Thiamethoxam and Imidacloprid Drench Applications On

Home , Thiamethoxam

E-mail: [email protected] Brazil, 14807040,Phone:+55163301-7025. Departamento PesquisaeDesenvolvimento, Av.A *Correspondence to:MarceloPMiranda,Fundo de Entomologia eAcarologia,Av.Pádua b a Adhemar PereiradeBarros201,Araraquara,SP,Brazil,14807040 João RSLopes Marcelo PMiranda feeding andsettlingbehaviorof Thiamethoxam andimidaclopriddrenchapplicat Escola Superior de Agricultura“Luiz deQuei Fundo deDefesadaCitricultura,Fundecitrus,

This article isprotected bycopyright. Allrights reserved. Accepted Article

article as doi: 10.1002/ps. 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 b

a *, Pedro TYamamoto 4213 Diaphorina citri Dias11,Piracicaba,SP,Brazil,13418900 b roz” /UniversidadedeSãoPaulo,Departamento , RafaelBGarcia Departamento PesquisaeDesenvolvimento, Av.

(Hemiptera: Liviidae) and the Versionof Record. Please cite this dhemar Pereira deBarros201,Araraquara,SP, dhemar Pereira ions on sweet orange nurserytrees disrupt de DefesadaCitricultura,Fundecitrus, a , João Pedro Ancona Lopes , JoãoPedroAncona a ,

pest management. Keywords: neonicotinoids beforeplantingnurserytrees ingestion phase.Thesefindingreinforceth on feedingandsettli ThisstudyclearlydemonstratesCONCLUSIONS: th resulting insignificant dispersal of 35 and90daysafterap 90%) inthe durationof this activity compared to probing, mainly forEPGvariablesrelatedtophlo RESULTS: Thedrench applicatio settling behaviorafterprobingonci nursery trees,usingtheelectr neonicotinoids (thiamethoxam andimid The objectivesofthisstudywere:todetermine the vectorofphloem-limite cont BACKGROUND: Chemical Abstract

This article isprotected bycopyright. Allrights reserved. Accepted Article Asiancitruspsyllid; Huanglongbing;neoni ng behaviorof plication). Moreover, both insectic ical penetrationgraph(EPG) d bacteriaassociated rol isthemost usedmethod formanagement of ns ofneonicotinoidsoncitr psyllids from treated plants. trus nurserytreesthatreceived D. citri acloprid) ontheprobingbehaviorof as ausefulstrategyforHLBmanagement. on citrusnurserytrees, e recommendationof drenchapplicationof em sapingestion,with untreated plants, inall the influenceofsoil-drenchapplications withcitrusHuangl e interference ofsoil- cotinoids; electricalpenetration graph; ides havearepellenteffecton technique; andtomeasure the us nurserytreesdisruptthe theseneonicotinoidtreatments. mainly during the phloem mainly duringthephloem a significantreduction( ongbing (HLB)disease. assessment periods(15, applied neonicotinoids D. citri Diaphorina citri

oncitrus D. citri D. citri D. citri ≈ , ,

plant, longresidualactivity acetylcholine receptor (nAChR)agonists],broad-sp piercing-sucking insects. Thesuccess ofthischemical HLB-symptomatic trees and and trees HLB-symptomatic preventive measures suchas:plantinghealthyc curative measures availabletocontroltheHLB.Th producer, around38million treeswere is present in themajor citrus-growing regionsof young trees),yieldreduction serious anddevastatingdiseaseof neonicotinoids imidacloprid andthiamethoxam are longer psyllidcontrolperiodthanfoliarapplica citrus treesinordertoprotec and bearingcitrustrees,syst The currentrecommendation istospraycontact ar HLB-measures mainly when which areassociatedwithcitr the phloem-limited bacteria 1 INTRODUCTION

This article isprotected bycopyright. Allrights reserved. Accepted Article The groupoftheneonicotinoidsismain ch The chemical controlisthemost used The Asiancitruspsyllid, Candidatus (fruit drop)andnoresist D. citri and applicationversatility t thenewflushes.Theselasttw us Huanglongbing(HLB)disease. e appliedonaregionalscale. Diaphorina citri emic soil-drenchortrunkapplic citrus in theworld,dueto control. Liberibacterasiaticus(Las)and already eradicated inthelast and effectivemethod formanagement of 4 tion andwithlessimpact onbeneficialinsects. However,diseasesuppr itrus nurserytrees,inspectionanderadicationof Kuwayama (Hemiptera: Liviidae), is avector of the world,and inBrazil, thelargest orangejuice insecticides (foliara erefore, diseasemanagement isbasedsolelyon the most commonsystemic insecticidesused ectrum ofefficacy,systemic actioninthe classisduetoitsmodeofaction[nicotinic ant citrusvarieties. emical classofinsecticides forcontrolling (e.g. foliar,soilandtrunk). 5

