimilarineffective-
Eichhoff, X. crassiusculus Motschulsky and Xyleborinus ness. MeSA is less expensive than verbenone; the
8 ©2016BlackwellVerlagGmbH
M.A.Hughesetal. EvaluationofX.glabratusrepellents
1 : 1 blend of MeSA and Verbenone is ca. 45% less AmmanGD,ThierRW,WeatherbyJC,RasmussenLA,
expensive than verbenone alone. Further research is MunsonAS,1991.Optimumdosageofverbenoneto
needed to determine whether this blend could allow reduceinfestationofmountainpinebeetleinlodgepole
foramorecost-effectivetooltomanageX. glabratusin pinestandsincentralIdaho.USDAForServResPap
residential landscapes, forests or avocado groves than INT-446.
verbenonealoneinSPLAT. AukemaJE,McCulloughDG,HolleBV,LieholdAM,Brit-
This initial investigation did not address dosage and tonK,FrankelSJ,2010.Historicalaccumulationofnon-
indigenousforestpestsinthecontinentalUnitedStates.
environmental factors affecting the longevity of dis-
Bioscience,60,886–897.
pensereffectiveness.Itwillalsobeimportanttodeter-
BatraLR,1985.Ambrosiabeetlesandtheirassociated
mine whether these repellents affect other Xyleborus
fungi:researchtrendsandtechniques.ProcIndianAcad
speciesinFlorida.XyleborusvolvulusFabriciusandXyle-
Sci,94,137–148.
borus ferrugineus Fabricius also transmit R. lauricola to
BordenJH,ChongLJ,GriesR,PierceHDJr,2001.Poten-
avocadoinno-choiceexperimentaltests(Carrilloet al.
tialfornonhostvolatilesasrepellentsinintegratedpest
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BrandJM,BrackeJW,BrittonLN,MarkovetzAJ,Barras
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lentsidentifiedinthisstudyto‘push’thevectoraway BrarGS,CapineraJL,McLeanS,KendraPE,PloetzRC,
from susceptible hosts, in combination with the best Pen~aJE,2012.Effectoftrapsize,trapheightandageof
available attractant (i.e. 50% a-copaene lure; Kendra lureonsamplingXyleborusglabratus(Coleoptera:Cur-
et al. 2016a,b) to ‘pull’ them to areas of non-hosts, culionidae:Scolytinae),anditsflightperiodicityandsea-
may increase the effectiveness of these semiochemi- sonality.FlaEntomol,94,1003–1011.
cals than if used alone. Also, combining repellents BrarGS,CapineraJL,KendraPE,McLeanS,PloetzRC,
with application of fungicides may improve manage- Pen~aJE,2013.Lifecycle,development,andcultureof
ment. For example, well-timed application of repel- Xyleborusglabratus(Coleoptera:Curculionidae:Scolyti-
lents in concert with application of propiconazole nae).FlaEntomol,96,1158–1167.
couldextend theduration oftree protection.Integra- BruhnJN,HeydRL,1992.Biologyandcontrolofoakwilt
tion of several tactics will be necessary for manage- inMichiganredoakstands.NorthJApplFor,9,47–51.
BurbanoEG,WrightMG,GilletteNE,MoriS,DudelyN,
ment of X. glabratus and laurel wilt in the
JonesT,KaufmannM,2012.Efficacyoftraps,lures,and
south-eastern USA, and the application of behaviour
repellentsforXylosandruscompactusandotherambrosia
modifying chemicals for the vector warrants further
beetlesonCoffeaarabicaplantationsandAcaciakoanurs-
investigation.
eriesinHawaii.EnvironEntomol,41,133–140.
CameronRS,HanulaJ,FraedrichSW,BatesC,2015.Pro-
Acknowledgements gressionoflaurelwiltdiseasewithinredbayandsas-
safraspopulationsinsoutheastGeorgia.SoutheastNat,
We thank Wekiwa and Ichetucknee Springs State
14,650–674.
Parks (FL) and the Historic Haile Plantation (FL) for
CarrilloD,DuncanRE,Pen~aJE,2012.Ambrosiabeetles
permission to utilize their property as experimental
(Coleoptera:Curculionidae:Scolytinae)thatbreedin
locations.TheauthorswouldalsoliketothankPatrick
avocadowoodinFlorida.FlaEntomol,95,573–579.
James, Ode Akpoji and Adam Black (University of CarrilloD,CraneJH,Pen~aJE,2013.Potentialofcontact
Florida, Gainesville) for their assistance in the field insecticidestocontrolXyleborusglabratus(Coleoptera:
collections.Fundingforthisworkincludedacoopera- Curculionidae),avectoroflaurelwiltdiseaseinavoca-
tive agreement with the USDA-Forest Service, Forest dos.JEconEntomol,106,2286–2295.
HealthProtection,Region8. CarrilloD,DuncanRE,PloetzJN,CampbellAF,PloetzRC,
Pen~aJE,2014.Lateraltransferofaphytopathogenic
symbiontamongnativeandexoticambrosiabeetles.
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©2016BlackwellVerlagGmbH 9