Barbosa This article isprotected bycopyright. Allrights reserved. E-mail: [email protected] 040, Araraquara,SaoPaulo,Br Pereira deBarros,201,V *Correspondence to:HaroldoXLVolpe,Fundode Paulo, Brazil Exact Sciences,viadeAcessoProf.PauloDo d Luís, Km 235,s/n,13565-905,Sã c b Araraquara, SaoPaulo,Brazil a Haroldo XL Volpe Efficacy ofessential oilof Diaphorina citri

Accepted Chemistry Department, FederalUniversityof Article Embrapa Acre,P.O.Box321,69908-970,RioBranco,Brazil Faculty ofAgriculturalandVeterinarySciences Fundo deDefesadaCitricultura,Av.Dr article as doi: 10.1002/ps.4143 differencesbetween version lead to this may been through the copyediting, typesetting, pagination andproofreading process, which This article hasbeen accepted for publication andundergone full peer review but has not d andMarcelo PMiranda

a *, MuriloFazolin ila Melhado,ViadeAcesso Prof. azil. Phone:+55163301-7025. o Carlos,SaoPaulo,Brazil a aduncum b , RafaelBGarcia

and the Versionof Record. Please cite this . Adhemar Pereirade nato Castellane,s/n,14884-900,Jaboticabal,Sao SãoCarlos,RodoviaWashington ofJaboticabal–FCAV/Unesp,Department of Defesa da Citricultura, Av. Dr. Adhemar daCitricultura,Av.Dr.Adhemar Defesa againstnymphsandadultsof a , RodrigoFMagnani Paulo DonatoCastellane,s/n,14807- Barros,201,14807-040, a,c , José Carlos , JoséCarlos This article isprotected bycopyright. Allrights reserved. integrated pestmanagement. Keywords: Dillapiol-richoil isapromisingCONCLUSIONS: compound for 30%). 80%) inadults. However,theessential oilshow containing 76.6%and81.6%ofdillapiolat0.75% a RESULTS: Alltreatments cause dilutions plus0.025%ad Oils withthreeconcen Here, westudieditsefficacyagainst nymphs andadultsof environment andcontainsdillapiol,whichwaspr Insecticideapplicati BACKGROUND: Abstract Accepted Articleto psyllidresistance.The essentialoilof it causesenvironmental contamin Asiancitrus psyllid; HLB;botanical insecticide; active ingredient rotation; trations ofdillapiol(65.2%,76.6% juvant weretested. d 90–100%mortalityinnymphs. Topicaltreatments withoil ation, hasanegativeimpact onbe on isthemain waytocontrol Piper aduncum ed noresidual activityagainst adults (mortality oven tobeeffectiveagainstseveralcroppests. nd 1%dilutionswereeffective(mortality , and81.6%)at0.5%,0.75%,1.0% D. citri haslowtoxicity towards the D. citri underlaboratoryconditions. neficial organisms, andleads Diaphorina citri control.

. However, ≤ ≥

approximately 30%infield conditions. demonstrated tohaveanefficacyagainst ( the useofextractandessentialoilneem Studies ontheseinsecticidesforcontrolof applications of insecticides. nurseries, inspectionanderadicationofdis This article isprotected bycopyright. Allrights reserved. citri in the field usingneem extract. Neem and Acceptedof resistanceandcontributetosustainable and fenopropathrin(4.8). imidacloprid, followedbychlorpyriphosphos( selective pressure. InFlorida, developed resistanceagainst chemicals dueto th the primary method usedformanagement oftheinsectvector. Articlecitri continents. Huanglongbing (HLB)thataffectscommercialci Liberibacter americanus have been associated w particular, thephloem-infectingbacteria Pathogens spread byinsectvectors arelimiti 1 INTRODUCTION nymphs andadults Kuwayama thathastheabilitytotransmit both bacterialspecies. One suchalternative that requires additional Management ofHLBincludestheusecitr 1–3 ThespreadofHLBinorchardsmainly occursviathecitruspsyllid

by upto4-foldcompared tount 9 Theuseofdifferentcontroltact 6 Chemical control, mostlybythe D. citri was reported to have a resistance ratio higher than 30 for wasreportedtohavearesistanceratiohigherthan30for 10,11 use of insecticides in eased plantsinorchards,andcontrolof Khan etal. D. citri Datura Candidatus D. citri 17.9), thiamethoxam (15.0),malathion (5.4), Azadirachta indica ng factors forthecultivation of citrus. In ith the destructive citrus

are incipient andmost ofthem arefocusedon eir frequent use,which leadsto agreater alba trus varietiesonthe us trees produced in screened vector-free us treesproducedinscreenedvector-free studies istheuse of botanicalinsecticides. 12 nymphsof92% reated areasinfieldconditions. reported 80% mortality of adultpsyllids Nees extractsreducedthe number of ics canslowdownthedevelopment Liberibacter asiaticus and active ingredient imidacloprid, is themanagement of 7,8 A. Juss.). Neem has been A.Juss.).Neemhasbeen 4,5 However,insectshave

American andAsian greeningdiseaseor in greenhouseand D. citri D. citri Diaphorina 13

with . 8 Ca.

D. This article isprotected bycopyright. Allrights reserved. currently, therearenoreports Tenebrio molitor P. aduncum aduncum viability in respectively, bothatac and 80%for artificial dietat100 Ostrinia nubilalis substances initsdigestivetract. xenobiotics presentinthehostplan products. which transform lipophiliccompounds intomore dillapiol beingthemajor compound especially monoterpenes, properties nymphs andadultsof 2.1 OPAextractionand quantificationofchemicalcompounds METHODS 2 EXPERIMENTAL Acceptedof activeingredients for cont Article Dillapiol potentially inhibits Piper aduncum The objective ofthepresentstudywastoevalua leaf extract.Previous studies havedetermined theinsecticidal effect of essential oilof 21 14 Throughinhibitionofmonooxygena becauseitcontainssecondarymetabolite Aleurocanthus woglumi (OPA) ondefoliatingpestssuchas Aetalion (Hübner) usingdillapio L., μ g/g. Inastudyonsuckinginsects,Silvaetal. 27 D. citri andstored-grainpest sp.adultsbyusing L., aplantabundantin 15 oncentration of30mg mL sesquiterpenes, rolling thisinsectvector. inoursearchforanewmode ofac onthetoxicactivityof 22,23 . theactivityofcytochrome t isreduced,resultingindeat 18–20 Ashbyeggsaftertopicala Bernardetal.