o applicationmethods providea ation insecticid 1-3 severe treedecline(mainly in HLBisconsidered the most nineyears.Thereareno pplication) onnonbearing Ca ession willbeachieved 1 Currently, thisdisease . L.americanus (Lam), es onnonbearing D. citri 11,12 The 9,10 . 6-8

the development ofelectricalpe examined becausethe study offeedingbehavior behavior withpotentialimpacts onpathogentr control efficacybymeasuringinsect mortality. In (soil andclimate). Moststudie be relatedtotheratesofinsec citri applied asasoildrench,oncitrus greenhouse conditions,whileinthe mortality (>80%) wasobserved when behavior of increased importanceofdiseases associated with (acquisition andinoculat probing behaviorofinsectvect insecticides. characterize theprobing be seedlings as well. effective control (mortality sharpshooters,aphidsandleafmicontrol ofpsyllid, apply theseneonicotinoidinsecticid in citrus worldwide.InBrazil, oneof themost

This article isprotected bycopyright. Allrights reserved. Accepted Article populationfor6–11weeksafterapplication. Detailed studiesoffeedingbehaviorpier Psyllid feeding behavior studies gained greater emphasis inthelastyears, due to the 20-26 D. citri Thiskindofworkisimportant toelucid 14 wascharacterizedbythe description However,thispercentageofmort ion) ofphytopathogens. havior ofseveralhemipt ≥ ticides, differentcitrusvarieties 80%) upto90daysinthefield. s involvinginsectvect ors andconsequentlyhowthey netration graph(EPG)systems. grovesaround2yearsold,were es bydrenchonnurserytreesfe field itwasjustkeptfor 60 neonicotinoidinsecticideswere depends ontechniquesthataremore specialized. ansmission, buttheseeff ordinary practicesused 15-17 ner, and this kind of application canprovidean kindofapplication ner, andthis erans, mainly foraphids cing-sucking insectswere Candidatus secticides may alsoaffectvectorfeeding Thisvariation inth ors andinsecticidesarefocusedonthe ate howtheinsecticides interfere inthe ality wasmaintained for90daysin of fiveEPGwaveforms using theDC used andenvironmental conditions Liberibacter spp.Theprobing 18,19 affect thetransmission process 13 abletoreducesignificantly days. Theseinsecticideswhen w daysbeforeplantingforthe InVietnam, ahighpsyllid Thistechniquewasusedto appliedbydr e periodofcontrolmay by citrusgrowersisto , onplantstreatedwith ects havebeenrarely made possiblewith ench oncitrus D.

al controlled room at25±2°C,photoperiod of14:10 2.1 Insectsandplants ANDMETHODS 2 MATERIAL test plant. settling behaviorof first studythatinvestigatesthe different andconsequentlya plants like seedlings compared tolargerplants su treatments. Thisstudyisimportant becausethe di behavior of of drench applicationsofneonicotinoids(thiam planting inthefield. are commonlytreatedwiththeseinsecticides probing behaviorof seedlings disruptsthe observed thatsoil-drenchapplication technique, studiesweredonewithpy system. . 28 D. citri This article isprotected bycopyright. Allrights reserved. Accepted Article Regarding the effectofinsectic Adults of Therefore, theprimary objectiveofthisst 27 More recently, twonewwaveforms related on citrusnursery trees,using theEPGt D. citri D. citri D. citri D. citri afterprobingoncitrus probing behaviorof wererearedon , usingawidelyplanted sweetorangecanopy cultivar (´Valencia´) as havenotyetbeenstudiedoncommercial citrusnurserytrees,which ffect theprobingbehaviorof combined effects ofsoil-applie combined effects metrozine, imidacloprid andaldicarb. ides ontheprobingbehaviorof of imidacloprid onsourorange( Murraya paniculata D. citri ethoxam ontheprobingbehavior andimidacloprid) nursery treesthatreceivedthese neonicotinoid to avoidearlyinfectionbythepathogenafter echnique; andsecondlytomeasure thesettling ch as citrusnurserytreescouldbe completely . However,theeffectof udy wastodetermine theinfluenceofsoil- (L:D) and60±10%ofRH toobtainLas-free stribution insecticides ofsystemic insmall to pathway phaseweredescribedbyCen D. citri L. (Rutaceae) plants in aclimate- d neonicotinoidsonprobingand inadifferentway.Thisisthe D. citri neonicotinoidsonthe Citrus aurantium 29-31 Serikawa usingtheEPG et al L.) L.) . et 30

of 0.35 g active ingredient/plant;andthiamethoxamof 0.35gactive (Actara using Stylet+software(EPG Syst ADuocore Akron, OH). analogic EPGsignalwasconvertedto description wasmade byTjallingii. device, (ModelGIGA-8,EPGsystems, Wageningen, 2.3 ElectricalPenetration Graph(EPG)recording group ofuntreatedplantswa were keptin agreenhouse undersimilar lightandtemperature conditions. Foreach experiment, a the same approachusedbycitrusgrowersbefore substrate; bythetime of application, thesubstrate rate of0.25ga.i/plant.Theinsec sweet orange [ removed fromtherearing colonyandmaintained psyllids of similar agefor theassays.Adults on Swinglecitrumelo [ following insecticides: imidacloprid (Provado 2.2 Insecticideapplication all experiments. in plasticbags(4L)with