l extractedfrom 16 and se, theabilityofherbivoreinsectstoexcrete P. aduncum Cerotoma tingomarianus Sitophilus zeamais phenylpropanoids, theAmazon region,displaysinsecticidal soluble (hydrophilic)a 18 P. aduncum -1 observeda95%increasedmortalityof . Castroetal. te theefficacyofdillapiol-richOPAon s thatshowtoxicitytowardsinsects, extractsfrom leavesandroots, P450-dependent monooxygenase, tion tobeadoptedintherotation pplication of a 4%aqueous pplication ofa (dillapiol) towards h duetoaccumulation oftoxic 24 17 reported mortalities of72% 25 withthephenylpropanoid Motschulsky. observed54.8%lossof L. incorporatedinan Bechyné, nd easilyexcretable 28 D. citri 26 However, flourpest . P. 200°C; thecarriergaswasHe injector anddetectorinterf to 250°C,andthenheldfor1.10minutes. This This article isprotected bycopyright. Allrights reserved. obtained massspectra with thos range from Acceptedwere dilutedinmethanol (injectionof0.5 followed byatemperature ramp of4°Cmin 0.10 (Shimadzu, Japan)equippedwithacapillaryco using agaschromatograph (GC)coupledto collector withadispensingsystem under–760-mmH cooling watercirculationtoc Articlemm Raschigrings.Thetopofthecolumn was absorption towerconsistingofasingleglassco by usingaheatingmantle upto150°Canda with ayieldaroundof2–2.5%.The 20–30% ofmoisture.Essentialoilwas extracted and finestems wereseparatedfo ground surface. wereharvestedevery12 m Rio Branco,Acre,Brazil(10°1 Three-year-old adult μ m filmthickness).TheGCtemperature program For theidentificationandqua m/z 40 to 350. Chemical characterization 40to350.Chemical P. aduncum ace temperature were250°Cand ondense thevolatiles.Thecondense (column flow0.64mL min ′ 21.36 r processing.Theplantmass wass plantswerecollectedintheproductionfieldofEmbrapaAcre, e availableintheGC-MS spectradatabase from National essential oilwasredistilledth ntification of chemical compounds,ntification ofchemical theOPAwasanalyzed ′′ S,67°42 μ L). Mass spectra wererecord -1 to190°C,followedbyanotherramp of47°Cmin amass spectrometer Plus (MS)GCMS-QP2010 lumn of50×600mmcompletelyfilledwith6–8 lumn (RestekRxi-5MS, 3000-mL flask. Theflaskwasconnectedtoan ′ by steamdistillationasdescribedpreviously gave atotalGCrun 31.70 connected toa40× onths foressential oil extraction. The leaves g pressurecreatedbyusingavacuum pump. consistedofstarttemperature at40°C, ′′ was performed bycomparison ofthe W)bycuttingthem at0.4m abovethe − 1 , splitratio1:500),andthesamples the ionsourcetemperature was r wasconnectedtoafraction ubjected todryingachieve rough fractionalrectification time of40minutes. The ed at70eV,withamass 10m X0.10mm IDX 300-mm condenserfor 29 -1

v dillapiol (65.2%,76.6%,and81.6%) Paraíso, Holambra, SãoPaulo,Brazil). was injected intothechromatograph substrate (80% young shootspergraftedtreewere Swingle citrumelo [ and relativehumidity (71.62% greenhouse (1.60×7.906.0m) underambient te This article isprotected bycopyright. Allrights reserved. adjuvant inwater,andthen addingtheOPAaccord 100%) (Momentive, Itatiba, Sao Paulo, Brazil). Accepted da Citricultura),Araraquara,SaoPaulo, Brazil(21°48 Articlefour differentconcentrations.Theexperiment wa and adults,weevaluatedthephytotoxiceffecton Before selectingtherange ofOPAconcen concentrations 2.2 PhytotoxicityoftheOPAagainst basis ofGC-MSpeakareas. indices Institute ofStandardsTec -1 inwater,withadditionof0.025%Silwet 30 For thepreparation of theinsecticide sprays, OPAwithdifferent concentrations of Forty-two nursery trees (1-year-old

. ForthedeterminationofKovatsreten Pinus Citrus paradisi sp. bark,15%vermiculite, and5% hnology (NIST),datafrom theli on average)duringtheenti Macf.x selected.The plantswere 31 . Component relative percenta trations toassess itsefficacyagainst Citrus sinensis Poncirus trifoliata Citrus sinensis tion rates,amixture oflinearalkanes(C ® 99.9%weredilutedto s carried out at Fundecitrus (Fundo de Defesa s carriedoutatFundecitrus(FundodeDefesa sweet orangeshootsusingthreedilutionswith adjuvant(polyesterco ing tothe concentrationsestablished foreach mperature (27.13°Conaveragetemperature) The sprays wereprepared by dilutingthe charcoal) (MultiplantCitrus ′ re experimental period. 32.35 (L.) Osbeckvar.Valenciagraftedon (L.) Raf.]) with three 15–18-cm (L.)Raf.])withthree15–18-cm anddefinitionofworking ′′ grown in20-Lpotscontaining Sand48°9 terature, andKovatsretention ges werecalculatedonthe 0.5%,0.75%,and1.0% polymer andsiliconeat ′ 50.82 D. citri ® ′′ , Terrado W) in a 8 nymphs toC 20 ) This article isprotected bycopyright. Allrights reserved. shoots weretransferredto acrylic photoperiod of14h,andrelative Acceptedmaintained on The insectswereobtained from a 2.3.1 Insects, plants,and testingconditions 2.3 Assessmentoftheefficacy OPAagainst treatments (score3)wereexcludedfrom theseexperiments. treatments thatresultedinmore than30% 1), orinducedmoderate toxicity shoot wasconsideredareplicate. Each treatment consistedofth Articleshoots. We usedarandomized blockexperimental plants withnecroticspotslarg toxicity), plantswith1–3-mmspotsontheleav (mild toxicity),plantswithupto1-mmnecrotic application (DAA)andscoredas Paulo, Brazil). (7.0 mL) theaidofaBrudden with adjuvant wereincluded(Table1).The youngshoots treatment. Inaddition, twocontroltreatments For efficacystudieson Phytotoxicity wasvisuallyassessedin Murraya paniculata ree nursery citrustreescont er than3mm ontheleavesa D. citri cages(20×2155cm) withan follows:score0(notoxicity),asymptomatic plants;score1 humidity of65±10%).To (score2)inupto D. citri ® (L.)ina climatized room S-600manually operatedsprayer(Brudden,Pompéia,São ,

doses thatcausednoormild phytotoxicity(scores0and rearing establishedatF consisting ofpurewaterandwith0.025% of plantswithscore spots(burning)ontheleaves;score2(moderate the youngshootsat1,7, es andbranches;sc designwith 14treatmen D. citri were sprayedtoapointjustbeforerunoff 30%oftheplants aining threeyoung shootseach;each nd/or completenecrosisofyoung obtain eggs,plantswithyoung anti-aphidscre (temperature of25±3°C, undecitrus. Therearingwas 2 andhighlyphytotoxic ore 3(hightoxicity), ts and9replications. and15daysafter were selected. The were selected.The en andexposed This article isprotected bycopyright. Allrights reserved. nymphs and8fortheexperiment withadu and eachseedlingrepresentedareplicate.Seven Acceptedthe testingofresidualcontact. outbreak, thenymphs developonthesame branch antennae. Fornymphs, theefficacy oftheOPAwas and adultswereconsidered deadwhenthey runoff. Thesame treatmentsasdescribedfortopical pervious tosprayingandthat 10 insectsatdaysafteremergence wereconf spray treatmentsandmaintained in aclimatized nymphs withtheaidofasoftpaintbrush.Thein OPA againstnymphs, eachseedling withone youngs ArticleCropScience AG,Dormagen,Germany)asapositive results (subsection2.2).We usedtr The OPAtreatments usedintheseexperiments 2.3.2 AssessmentoftheefficacyOPAon psyllids. temperature, photoperiod, andrelativehumidity conditions asdescribedfortherearingof in screenednurseries,wereus adults. To evaluatethe efficacyofthe OPA,seedlings of to adultsfor 7 days.Theplantscontaining eggs werekeptinthecagesuntil emergenceof The number ofdeadinsects(nymphs ora coveredthewholeshoot.Theshootsweresprayeduntilproduct ed. Alltestswereconductedin For bothtests,acompletely ra eatment withimid D. citri didnotpresent mobility of legs,wings,and ined oneachshootbyusingsleevecagesthatare room. Totest the efficacy ofOPAagainstadults, fested plantsweresubjectedtothevariousOPA dults) was counted at 1, 3, and 7 DAA. Nymphs dults) wascountedat1,3,and7DAA.Nymphs lts. Additionally,a3 × 3factorialdesign were selectedbasedonthephytotoxicitytest untiltheemergence of applicationonnymphs wereused(Table1). plants were usedfor theexperiment with nymphsandadultsbytopicalapplication tested onlyfortopical applicationasafter control (Table1).Totesttheefficacy of hoot wasinfestedwith10 third-instar C. sinensis acloprid (Provado ndomized blockdesignwasused the laboratoryundersame var. Caipira,grownintubes adults, not justifying ® 200 SC,Bayer Tukey’s test( variance (ANOVA) withrepeatedmeasures overtim analysis toreduceheteroscedasticity andachieve This article isprotected bycopyright. Allrights reserved. 3.1 Chemicalconstituents ofessentialoils Accepted3 RESULTS ± standard errorofthe mean (SE).Theda insects forallefficacytestswasexpressed asape from thescoresattributedtodamage in The resultsoftheassessment ofthephytotoxice 2.3 Dataanalysisandstatistics Articleand experimentaldesignweresimila application test(mortality described above. Thetreatments used inthis test residue (2hafterspraying),usingthesame ins Ten adultsat10 daysafteremergence were 2.3.3 AssessmentoftheefficacyOPAonadults insect mortality. there wasan influence of increasing dillapiol/O (concentrations ofdillapiol×dilutionsOPAus