This article isprotected bycopyright. Allrights reserved. Accepted Article The monitoring oftheprobingandfeeding One-year oldsweetorangenurserytr Nursery treesfrom thesame lotwerepruned Citrus sinensis Citrus paradise ® Pinus desktopcomputer wasusedfor s includedasacontrol. (L.)Osbeck]. substrate(MultplantCitrus®;Holambra,SP,Brazil)andusedin ems, Wageningen,ems, TheNetherlands). ticides weredilutedin 19,32 Macf.× Therecordingswereperformed with100×gainandthe digital throughaDi-710A/Dcard(Dataq ees (70-80 cm tall),‘Valencia’( ® 200SC;Bayer,BelfordRoxo, RJ,Brazil)atarate Poncirus trifoliata with 10to15daysafter theiremergencewere planting. Afterinsecticideapplication,theplants fora24-hacclimation periodonseedlings of in thebagswasdamp butnotsaturated.Thisis and immediately treatedby drench withthe behavior wasperformedwithaDC-EPG The Netherlands),whoseoriginaldetailed 50ml ofwater/plant ® 250WG;Paulinia,SP,Brazil)ata EPG dataacquisitionandanalysis L. (Raf.)] rootstock, were kept L.(Raf.)]rootstock,werekept before drenching the before drenchingthe C. sinensis ® Instruments, ), grafted

Bonani replaced andrandomly arrangedinthecage. located in aFaradaycageperEP per periodofapplicationwas artificial lightprovided electrode was inserted inthewater inside thebottle. electrode was insertedinthes placed inplasticbottles(250ml theshootsimmersedinwaterto was madewith duetotheplantsize,shootsweredetached DAA, at 90DAAasecondflushoccurredandshootssim were used.Afterthat,theplantstr not fullyexpandedleaves;at35DAA,shootswith after application(DAA).At15DAA,theplants psyllid wasplacedonthe abaxial events per insect(NWEI),wave were separatedintonon-sequent recorded datausingtheEPG-Excel (probably xylem sapingestion) with phloem);

This article isprotected bycopyright. Allrights reserved. Accepted Article The recorded EPGdatawereanalyzed according tothefollowingwaveforms describedby The processofmonitoring wasperformed ina Adult females wereattachedtoth et al . 27 : E1 C (putativesalivation (salivary sheathsecretion and ot by sixfluorescentlights(240W). Thenu ubstrate closetothetrunkofth ) fullofwater(oneshootper 20 recordings. Twoplantsfrom eachtreatment wererandomly . RelevantEPG responsevariab form durationperinsect(WDI), ial: number ofprobesperinsect(NPI), number ofwaveform G recording. Aftereachrecording, the plants and insectswere surface of theleafandmonitored for6h,at15,35 and90days DataWorkbook developedbySarria in phloemsievetubes); ansplanted toplasticpots(20L)with e EPGdeviceasdescribedbyBonani avoidcavitationandthen,theywereimmediately presentedyoungshoots(15-20cm length)with 20-25 cminlengthwith from the plants,andasecondcutinthestem from the her styletpathwayactivities); ilar to 15 DAA were used.Inthecaseof90 ilar to15DAAwere climate-controlled room (25±2°C) with bottle). For15and35DAAtheplant e plant; and for 90 DAA the plant e plant;andfor90DAAtheplant E2 mber ofreplicatespertreatment (phloem sapingestion);and waveform durationper event les werecalculatedfrom the et al fully expandedleaves Pinus . 33 Thesevariables D substrateand (first contact et al . 27 Each G

15-20 cm ofbothtreated anduntreated length) marked withdifferentcolors(yel by plastic vial(5cmdiameter kept onsweetorangeseedlings Nurserycitrustreeswereprune insecticides 2.4 Settlingbehaviorof Bioestat 5.0software. variable, thedifferenceamong treatments analysis wasperformed usingthe were compared usingthe FisherLeastSignificantDifference(LSD)test(P pearson’s testandweresubjectedtoconventio variability. Aftertransformation thedatashow transformed andsquareroot-transformed, resp (yellow orpink,DayGlo,Cleveland,OH,US Prior totheexperiment, adult observation cagehadanuntreatedandatreated facilitate observation,exceptthecagetop,wh (100 ×100cm), withanaluminum structure greenhouse (20-30°C,photophaseof with thiamethoxam orimidacloprid asdescribedin sequential: time toeventperinsect. (WDE) andproportionofindividualsthatprodu

This article isprotected bycopyright. Allrights reserved. Accepted Article

D. citri 6 cmhigh)fora1-hstarvationpe psyllids ofmixed genderwere marked withfluorescentpowder for a24-hacclimation period.Marked afterprobingonnurserycitrustreestreatedwithsystemic low orpink)wereconf Statistica 7.1software 27,34,35 11 h and 50 ± 10% of RH), using observationchambers 11 hand50±10%ofRH),using d 30daysbeforetheexperime was compared byaChi-square3x2(P Thedataofwaveform dur A) usingthemethodology describedbyNakata ed anormal distribution accordingtoD’Agostino- plants usingasleevecage. After30min, enough nal analysis of variance (ANOVA). Themeansnal analysisofvariance(ANOVA). ectively, toimprove homogeneity andreduce ich wascoveredwithan tree witheitherthiamethoxam orimidacloprid. item 2.2.Thisstudy ced aspecificwaveform type(PPW); and and transparentwalls(PVCfilm) inorderto (StatSoft, Tulsa,OK,USA).ForthePPW ined onasinglebran riod; thengroupsof50insects nt andpartofthem treated ation andcountwerelog- psyllids wereplacedina was performed insidea ti-aphid screen.Each ch (newshootwith ˂ 0.05). Statistical ˂ 0.05) usingthe 37 and and

sleeve removal (0.5,1,2,3,4,5,6,7,8,24,27,32 number ofpsyllids settledwascounted,between cage (aswellasinsects thatremained onthe time for original plant;2)moved from theoriginalplan plant (treatedanduntreated)todetermine thepe waveform compared withuntreatedplants.At90DAA(Table3), ingestion ( between imidacloprid andthiamethoxam treatment phloem sievetubes)wassignificantlylowerontreat other styletpathwayactivities), (Table 1).However,at35DAA, waveform eventsperinsect (NWE 3.1 ElectricalPenetration Graph(EPG)recording 3 RESULTS software (StatSoft,Tulsa,OK,USA). homogeneity andreduce variability, insecticide treatment. Thepercentage data we chamber orceiling;4)died.Twelve walls

This article isprotected bycopyright. Allrights reserved. Accepted Article At 15DAAtherewerenon-signifi D. citri E2 C eventsonuntreated thanontreatedplants ) eventswas significantlyloweronthia individuals to settle onthe the mean number ofwaveform D I) betweentreatedand untreated (firstcontactwithphloem) and and thensubjected tot-test (P cant differencesinmean shoot andstartprobing(Bonani sleeve cage) ofeachplantwasremovedandthe replicates (observationch t tothe other plant; 3) re arcsin-square root 8:30 and9:00am. Atvariousperiods following rcentage of insectsthat:1)remained onthe s (Table2).Themean number ofphloem sap ed thanonuntreatedpl and 48h),assessments methoxam treatedplants andimidacloprid . D. citri In relation towaveform C ˂ producedsignificantlyfewer (salivarysheathsecretion and plants for allwaveform types 0.05), usingthe number ofprobes(NPI)and E1 landed ontheobservation transformed to improve (putative salivation in ambers) were used per ants, butdidnotdiffer were doneforeach et al et .,) Statistica 7.1 27 thesleeve G (xylem (xylem

and (Table 1and3),therewerenon-significantdi untreated plantsinallassessment periods( non-probing ( periods. ingestion), therewerenon-signi DAA thedurationof waveform DAA differences innon-probing( of performed activity(5-20%).Inallassessmen this assessment periodstherewerejustfew eventspe For waveform 90 and87%atDAAforthiamethoxam andimidacl than onuntreatedplants,withareductionof the mean durationofphloem sapingestion( imidacloprid treatments thanonuntreatedplants than onuntreatedplants.Themean durationofwaveform waveform waveform G This article isprotected bycopyright. Allrights reserved. Accepted Article E1 perinsectwaslower than10min, while D. citri , whereasat35DAA (Table2),waveform Considering themean waveform durationperinsect(WDI), Regarding themean waveform durationper C D D therewerenon-significant wassignificantlyshorter ontreatedthanuntreatedplants.Forwaveform , significantdifferenceswereobserved remained longerin Np G, ) activitiesontreatedplan thevariable WDI wasnotstatistically C wassignificantlyshorterontreatedth Np ) activitiesamong treatments at ficant differencesamong treatments foranyoftheevaluation Np differences among treatments at15and35DAA,but90 ontreatedthanuntreated 15, 35 and 90 DAA) (Tables 1-3). At 15 and 90 DAA 15, 35and90DAA)(Tables1-3).At15DAA 95 and 91% at 15 DAA, 86 and 81% at 35 DAA and 86and81%at35DAA 95 and91%at15DAA, on untreateditwas fferences amongthe treatments forwaveforms E2 ts withthiamethoxam a at35DAA.However,inallassessmentperiods, ) perinsectwassignificantly shorterontreated r insect (1-5)andlowproportionof psyllids that t periodsforbothinsecticidesthemean duration C durationswassignifi only at 90 DAA, in which the duration of inwhichtheduration of only at90DAA, oprid treatments, respectively(Tables1-3). event (WDE), therewerenosignificant compared, becauseinalltreatments and E1 an onuntreatedplants.Inrelationto wasshorteronthiamethoxam and 15 DAA; however, at 35 and 90 15 DAA;however,at35and90 longer than10min. D. citri plants (Tables 1-3). For plants(Tables1-3).For nd imidacloprid thanon cantly shorterontreated remained longer in E1, there C , D