P

≤ 0.05).Allanalyseswereperforme ≥ 80%)(Table1)asdescribedinsubsection2.3.2.Theassessments r tothoseofthetopicaltests.

all youngshootspertreatment. Thenumberofdead ta weretransformed intoarcsine(x/100) placed onthe shoot ofeachseedling onthe dry ect confinement method andsprayapplication as normality. Meansweresubjectedtoanalysisof PA andOPA/dillapiol concentrationratioson were those classified as effectivein thetopical ffect wereexpressedaspercentagescalculated rcentage. All datawereexpressed as themean ed) wasusedforthe adultinsects toverify if d usingtheAgroEstatsoftware. e, andincaseofsignificance,compared by D. citri byresidualcontact 32

0.5 priorto This article isprotected bycopyright. Allrights reserved. and 1.0%causedmild toxicityto22.22% Accepted79.9 at0.5%and1.0%wasnontoxicfor66.6%a effect in100%oftheplants.OPA-69.3at 79.9 at0.75%dilution,andOPA- DAA) remained constantduringtheentireexperimental period. The proportionofplantswithth 3.2 PhytotoxicityofOPA against obtained fractions. 99.5 hereafter,forOPA1,2,3and4,respectively,c Article99.5%. ThedifferentOPAfrac OPAs obtainedbyfractionaldi apiol werepresent inallfourOPAs.Dillapiol compounds comprising myristicin, and 6components wereidentifiedinOPA01, compounds of theseoilswiththei fraction todetermine theaveragepercentage spectra oftheOPAscomponentsfrom adata experiments weredetermined byGC-MScompari The compositions ofthefourOPAsobtainedby OPA-69.3 at0.5%causedmild toxicityto Treatment of C. sinensis tions aretermed OPA-69.3,OP stillation andthepercentage plantswithOPA-69.3atd e same degreeoftoxicityobser r relative percentages are listed in Table 2. A total of 40, 39, 30 r relativepercentagesarelistedinTable2.Atotalof40,39,30 85.4 at0.5%,0.75%,and1.0%d C. sinensis z -isoelemicin, caryophylleneoxi and 33.33%oftheplants, respectively. 0.5% wasnontoxicto88.88%oftheplantsand OPA- wasthemost compound identifiedfrom abundant and definitionofworking concentrations OPA 02,03and04respectively.Six library. We 3samplesfrom injected eachOPA nd 33.33%oftheplants,respectively. ± SEforeachfraction ofoil.Characterized ng theirrelativeretentiontimes andthemass 11.11% oftheplantsandOPA-79.9 at0.5% onsidering thepercentage fractional distillation used inthepresent ilutions of0.75%and1.0%,OPA- s were69.3%,79.9%,85.4%,and A-79.9, OPA-85.4,anddillapiol- ved inthefirstassessment (1 ilutions causednophytotoxic de, globulol,dillapioland ofdillapiolinthe This article isprotected bycopyright. Allrights reserved. all time points(1DAA:F=44.10;df =11, average mortality obtained bythetreatments signif Acceptedshowed asignificantlyhighermortality at7 the positivecontrolimidaclopri citri DAA inalltreatments indicates in thefinalassessment (7DAA)(Table3). assessment (1DAA),between91.42and100%at mortality obtainedbythetrea The nymphs of contact of 3.3 AssessmentoftheefficacyOPAagainstnymphs andadults Articletests on plants andwastheonlytreatment thatcausedhi plants of plants. Thetwocontroltreatments, withorwi toxic to66.6%oftheplants,while0.75% plants. of theplants,respectively.Dillapiol-99.5at0.75% respectively, whilethatofd . Theaveragemortality didnotsignificantly OPA-79.9 at 0.5%and1.0%wasmoderatelyt OPA-79.9 at Only dillapiol-99.5washighly D. citri C. sinensis . D. citri . Becausedillapiol-99.5 presentedmoderate displayed high sensitivity to to sensitivity high displayed illapiol-99.5 at0.5% and 1.0%caused moderate toxicity to 33.66% tments variedbetween90.00%and98.57%onthefirstdayof d. Onlythelowestconcentra a knockdowneffectoftheOPA toxic toplants.The0.5and P The highmortality (90.00–

˂ DAA compared to1and3DAA.However, the DAA compared 0.0001;3DAA:F=35.80; df=11, thout adjuvant,weren dilution causedhightoxici 3DAA,andbetween97.14%100.0% gh toxicity,itwasexcludedfromtheefficacy differ among thetreatmentsdiffer among andcompared to icantly differedfromthecontroltreatment at causedmoderatetoxici all treatments containingOPA oxic to11.18and33.33%oftheplants, tion evaluated,OPA-69.3at0.5%, toxicity tomore than 35%ofthe for third-instarnymphsof 1.0% dilutionswerehighly ontoxic to 100% of the ontoxic to100%ofthe 98.57%) observedat1 ty to 33.33% of the ty to33.33%ofthe ty to66.66%ofthe D. citri . The average P bytopical

˂ 0.0001, D. This article isprotected bycopyright. Allrights reserved. (Table 4). 11; Acceptedbetween 62.5–96.26%;however,theysi and 1.0%did notsignificantlydiffer from imid controls (2.5%),butwerelesseffective they caused significantly highermortality(55% ˂ by imidacloprid, butweresignifican higher valuesofmortality(78.75–97.50%).Again,th 1.0% OPA-85.4dilutionsustainedtheefficacyobser not differfrom thecontrols(Table4).At 11; and thehigherOPAconcentrations(F=18.21;df Articlehigher thanthatinducedbythe 69.3 ataconcentrationof1.0%showedintermed did notdiffersignificantlyfromofthec that 18.21; df=11; those obtainedbyimidacloprid, butweresignificantly mortality (>72.5%)at1DAA,i 3). (Table was significantlyhigherthancausedbythecont F=35.87;df11, and 7DAA: 0.0001).TheefficaciesofOPA-69.3at1.0%and P

˂ At 7 DAA, OPA-69.3 at 1.0%,OPA-79.9 at At 7DAA,OPA-69.3at In adults,OPA-79.9 at0.75%and1.0%dilutionsOPA-81.6inducedthehighest 0.0001)(Table4).Theothertreatments di P

˂ 0.0001).Intheadultinsect P control (46.25%),butlowerthan ndicating aknockdowneffect.The ˂ tly differentfrom thoseofth 0.0001).Themortality inducedbythecontrolwithadjuvant than imidacloprid ( gnificantly differedfrom th 3 DAA,OPA-79.9at0.75%and ontrol containingadjuvant.Treatment withOPA- s, theeffectof thecont and57.50%,respectively) compared tothe acloprid, withtheaverage mortality ranging rol containingwateraloneatalltime points iate efficacy;themort OPA-85.4 at0.75%were intermediate, since d notsignificantlydifferfrom thecontrols 0.75%and1.0%,OPA-85.4at e effectsdidnotdifferfrom thoseobtained differentfromthoseofthecontrols(F= ved inthefirstassessment, displaying P 98.75%) (Table4).