they diedthroughouttheexperiment (Fig.1Dand2D). 1B and2B)ortotheobservation chamberwallsor psyllids on treatedplantswaspartially due to m the percentageofpsyllidsthat At thefinal assessment (48 hafter release) most since 0.5and1hafterreleasefo decreased rapidlywithti insecticides 3.2 Settlingbehaviorof observed among treatments inallperi events related tothephloem ( (Table 4).Likewise, forthesequen the phloem ( number ofevents available. percentage was93%.Forwaveform thiamethoxam- and imidacloprid-treated plants,re E2 plants witheitherthiamethoxam orimidacloprid duration ofphloem ingestion( were non-significantdifferences amongtreatments inallassessment periods.Incontrast,themean

This article isprotected bycopyright. Allrights reserved. Accepted Article in allassessment periods,66and The percentageof Regarding theproportion ofpsyllidsthatproducedwaveform D + E1 and me, withsignificantdifferencescomp E2 D. citri D. citri ), thetreatments didnotdiffer(P> D E2 stayed ontreatedplantswasnear r thiamethoxam andimidacloprid, respectively(Fig. 1Aand2A). , afterprobingonnurserycitrustreestreatedwithsystemic ) eventswassignificantlylongeronuntreatedthantreated E1 tial variables, thetime toperform thefirst probe and waveform settledonthiamethoxam- andimidacloprid-treated plants e ods ofassessments (Table5). G E2 , WDEwasnotstatisticallycompared duetothelow 50% wereabletoproducean ) fromthestartofrecording,nodifferenceswere (Tables1-3). Among thepsyllids that performed ovement of these insects totheother plant (Fig. psyllidsremained onuntreated plants,whereas spectively, whereasonth ceiling(Fig. 1Cand2C),butmainly because ared to untreated plants (P < 0.05) ared tountreatedplants(P<0.05) 0.05) inallperiodsofassessment ly zero.Thislowpercentageof C and waveforms relatedto andwaveforms E2 e untreatedplantsthe >10minutes on

disrupt theprobingand showed thatdrenchapplicationsofthiamethoxa phase represents the predominant activity of that occursafterphloemcont that occursinnon-vasculartissues ANDCONCLUSIONS 4 DISCUSSION experiment, whereasforuntreatedplants for insectsfrom thiamethoxam- andimidacloprid-tre imidacloprid, respectively. Thepercentage of mortality increased over time reaching 83and56% treated anduntreatedplants higher thanthatobservedforindi treated plants, however,thepercentage of psy first untreated plantsinonlyfouroftheeight to theobservationchamber wallsandceilingwas (Figure 1Band2B).Thepercentage that moved from untreatedtotreatedplants psyllids that moved from treatedto untreatedpl observed after6(imidacloprid) an untreated plantsandviceversawassimilar. Ho

This article isprotected bycopyright. Allrights reserved. Accepted Article The probingbehaviorofphloem-feedinginsect Regarding psyllidmortality, significantdiffe During the first assessments, thepercentage of psyllids thatmoved from treated to settling behaviorof act (activities related withsalivation andingestion). since thefirstandsecondassessment forthiamethoxam and viduals from untreatedplantsin (activitiespriortofirstphlo d 8h(thiamethoxam) ofreleas ofpsyllidsthatdispersedfr itwasaround20%(Fig.1Dand2D). D. citri. llids dispersed onthechamber wallsand ceilingwas assessments (Fig.1C).Inthe case ofimidacloprid- decreased, mainly in the case of thiamethoxam decreased, mainlyinthecaseofthiamethoxam m andimidacloprid onsweet orangenurserytrees D. citri ants increased, whilethe percentage of psyllids wever, significantdifferences(P<0.05)were significantly higher(P< rences (P<0.05)wereobservedbetween This wasespeciallytrue forEPGvariables ated plants,respectively,attheendof s canbedividedmainly inapathwayphase probingbehavior. em contact)andaphloem phase most assessments (Fig.2C). om thiamethoxam-treated plants e, becausethe 27 0.05) thanthatfrom Thepresentstudy 38,39 percentage of Thephloem

treatments. Unlikeourstudy,Serikawa proportion ofindividualsthat imidacloprid aswell. reach phloem vessels(waveforms This isevidenced bythe similarity inthetime necessary for initiation and some variablesrelated tostylet pathwayphase, regardless of theinsecticide used. were reported for thepotatopsyllid significant. Thereductionofphloem sapi seedlings treated withimidacloprid by drench, but were 0.25and0.35gper plant,respectively.Ther trees about70-80cm tallwereus imidacloprid wasappliedatarateof0.32gperpl treated plants.Inthatstudy,sourorangeseed related with pre-phloem level, andmostof the et al act asfeeding deterrents for treated compared tountreatedplants.Thus, This factcanexplain,in phloem andafterashortperiodofingestionmost ps insecticides. Thediscrimination of treated plants occurs mainly when reduction inthedurationofthisactivitywasob related to phloem sapingestion (waveform

This article isprotected bycopyright. Allrights reserved. Accepted Article . 30 observedareductioninthephlo In general,drenchapplications 40

part, thelongertime spent D. citri produced waveforms relatedtophloemwassimilar among the ed andtheratesofthiamethoxa D whensoil-appliedoncitrusnur , Bactericera cockerelli E1 oncitrusnursery treesseemed em ingestiondurationswhen et al and ngestion durationandlongerperiodsof these compounds (thiamethoxam andimidacloprid) . 30 E2 E2 observed more differences in the EPG variables observedmoredifferencesintheEPGvariables served on plantstreate lings withheightof20-30cm wereusedand ) ontreatedanduntreatedplants.Moreover,the ) and,inallassessment periods,asignificant psyllids werenotable ant, whereasinourstudy,sweetorangenursery by thisinsectinnon-probing( in that case, the reduction wasnot statistically efore, thedifferences yllids withdrawtheirst (Sulc)onpotatoplantstreatedwith D. citri m andimidacloprid applications sery trees.Likewise, Serikawa toperform thefirstprobeand D. citri nottointerfere withprobe D. citri d withtheseneonicotinoid to reachthephloem on observedbetweenthe feeds on sour orange feedsonsourorange ylets from theplant. starts feeding inthe Np ) activities on Np activities

interfere onLasandLam acquisitionandinoculationby imidacloprid havearepellenteffecton from thesettlingbehaviorexperiment showth understand theeffectsofinsecticidesonfeedi which canlimit itsmovement. Thus, additionalstudi affected theprobingbehaviorof that theinterferencefactorsonprobingbehaviorof throughout theexperiment. Therefore,thissettling significant increase inthepercentage ofpsyllids treated tountreated plants andvice versa wassimilar during the first assessments, with a confinement (sleeve) cage wasremoved. Moreover, the treatedplantwereobservedonehour(imi dispersal did notoccur immediately; significant diffe treated plants totheobservation chamber walls a used bySerikawa studies couldberelatedtothesi potato may causebotha feeding deterrent abandoned thetreatedplantsandth insecticides whentheystartedingesting inth behavior datasuggestthatmost neonicotinoids weremorerelatedtothephloem

This article isprotected bycopyright. Allrights reserved. Accepted Article The resultsofthispaperareimportant to One of thelimitations of EPGtechniqueisthe et al . 30 the insecticide concentration wa D. citri ze of theplants andrates used; on the smaller plants (seedlings) D. citri e othersdied.Likewise,soilappl inadifferentway. D. citri, and arepellenteffect onthe psyllid individuals wereabletodetect thepresence of the dacloprid) andtwohours(thiamethoxam) the after ingestion phase.Altogether,ourEPGandsettling e sieveelements, andafterthat, partofthem resulting insignificant dispersal of psyllids from nd ceiling orto the untreatedplant. However,the ng behaviorofpiecing-suc understand howneonicotinoidsinsecticidescan thatmoved from treatedtountreated plants behavior experiment pr at drenchapplications the percentage of psyllids that moved from es using freeinsects are important to further rences inthe percentage of psyllids that left D. citri fact thatthe insectisconnected toawire, s probablyhigher,whichmay have D. citri oncitrusnursery incitrus.Bonani ications ofimidacloprid in ovided furtherevidence of thiamethoxam or ofthiamethoxam or king insects.Thedata B. cockerelli trees treated with . et al 41