˂ 0.0001).Theothertreatments did e controls(F=18.19;df11; thatinducedbyimidacloprid e controls(F =17.25;df effects didnotdifferfrom rol containingwateralone ality wassignificantly 1.0% dilutionsandthe P This article isprotected bycopyright. Allrights reserved. and 1.0%thanat0.5% dilu = 3.09;df2; Accepteddilutions of0.5%and1.0%,butnosignificantdi for eachoilextractdilution,weobservedthat diluted oilextracts (Table5).When comparing the citri 0.0001) dilutions.However,forthe0.5%dilutio mortality ofadultsforth dilutions used inthetreatments. Anincrease in 50%) (Table4). df =2; increased mortality,duringtheexperi Article85.4 treatments alsodisplayed asignificant increa intermediate effectincompar indicating occurrenceofaleth mortality (F =15.67;df2; at 0.75%significantlyincreasedov the othertime pointsofassessment. Incontra induced high mortality in 1.75; df=2; that themortality didnot significantlydiffe adultswasobserved(F=2.81;df2; Comparison of themortality caused atdiffe Next, wetestedtheinteractions betweenthe P = 0.004);however, theirefficacyremained lowinthefinalassessment (mortality < P P =0.1767)and1%OPA-85.4(F0.17;df2; =0.0509).OPA-79.9causedsignificantlyhi e 0.75%(F=11.05,df=2; D. citri tion (F =17.54;df2; ison tomore effectivetreatm P al actionafteralongerperiod,

˂ adults (>88.75%)at1DAA,not 0.0001andF=5.14;df2; er theexperimental periodas mental period(F=5.25;df2; P r betweenthetime pointsfor1%OPA-79.9(F= st, theeffectsofOPA-69.3at1.0%andOPA-79.9 =0.0659)withincreasing dillapiol content inthe OPA-69.3 causedsignificantly thedillapiolcontentwasreflectedinahigher fference wasnotedbetween0.5%and0.75%(F n, nosignificantdifferenceinmortality in se inefficacy,asindicatedbythesignificantly effects ofdifferent concentrations ofdillapiol concentration of dillapiolintheOPAand rent time points byeachtreatment revealed P

P ˂ 0.0001)and1%(F=9.88;df2;

˂ ents. The0.5%OPA-79.9andOPA- 0.0001).Finally,OPA-85.4 caused gher mortality at P which may beconsideredan =0.8443).Thesetreatments significantlydifferingfrom indicated bytheincreasing P P =0.0068),respectively, = 0.0061 and F = 8.30; =0.0061andF8.30; highermortalityat dilutions of0.75% P D. D. = C. sinensis 4 DISCUSSION remained low,evenat7DAA(averagemortality and F=9.14;df=2, mortality overtheassessment period(F=9.34;df2, This article isprotected bycopyright. Allrights reserved. insecticides. corrobates withother studiesshowing that Acceptedpromising resultsrevealeditsgreatpotentialfo imidacloprid, awidelyusedandeffec for thecontrolof purity (99.5%), thedevelopment ofnewformulati Article= 5; 1, 3,and7DAA(F=60.20;df5; of dillapiol in the OPAanddilution used,wassignificantly lowerthan thatof imidacloprid at The residualefficacyofallthetreatments againstadult 3.4 Assessmentoftheresidual 5). (Table significantly increasedmortalitywitheach P The treatments withtheOPAwerehighlyeff The phytotoxicityexperiment indicatedthat ˂ 0.0001,respectively).Thetreatments with , independentlyofthedilution 10,33 Thetoxicityofthe adjuvant (0.025 D. citri P = 0.0003 for 1, 3, and 7 DAA, respectively);however,theefficacy =0.0003for1,3,and7DAA, nymphs intopicalapplications,presentingsimilar efficacyas efficacy oftheOPAon P ˂ 0.0001,F=71.40;df5; tive activeingredientforcontrolof used. Thus,tofacilitate the decrease indilution (F=29.22; df=2; nymphs aregenerally sensitive tobotanical ≤ ons wouldberequiredtomitigate this problem. r themanagement ofthisinsectvectorand 30%)(Table 6). dillapiol at99.5%washighlytoxicagainst ective (mortality >90%assoon1DAA) P %) tonymphs observedinthisstudy OPA showedasignificantincreasein D. citri D. citri = 0.0002,F9.74;df2, , regardlessofth adults P ˂ 0.0001,andF=64.56;df use ofdillapiolatahigh D. citri e concentration P P

˂ = 0.0002, 0.0001) . These leaves by up to4-foldcompared tountreatedareas. This article isprotected bycopyright. Allrights reserved. ofaction, whichcanleadtoselec Acceptedinsecticide spraying and theuseofareducednu dillapiol. additive orevensynergistic insecticidal eff smaller molecules the essential.However,ac terpenoids, andaromatic andaliphaticconstituen normally hasgreaterstability fractional rectification,whicha reported aftertopicalapplicationat adults of the sucking insects Articleareas. 80% mortality andreducedthenumberofadults (mortality between 70–98%). OOtherstudiesshowed concentrations ofdillapiolinOPAatdiffere immersion of nymph-infested bran mortality canbeattributedtotheapplicati authors useda5-foldlowercon (approximately 50%mortality), corroborated theresultsof Srinivasan etal. 12,13 It isimportant toemphasize thatinour Regarding theefficacyoftopicalapplicati 18,22,36 Similarly, a1%dilutionof Because 35 such asapiol andmyristicinthat occur inminor quantities and canexert D. citri Aetalion tivity ofthemajorcomponents might bemodulated byother centration thantheonetested than botanicalextracts.The llows more accuratequalitative tion ofpsyllidpopulationresistan isaninsectvector,itsmana ches inthead adoseof3%and8%,respectively. sp. and D. alba on method usedbytheseau ects ontheknown insecticidalcompounds suchas E. herus nt dilutionswereeff extract reducedthenumberof juvant solution. ts) usually determine th mber ofactiveingredientswithdifferentmodes study, weusedtheessentialoilobtainedby on leavesupto4-fold compared tountreated on against adults, weobservedthatvarious 13 of80%and100%,re Efficaciesof that neemextractat1%dilution caused in ourstudy(0.005%),thehigher major compounds(terpenesand gement requiresfrequentfoliar and quantitative profilingand t tothe insecticidescommonly ective forcontrolof P. aduncum 24,33 e biologicalpropertiesof

thors, whichinvolved spectively, havebeen D. citri 34 extractagainst Althoughthe adults on D. citri