. 27

nursery treesisclosetozero. acquisition andsubsequent transmission of these b applications ofthiamethoxam orim died within48hoursfollowing insecticides couldreduce thepr drench onsmaller citrusplants(seedlings).Ther variables (number insect werereduced.Likewise,Serikawa duration ofthiswaveform thenumber perevent,exceptat35DAA,when anddurationof significant reductioninthenumber ofps associated withtheinoculationofLsoby whitefly andaphidsduringsaliv This waveform wascorrelatedwiththeinoculat week beforethepsyllids become infective. psyllids areabletoacquire bacteria, th trees with neonicotinoids isefficient toprevent the acquisition ofLas orLam. In casesome regardless of theinsecticide used. Therefore, our ingestion. on tomato well;however,inthis as casethethreshold toacquire Lsowas6.9min ofphloem sap E2 phloem (waveform observed thatLasacquisitionoccurredexclusivelywhen wasassociated with This article isprotected bycopyright. Allrights reserved. Accepted Article For 42 Inourstudy,most psyllids D. citri and durationof thewaveform E2 ) foraperiodof1honinfectedplants Candidatus obability ofLiberibacter inoc ation inthesieveelements. E1 E1 Liberibacter solanacearum (Lso)acquisition by idacloprid, itisreasona idacloprid, exposure toyoungshootofplan may beassociated withsaliv perinsect) for didnotperform awaveform ere isstillalatencyperiodof Lasin et al yllid adultsthat 43 B. cockerelli Consideringthatmost in . 30 ion ofcirculativeplant efore, theinterference in theseEPGvariables by foundasignificantreductioninthesetwoEPG results suggest thattreatment of citrusnursery acteria by psyllidsthat D. citri . 42 ble tospeculatethat Inourstudy,weneitherobserveda D. citri , witha6%efficiency.Thewaveform produced waveforms ulation incitrusplantsby whenimidacloprid was applied via 44,45 Waveform was able toingest from the ation inthesieveelements. E2 >10min ontreatedplants, sects inthepresentstudy viruses inthephloem by ts thatreceiveddrench landontreated citrus D. citri theprobabilityof E1 E1 wasrecently ofatleasta B. cockerelli B. cockerelli norinthe D. citri E1 per per . In 27

pathogens byotherhemipteran vectorshasalsobeenreported. efficacy ofsoil-appliedneonicotinoids inpreventi days. citrus treeshasgoodefficacy(mortality>80% and until90daysaftera acknowledge Dr.HaroldoVolpeforhiscont Desenvolvimento Científicoe Tecnológico Foundation, andthelastauth This researchwasfinanciallysupportedbyFund ACKNOWLEDGEMENTS spread inyoungcitrusorchards. can beappliedvia drench incitrus nursery tr in generalweresimilar tothoseobservedfor this isthefirstreportshowingeff neonicotinoids beforeplantingnurserytreesas and settlingbehaviorof pathosystem. neonicotinoids onvectorinoculatio Candidatus potato, theuseofimidacloprid appliedtoth

This article isprotected bycopyright. Allrights reserved. Accepted Article 9,13 This studyclearlydemonstrates theinterferen Therefore, our results reinforce the Liberibacterpsyllaurousby D. citri pplication. InSãoPauloState,Brazil on citrus nursery trees, mainly or receivedafellowship n shouldbeinvestigatedforthe ects ofthiamethoxam onprobingbehaviorof B. cockerelli ribution inthesta ees aiming atareductionin theprobability of HLB imidacloprid. Thus,bothne a usefulstrategyforHL " (CNPq)/Brasil(Proc. 309883/2011-3).We ) onpsyllidcontrolforaperiodofaround100 ng orreducing inoculation e soilsignificantly reducedtransmission of ecitrus andCitrusRes recommendation ofdrenchapplication ce ofsoil-appliedneoni whencompared tountreatedplants. tistical analysis. , thiskindofapp during thephloem ingestionphase from "Conselho Nacional de 46-48 Thus,possibleeffectsof D. citri D. B management. Moreover, earch andDevelopment onicotinoid insecticides of vascular-restricted cotinoids onfeeding -Liberibacter-citrus lication onnursery D. citri , which 41 The The

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96 :1334-1342 (2013). Entomol ExpAppl

70 143 ord P, Beattie GAC,Spooner-HartRN ord P,Beattie :836–840 (2013). DOI:10.1002/ps.4041(2015). l penetrationgraphsof :13–22 (2012).

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This article isprotected bycopyright. Allrights reserved. Accepted Article Aphis gossypii Entomol ExpAppl . Appl Entomol Zool Entomol ExpAppl : behaviouralanalysisandchemi Ann EntomolSocAm , ed.byMinksAKandHarrewijn

rseick MRandSerranoMS, 85 rsion 5.0,Belém,PA(2007). :33–44, (1997). 67 allingii WF, Analysisofresist