cyhalothrin, andprofenophos.Tomar etal. resistance of reported thatdihydrodillapiol This article isprotected bycopyright. Allrights reserved. required toprovethishypothesis. The present st potentially inhibittheactivity of Accepteddillapiol might helpdecrease resistance in folds whencompared topyrethrum aloneagainst synthesized bychemical transfor factor (LC synergistic activity towards pyrethrum against including pyrethrum. .Mukherjeeetal. more effectiveforthecontrolof combination withpyrethrum extractpurifiedfrom Articlebotanical insecticides inagricultural pest control. susceptibility of thisinsect toinsectic insecticides in glutathione toxic toinsects. against the detoxifyingenzymes responsiblefor to controlthisinsectvectorisaconstantneed. used fortheir control. Studies havedemonstrated thatdillapiolac The OPAismainly composedof dillapiol 50 S -transferase, andcytochrome P450enzymes responsibleforthedetoxificationof are for pyrethrum/LC Spodoptera litura D. citri 21,22 Previousstudieshavedemonstrated 37,38 nymphs and adults Therefore,searchingfor newactive 50 mation (1:5)showedasynergism combined withpyrethroids pyrethrum plussynergist)of2.3–4.0.Shankarganeshetal. Leptinotarsa decemlineata detoxifyingenzymes ininsects. (F.)thatis currentlyresi ides frequentlyusedfortheircontrol. 44 showed thatacylderivatives D. citri 46 observed that amixture of pyrethrum anddillapiol , Tribolium castaneum and theseenzymes areassociated withlower udy clearlydemonstrated thehighefficacy ofthe the elimination ofplant metabolites potentially , 14,19,20 populations, Chrysanthemum cinerariifolium 42,43 T. castaneum. ts asapotent synerg Liu etal. whichhasapotentialinhibitory activity (Say) larvae resistant toinsecticides that elevatedlevelsofesterases,

because thisactiveingredient can caused significantreductionin ingredients tobe used inrotation stant tocypermethrin, lambda However,furtherstudiesare factor varyingfrom 2.0to5.0 43 Thus, essentialoilsrichin observed that dillapiolin (Herbst.) withasynergism ofdihydrodillapiolhavea 39–41 ist ofsyntheticand

was9.1-fold 45

This article isprotected bycopyright. Allrights reserved. Accepted REFERENCES 442926/2014-6). support forthisresearchintheform ofai aduncum for technicalsupportandallow Microorganismos /Department ofChemistry from ArticleRodrigues FilhofromLaBioMMi–Labor project bymeans ofresources first author, theEmpresa Brasileirade PesquisaAg ACKNOWLEDGMENTS citri of thisstudy willcontribute to used incitriculture, itco OPA inthecontrol of 2. . 1.

We aregratefultotheFundodeDefesada Teixeira DC,AyresAJ, Kitajima EW, Tanaka Saillard CandBovéJM, Firs (" MA, Amaral AMandMullerGW, Firstre Coletta-Filho HD,Targon MLPN,Takita MA,

and theNationalCouncilfor Scientificand Candidatus

Liberibacterasiaticus")inBrazil. D. citri uld beusedinrotationfo the futureadoption ofdillapiol-rich oils as acontrol strategyof . Asthemode ofactionisunlik obtained from theNational Treasury, theprof. Dr.Edson the useofGC-MS fortheanal t reportofahuanglongbing-like di d toresearch(MCTI/ atório deBioquímica Micromolecularde r effectivemanagement of ropecuária (Embrapa) forfinancingpartofthe FederalUniversity of Citricultura (Fundecitrus) foragranttothe port ofthecausalagentHuanglongbing Technological Developm De NegriJD,Pompeu JuniorJ,Machado Dis FAO,DanetJL,Jagoueix-Eveillard S, e that of insecticidescommonly

88 yses ofessentialoils :1382 (2004). CNPq/Universal, proc. SãoCarlos–UFSCar sease ofcitrusinSao D. citri. ent (CNPq)forthe The results The results D. P. This article isprotected bycopyright. Allrights reserved. Accepted Article 3. 9. 8. 7. 6. 5. 4.

Paulo State,Brazil,andassociati Bové JMand AyresAJ, Etiology of threerecent Liberibacter americanus, withthedisease. Firmino AC,NegrisoliE,SouzaES, Pr greening noBrasil,in Sudden death,variegatedchlorosisandhuanglongbing. populations ofAsiancitrus Tiwari S,MannRS,RogersME andStelin Huanglongbing diseaseincitrus. Boina DRandBloomquistJR,Chemical cont Botucatu, pp.143-163(2011). Miranda MP,NoronhaJr.NCandMarquesRN, Technology Controle dohuanglongbingnoestadodeSãoPaulo,Brasil. Belasque Jr.J,Yamamoto PT,MirandaMP,BassaneziRB,AyresAJandBovéJM, Huanglongbing –GreeningInternationalWo Psyllidae): vectorofthebacteriu APR, SousaMC,AbrahãoDPandBrazJD, Yamamoto PT,FelippeMR,Garbim LF,CoelhoJH,XimenesNL,MartinsEC,Leite greening diseaseofcitrusinIndia. Capoor SP,RaoDGandViswanathSM, (2007). , 31: 53-64 (2010). Avanços em Fitossanidade psyllidinFlorida. Pest ManageSci Ind AgricSci on ofanewliberibacter , m CandidatusLiberibacteramericanus. ado EPandMarubaya Diaphorina citri Plant Dis rkshop, RibeirãoPreto,p.96(2006). rkshop, Diaphorina citri(Kuwayama) (Hemiptera: Pest ManageSci

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This article isprotected bycopyright. Allrights reserved. Accepted Article 14. 15. 13. 12. 11. 17. 16. 10.

CN, Constituintesof Maia JGS,ZoghbiMGS, AndradeEHA,Sa Region. orchards. bark of Zapata NandSmaggheG, Repellencyandtoxicity insecticides, andpredators tocontrol Khan AA,AfzalM,QureshiJA,AMandRazaAM,Botanicals,selective 44: citrus psyllid Khan I,ZahidMandGZ,Toxicityof psylla ( Rao CNandShivankarVJ,Relative efficacyof Crops Prod Entomol Majaliwa JGM, Ogendo JO,DengAL, KostyukovskyM,RavidU,MatasyohJC,Omolo, EO and 4: Illicium simonsii SL,JiangGHandLiu ZL,Compos Chu SS,Liu mortality, oviposition,anddevelopment of Weathersbee AAWandMckenzieCL,Effect 205-210 (2010). 197-201 (2012). Diaphorina citri Flavour andFragranceJ Laurelia sempervirens 88: Insect Sci

32: 401-407 (2005). Diaphorina citri 405-410 (2010). Fumigant toxicityoffive essential Maxim (Illiciaceae) fruit against themaize weevils. 21: the essentialoilof 717-726 (2014). ). Ind JAgriSci Kuwayama and and

13: Drimys winteri Diaphorina citri 269-272 (1998). 81 :673-676 (2011). Piper aduncum Diaphorina citri botanic andsyntheticpe ntos AS, Silva ML, Luz AIR and Bastos ntos AS,SilvaML,LuzAIRandBastos Chysoperla carnea of a neem biopesticideonrepellency, ofaneem certain bio-rational inse oil constituentsagainstmajor stored- ofessentialoilsfromtheleavesand ition andtoxicityofessentialoil against (Hemiptera: Liviidae) incitrus L.growingintheAmazon (Homoptera: Psyllidae). Tribolium castaneum Stephens). sticide residues to cticides tocitrus Rec NatProd Pak JZool . Fla Fla Ind Ind

This article isprotected bycopyright. Allrights reserved. Accepted Article 19. 23. 22. 21. 20. 18.

Guerrini A,SacchettiG, RossiD,Paganett to syntheticandnaturalxenobiotics. european corn borer. other secondary metabolites oftheAstera Pharmacol Ruiz &Pavon()essential oilsfrom EasternEcuador. Maldonado MEandBruniR,Bioactivitiesof nubilalis mixtures of on" Buildingcapacityforfoodsecurity product insectpestsoffoodgrains. Li X,SchulerMAandBerenbaum MR,Molecu Piperaceae from from theNeotropics. P, HasbunC,PovedaL,SanRomán Land Bernard CB, Krishnamurty IHG,ChauretD, Bernard CB, ArnasonJT,Philogè Brazil. essential oilconstituents Potzernheim MCL,BizzoHR,SilvaJPand Bernard CB,ArnasonJT,PhilogeneBJR, Biochem SystEcol . Entomol ExpAppl

27:

allelochemicals onthe lifecycle oftheeuropean corn 39-48 (2009). Phytochemistry offourpopulations 42:

57: 25-31 (2012). 17-22 (1990). ne BJR,LamJandWaddell T,Effectoflignansand Proc. 2 Annu RevEntomol

28: J ChemEcol inAfrica",Entebbe,pp.325-332(2010). 1373-1377 (1989). nd ceae onthemono-oxygenase activity ofthe o G,MuzzoliM,AndreottiE,Tognolini RUFORUM Biennial Regional Conference RUFORUMBiennial Lam JandWaddelT,In-vivoeffectof Durst T,PhilogèneBJR,Sánchez-Vindas Vieira RF,Chemical Piper aduncum Piper aduncum lar mechanisms of metabolicresistance Arnason JT,Insecticidaldefensesof 21: 801-814 (1995).

52: 231-253 (2007). L. from Distrito Federal, Federal, L.from Distrito L. and characterization of Environ Toxicol

Piper obliquum borer, Ostrinia This article isprotected bycopyright. Allrights reserved. Accepted Article 27. 26. 28. 29. 25. 24.

Chrysomelidae). 2015]. óleos essenciaisde Fazolin M,EstrelaJLV,CataniV,AlecioMR aduncum Fazolin M,EstrelaJLV,CataniV,Lima MSandAlecioMR,Toxidadedoóleode http://www.sbpcnet.org.br/livro/61ra/ Manaus,AM(2009). Reunião AnualdaSBPC,UFAM, essenciais de Estrela JLV, Fazolin M, CataniV,Aléci nocturnum Agropecu Bras Agrotecnol pimenta longa(Piperhispidinervum). Pimentel FA,PereiraJBM andOliveiraMN, Acre (1998). importância econômica noAmazonas. aduncum Aleurocanthus woglumiAshby(Aleyrodi extratos aquososdefolhasPiperadunc Castro RS,PenaMR,SilvaNM,Vendramim JDandCostaIB, Silva WC,RibeiroJD,SouzaHEMand L.(Piperaceae)sobre

31: L. aadultosde (Barb. Rodr.)Bur.&K.Shum Piper aduncum 113-120 (2007).

41: Neotrop Entomol 217-222 (2006). Piper hispidinervum e Piper hispidinervum Piper

Cerotoma tingomarianus 34: Aetalion resumos/resumos/4179.htm [accessed04April Comunicado Técnico,97, 485-489. (2005). Acta Amazonica dae) emcondiçõesdelaboratório. o MRandLimaMS,Toxicidadedeóleos sp.(Hemiptera: Aetalionidae),pragade C. DC.; Corrêa RS,Atividadeinseticida de

Processo deextração andLimaMS,Propriedadeinseticidados sobre sobre um L.sobreamosca-negra-dos-citros, Tenebrio molitor Piper aduncum

em 37: 293-298 (2007). Sitophilus zeamais Bechyné (Coleoptera: Embrapa, RioBranco, Atividade ovicida de Atividade ovicidade óleoessencialde L. L., 1758. e Tanaecium Proc 61 . Pesqui Ciênc Piper Piper th

This article isprotected bycopyright. Allrights reserved. Accepted Article 31. 30. 33. 32. 34. 36. 35.

Corporation, CarolStream,Illinois, USA(2007). sabor. Thomazini MandFrancoMRB,Metodologia chromatography/quadrupole massspectroscopy. Adams RP, (2000). Piper aduncum Piton LP,TurchenLMandButnariuAR,Natu Ensaios Agronômicos. Version1.1.0.711,Jaboticabal,SP(2014). Barbosa JCandMaldonadoJrW, AgroEstat 44: the management oftheAsiancitruspsyllid, Silwet L-77andkineticaloneincomb Srinivasan R,HoyMA,SinghRandRogers Flavour FragranceJ. Guimarães EF, – Areview. Fla Entomol Bizzo HR,LopesD,AbdalaR.,Pimentel FA Bakkali F,AverbeckS,DandIdaoma 1915-1920 (2014). Boletim daSociedadeBrasileiradeCiênciaeTecnologiaAlimentos Food ChemToxicol

91:

87-100 (2008). ( Sarisan from leavesof Piperaceae Identification of essential oils components by gas Identification ofessentialoilscomponentsbygas

16: 113-115 (2001). ) inthecontrolof

46: 446-475 (2008). Piper hispidinervum ination withimidacloprid and abamectin for , deSouzaJA, PereiraMV,BergterL,and Diaphorina Citri ME, Laboratoryandfieldevaluationsof para análisedosconstituintesvoláteisdo - Sistema para Análises Estatísticas de stink bugbrownsoybean. r M,Biologicaleffect ral insecticide based-leavesextractof

4 th Ed. AlluredPublishing (Hemiptera: Psyllidae). C. DC (). C. DC(Long s ofessentialoils Ciênc Rural

34: 52-59

This article isprotected bycopyright. Allrights reserved. Accepted Article 37. 38. 40. 39. 43. 42. 41.

Grafton-Cardwell EE,Stelinski LLand Stansly Citrus Psyllid,VectoroftheHuanglongbingPathogens. IRAC. Comitê BrasileirodeAção (2013). online.org/documents/resistencia-de-psilideo-dos-citros/?ext=pdf [Acessed03122014] resistência depsilídeocitros a inseticidas 107 Entomol Soc Am Tiwari S,Pelz-Stelinski K,MannRand insecticides. infection onsusceptibilityofasiancitruspsyllid, Tiwari S,Pelz-Stelinski KandStelinski LL,Effect of pyrethrum synergistforcontrol Liberibacter asiaticus-infected anduninfected psyllids. five CYP4gensfrom Asiancitrus psyllidand theirexpression levels in Asiaticus-Infected anduninfected Asian cytocrome P450(GeneralOxidadase)activitylevelsin Liu SQ,ScottIM,Pelletier Y,Kramp K,Durs synergists derivedfrom dillapiole. Tomar SS,SaxenaVS,MaheshwariML, (2011d). Tiwari S,Gondhalekar AD,MannRS,Scharf :797-805 (2014). Pest ManageSci , 104 :297-375 (2011c). , 67: 94-99 (2011b). Indian J.Entomol of theColoradoPotato Beetle. aResistênciaInseticidas2014. Sarup PandMukherjeeSK, Newcarbaryl citrus psyllid(Hemipera: Psyllidae). Stelinski LL,Gluathione transferaseand t T,Sims SRandArnasonJT,Dillapiol: A ME andStelinskiLL,Characterization of

PA, BiologyandManagement ofAsian , 40: Candidatus Diaphorina citri 113-116 (1978). Annu RevEntomol Insect Mol Biol (2014) http://www.irac- Candidatus Liberibacter asiaticus J EconEntomol , toselected Liberibacter , , 20: Manejo de Candidatus Candidatus 58: 733-744 413-32 Ann Ann , This article isprotected bycopyright. Allrights reserved. Accepted Article 46. 45. 44.

dillapiole basedpyrethrumsynergists. Tomar SS, MaheshwariMLandMukerjeeSK Shankarganesh S,Dhingra K,Walia population of dihydrodillapiole onpyrethroidre dihydrodillapiole andfurapiole. Mukerjee SK, Walia S,SaxenaVSandTo Spodoptera litura (Fabricius). Agric BiolChem sistance associatedesterase J AgricFood Chem Subrahmanyam BandBa Pesticide BiochPhysiol mar SS,New pyrethrum synergistsfrom , Synthesisandsynergisticactivityof , 46 : 1277-1283(1982). , 27 : 547-550(1979). inhibitioninanIndian , 102 bu SR,Effectof : 86-90(2012). This article isprotected bycopyright. Allrights reserved. Accepted Article sprayed onsweetorange( Table 1. 3 2 1 Residual application onadultsof Topical applicationonnymphsadults of and Phytotoxicity against Treatments, dilutions,andpercentageofactiv 3. OPA69.3%dillapiol 9. OPA85.4%dillapiol 6. OPA79.9%dillapiol 2. Control (Water +Adjuvant) 1. 10. OPA85.4%dillapiol 8. OPA79.9%dillapiol 7. OPA79.9%dillapiol 5. OPA69.3%dillapiol 4. OPA69.3%dillapiol 15. Imidacloprid 14. Dillapiol 99.5% 13. Dillapiol 99.5% 12. Dillapiol 99.5% 11. OPA85.4%dillapiol

Control (Water) C. sinensis. ramns Dilutionv Treatments C. sinensis (2,3) (1,2,3) 0.004 20.0 (1) (1) (1) D. citri. D. citri. 1.00 0.75 0.5 1.00 0.75 0.5 ** ** (1,2) (1,2,3) (1,2) (1.2) (1,2,3) (1,2) (1,2) (1,2,3) (1,2) 0.5 0.75 0.408 0.5 0.5745 0.75 0.383 0.5 0.4890 0.3260 ) plants. ) plants. 1.00 0.766 1.00 0.6520 0.75 0.6120 D. citri. 1.0 0.8160 (1,2,3)

** ** e ingredientforeach -1 % a.i.L concentration ofmix -1 This article isprotected bycopyright. Allrights reserved. d c b a Table 2. aypyln xd 2.4 16 02 .1 .1±00 07 .4 tr 0.77±0.04 0.61±0.03 0.29±0.01 1565 21.549 caryophyllene oxide allo Expressed as area % mean number±SEfromGC-MSdata; RT =retention timeonthe Rxi-5MS (10mX0.10 mmIDX0.10 tr = Traces (<0.1 %). RI =retention indexasdetermine on an Rx

Accepted 1483 1.31 19.187bicyclogermacrene ± Article ( E ac-,-in 1.8 16 01 .1 r tr tr 0.10±0.01 1463 18.583 dauca-5,8-diene rmdnrn 1.4 12 02 .0 .0±00 tr 0.10±0.00 0.24 ±0.00 1424 17.441 aromadendrene α β ( ( n emceeD 870 47 .9±00 08 .2 0.21±0.01 0.80±0.02 0.99±0.01 1467 18.720 germacrene D emceeB 085 50 .0±00 t tr tr 0.10±0.00 1540 20.835 germacrene B α ylstvn 1.9 15 01 .1 .2±00 tr 0.12±0.00 0.17 ± 0.01 1354 15.297 cyclosativene -aypyln 1.7 10 1.4±01 71 .8 4.85±0.06 7.14±0.08 10.44±0.12 1405 16.879 )-caryophyllene z δ β E α α Z pelnrn 464 9 09 .4 0.30±0.03 0.90±0.04 997 4.634 -phellandrene aoaedee 804 46 0.24±0. 1446 18.084 -aromadendrene hmcaee 955 46 .2±00 03 .1 0.13±0.02 0.34±0.01 0.72±0.01 1496 19.595 -himachalene α α pnaeae 978 51 .6±00 13 .1 2.01±0.10 1.34±0.01 0.96±0.03 1501 19.718 -pentadecane α ptueo 2.5 16 0.12± 0.01 1562 21.459 spathulenol β α iolmcn 133 57 .5±00 01 .1 .2±00 tr 0.12±0.01 0.13±0.01 0.15± 0.00 1557 21.333 -isoelemicin -longipinene 15.193 1351 tr epnln .8 03t tr terpinolene 6.886 1083 tr Compound RT Compound aopee 970 46 .4±00 01 .1 0.46±0.05 0.19±0.01 0.24±0.01 1496 19.770 -amorphene cruee 980 56 .6±00 01 .0 0.18±0.00 0.17±0.00 0.26± 0.00 1506 19.870 -curcumene β α β muoee 942 40 .4±00 02 .1 0.15±0.01 0.24±0.01 0.34±0.01 1490 19.412 -muurolene clcrn 2.3 12 01 .0 .2±00 0.30±0.01 0.12±0.02 0.15±0.00 1529 20.534 -calacorene camphene 3.385 931 tr yitcn 007 54 .8±00 21 .2 .6±00 0.17±0.02 2.06±0.04 2.13±0.02 2.68±0.01 1514 20.097 myristicin )- p )- gruee 660 37 .9±00 t tr tr 0.19±0.00 1397 16.620 -gurjunene hmln 1.7 13 14 .2 .3±00 0.68±0.00 0.93±0.04 1.40±0.02 1439 17.872 -humulene ioee .4 12 04 .2 0.17±0.00 0.48±0.02 1021 5.241 limonene cbbn 6171 8 t tr -cubebene 16.127 tr 1381 cbbn 1.0 14 01 .0 r tr tr 0.10±0.00 1342 14.907 -cubebene α snaee 708 41 .9±00 01 .0 0.10±0.01 0.14±0.00 0.19±0.01 1411 17.058 -santalene β myrcene 4.422 tr 986 tr lbll 182 55 .7±00 04 .1 .2±00 tr 99.5±0.03 85.4±0.21 0.72±0.04 79.9±0.10 0.45±0.01 69.3±0.40 0.47±0.01 1622 1575 23.128 21.822 dillapiol globulol eeee 623 33 .7±00 t 0.10±0.01 tr 0.27±0.00 1383 16.203 -elemene -copaene 17.196 1416 tr cpee 564 35 .8±00 07 .1 0.63±0.01 0.73±0.01 0.98±0.01 1365 15.634 -copaene -cymene 5.149-cymene tr 1018 tr β β pnn 397 6 19 .9 .1±00 tr 1.01±0.06 1.92±0.09 962 3.957 -pinene pnn 313 1 10 .7 .5±00 tr 0.55±0.04 1.08±0.07 917 3.123 -pinene po 2.7 17 01 .0 .8±00 02 .1 0.15±0.00 0.23±0.01 0.18±0.02 0.15±0.00 1675 24.570 apiol oiee .1 13 07 .2 0.21±0.01 0.79±0.02 1035 5.616 -ocimene oiee .7 14 18 .5 0.46±0.03 1.87±0.05 1045 5.878 -ocimene Relative area (% ±SE) Thecomposition oftheessentialoils a (i. RI (min.) b i-5MS columnusingthehomologousseriesof

OPA 1 OPA 2 OPA 3 OPA 4 OPA4 OPA3 OPA2 OPA 1 d .2 .6±00 0.28±0.01 0.86±0.01 0.02 1 .7±00 0.13±0.01 0.17±0.00 01 Piper aduncum μ m) column; m) column; tr −

. − − − − − − − − − −

c n -hydrocarbons (C − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − −

8 -C 20 ); );

This article isprotected bycopyright. Allrights reserved. in letter capital Means followedbysame aduncum. application of different concentrations of d Table 3.

AcceptedOPA 85.4% dillapiol OPA 85.4% dillapiol OPA 85.4% dillapiol OPA 79.9% dillapiol OPA 79.9% dillapiol OPA 79.9% dillapiol OPA 69.3% dillapiol ArticleOPA 69.3% dillapiol OPA 69.3% dillapiol Control (water) Treatments Control (adj) Imidacloprid

Average mortality (±SEM)ofthirdinstarnymphs of Dilution v .2 73.1±1.3b 45.71 35.71±10.43Bb 7 0.025 .0 7100 .0a 0.0±00A 100.00±1.42Aa 100.00±0.00Aa 100.00±0.00Aa 7 0.004 .0 85 .2a 0.0±00A 100.00±0.00Aa 91.42±8.57Aa 100.00±0.00Aa 98.57±1.42Aa 91.42±8.57Aa 7 91.42±8.57Aa 97.14±2.85Aa 7 1.00 97.14±2.85Aa 100.00±0.00Aa 0.75 97.14±2.85Aa 100.00±0.00Aa 7 98.57±1.42Aa 98.57±0.00Aa 7 1.00 97.14±1.84Aa 100.00±0.00Aa 0.75 95.71±2.97Aa 100.00± 0.00Aa 7 100.00±0.00Aa 7 1.00 0.75 . 79.4±28A 9.4±28A 97.14±2.85Aa 97.14±2.85Aa 97.14±2.85Aa 7 97.14±2.85Aa 0.5 97.14±2.85Aa 94.28±5.71Aa 7 97.14±1.84Aa 0.5 91.42±5.53Ab 90.00±5.34Ab 7 0.5 * .2±14C 1.0±53C 10.00±5.34Ca 10.00±5.34Ca 1.42±1.42Cb 7 ** -1 the column and lowercase in the rowdonot lowercaseinthe and the column n A 3DA 7DAA 3DAA 1 DAA illapiol and dilutionsof the essentialoilof differTukey’s asindicatedby test( Mortality (%) 08B 47.14±10.62Ba ± 10.87Ba Diaphorina citri P

≤ 0.05).

in topical Piper This article isprotected bycopyright. Allrights reserved. in letter capital Means followedbysame different concentrations of dillapiol anddilutions of theessential oilof Table 4. OPA 85.4% dillapiol OPA 85.4% dillapiol OPA 85.4% dillapiol OPA 79.9% dillapiol OPA 79.9% dillapiol OPA 79.9% dillapiol OPA 69.3% dillapiol Accepted ArticleOPA 69.3% dillapiol OPA 69.3% dillapiol Control (water) Treatments Control (adj) Imidacloprid Average mortality (±SEM)ofadults Concentration v 0.004 8 0.025 8 1.00 8 0.75 8 1.00 8 0.75 8 1.00 8 0.75 8 0.5 8 0.5 8 0.5 8 ** 8 -1 the column and lowercase in the rowdonot lowercaseinthe and the column n 62 08 Cb 57.50± 36.25 ±10.84 BCDb 37 29 Ca 36.25± 33.75 ±12.94 BCDa 25 17 B 7.5±1.2ACb 86.25 ± 9.80 Aa 78.75±10.92 ABCab 72.50 ±11.76 ABb 62 49 C 5.0±1.6Cb 70.00±10.00 ABa 55.00±14.26 CDb 46.25 ±14.99 BCb 00 .5Cb 50 .6Da 33.75±9.43 BCDa 25.00±9.06 DEab 20.00 ±9.25 CDb 62 .4Ca 75 .3Da 21.25±7.42 Da 17.50±7.73 DEa 16.25 ±7.54 CDa 62 .5A 9.0±25 B 98.75±1.25 Aa 97.50±2.50 ABa 96.25 ±3.75 Aa 96.25±2.63 Aa 95.00±3.77 ABCa 88.75 ±6.39 Aa 50 .7A 9.5±12 a 100.00±0.00 Aa 98.75±1.25 Aa 95.00 ±3.77 Aa .0±37 b 37 .5Eb 22.50±8.60 CDa 13.75±7.05 Eab 5.00 ±3.77 Db .5±12 a .0±16 a 6.25±2.63 Da 2.50±1.63 Ea 1.25 ± Da .0±26 a 12 .9E 13.75±4.60 Da 11.25±4.79 Ea 5.00 ±2.67 Da A 3DA 7DAA 3DAA 1 DAA Diaphorina citri differTukey’s asindicatedby test( Mortality (%) 35 Ca 62.50±13.59 13.59 BCDa 32 E 43.75±12.94 13.22 DEa in topicalapplicationof Piper aduncum. P

≤ 0.05). ABCa BCDa

This article isprotected bycopyright. Allrights reserved. AcceptedValues inthetablecorrespond tofinal time point(7 DAA). Article in letter capital Means followedbysame application. aduncum Table 5. Influence oftheconcentra on thepercentage of mortality (± SEM)of ilpo % N Dillapiol (%) 85.4% 79.9% 69.3% the column and lowercase in the rowdonot lowercaseinthe and the column 2.0±86 c 25 35 b 98.75±1.25 Aa 96.25±2.63 Aa 62.50±13.59 Ab 70.00±10.00 Ba 86.25±9.80 Aa 22.50 ±8.60 Ac 8 33.75±9.43 Ab 21.25±7.42 Bb 8 43.75±12.94 Aab 8 tion of dillapiol anddilutio 0.5 0.75 1.00 Essential oil (v v differTukey’s asindicatedby test( Diaphorina citri -1 n of the essential oilof ) adults in topical P

≤ 0.05). Piper This article isprotected bycopyright. Allrights reserved. in letter capital Means followedbysame different concentrations of dillapiol anddilutions of theessential oilof Table 6.

Control (water) Control (water) Accepted ArticleDillapiol 85.4% Dillapiol 79.9% Dillapiol 69.3% Control (adj) Imidacloprid Imidacloprid Treatments Treatments Average mortality (±SEM) of Concentration Concentration .0 88.0±52 b 87 .5A 100.00 ±0.00 Aa 98.75 ± 1.25 Aa 87.50 ± 5.26 Ab 8 0.004 .2 800 .0B 25 .3C 5.00±3.27 Ca 2.50± 1.63 Ca 0.00± 0.00 Ba 8 0.025 v .0 62 .5B 2.0±55 a 30.00 ±8.01 Ba 17.50 ±4.53 BCa 22.50 ± 5.59 Bab 16.25 ± 5.95 Bb 8 18.75 ±5.49 BCa 7.50± 4.11 BCb 3.75± 2.63 Bb 8 10.00 ± 4.62 BCb 1.00 ± 5.00 1.88 Bb 8 1.00 1.00 * 00 .2B 1.8±41 C 17.14 ±4.51 BCa 14.28 ± 4.16 BCa 10.00 ± 4.62 Ba 8 ** -1 the column and lowercase in the rowTukey’s donotdifferand lowercase bytest( inthe the columnasindicatedby n 1 DAA 3 DAA 7 DAA 7DAA 3DAA 1 DAA Diaphorina citri Mortality (%) adults after residu Piper aduncum. al applicationof P

≤ 0.05).