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100 cel Workbook forautomaticparameter calculation :296–310 (2007). y monitored andvisuallyobservedprobing uorescent powderformarking citruspsyllid illaud JandFebvayG,Melonresistance tothe BioEstat: aplicaçõesesta designandanalysisinEPGexperiments with foranalysisofnon- Lygus Hesperus cal correlations with nitrogenous cal correlationswithnitrogenous ance intomato andsweetpepper P, Elsevier,Amsterdam, pp.98- of ElectronicMonitoring , ed.byWalker GPand Aphids: TheirBiology, (Hemiptera: Miridae) tísticas nasáreasdas sequential electrical

transmission by 47. MowryTM.andOphusJD,Effectsofsub-leth Study. coagulata 46. BethkeJA,BluaMJ,andRedakRA,E Press, SanDiego,pp.69-85(2001). graphs, in 45. TjallingiiWF andPradoE,Analysisofcirc Soc A Aleyrodidae) feedingbehavi 44. JiangYX,BlasC,BarriosLandFereres bacterium afteracquisition. 43. CanaleMC,Coletta-FilhoHDandLopesJRS, Psy solanacearum byitsvectortomato potatopsyllid. penetration activities linkedtoth 42. SandanayakaWRM, MorenoA,Tooman LK,Page-Weir NEMandFereresA,Stylet Liberibacter psyllaurous. behavior ofadult 41. ButlerCD,ByrneFJ,Keremane ML,LeeRF 142 cockerelli 40. ButlerCD,Walker GPandTrumble JT, This article isprotected bycopyright. Allrights reserved.

Accepted Article :247–257 (2012). . 93 J. Econ.Entomol :573-579 (2000). , byimidacloprid asmeasured by (Homoptera: Cicadellidae)andTransmission Virus-insect-plant interactions Myzus persicae Bactericera cockerelli

94 J EconEntomol :1031-1036 (2001). Citricultura Atual or andtransmission onTomato . Entomol ExpAppl e acquisitionandinoculationof (Hemiptera: Triozidae) andtransmission of

, ed.byHarrisKF,Smith OPandDuffusJE,Academic 104 : 586–594(2011). , electricalpenetrationgraphs. 105 Feeding disruptionofpotatopsyllid, ffect ofSelectedInsecticideson Entomol ExpAppl A, Correlationbetweenwhitefly(Homoptera: :10-11 (2015).

ulative transmission byelectricalpenetration 103 al imidacloprid levels and Trumble JT,Effect llis takemore thana :249–255 (2002). of OleanderLeafScorchinaGreenhouse YellowLeafCurlVirus.

151 :1–12 (2014). Candidatus on potatoleafrollvirus week totransmit HLB s ofinsecticideson Entomol ExpAppl Homalodisca Ann Entomol Liberibacter Bactericera Candidatus

leafhopper transmission ofchrysanthemum yellowsphytoplasma ( 48. Saraccoa P,MarzachìCand BoscoaD, Ac

This article isprotected bycopyright. Allrights reserved. Accepted Article Macrosteles quadripunctulatus Kirschbaum. tivity ofsome insecticidesinpreventing Crop Prot Crop Candidatus

27 :130–136 (2008). Phytoplasma asteris)bythe

This article isprotected bycopyright. Allrights reserved. Accepted Article event). neonicotinoid insecticides(15 Table 1.Means(±SE)ofEPGva c b a

Values in minutes. NPI (number of probes per insect), NWEI (number of waveform events per insect), WDI (waveform duration per insect) and WDE (wav thesame do sameletter,in row, the by followed Averages

WDE WDI NWEI NPI 31 A .0±28 4.0±1.1 ------0.122 --- 0.469 0.000 43 47.70±13.61 43 --- 38 0.507 2.208 0.769 3.70±2.89 35.925 292 --- 0.111 1.31±0.22a 161.27±29.32a 0.48±0.05a 0.680 290 12.80± 2.55b 0.306 3.15±NA --- 0.90±0.16a 0.280 13.20±2.65a 2.213 0.58±0.11a 0.000 7.64±2.19b 37 47.70±13.61 37 32.69±5.83a 15.88±3.64a 0.79±0.16a 37 0.42±0.06a 1.222 0.323 1.317 54.48 ± 8.23a 4.63±3.59 14.26±3.10a 34.558 0.254 1.40±0.35a 161.27±29.32a 57 0.54±0.11a 56.99±9.21a 0.404 57 13.87± 2.72b 0.552 3.15±NA 0.000 1.19±0.19a 1.151 0.449 57 0.77±0.21a 1.402 0.901 7.64±2.19b 57 57 65.35±17.22a G 57 0.919 0.85±0.17a 0.10±0.07a 0.969 E2 79.41± 15.67a 0.45±0.05a 57 0.059 9.190 E1 0.810 0.80±0.09a 68.44±14.28a 158.57±25.78b 57 D 0.104 0.25±0.12a 0.85±0.11a 261.52±16.61a 0.90±0.12a C 284.94± 14.02a 0.65±0.13a 0.314 4.95±0.55a Np 0.05±0.05a 0.80±0.17a 0.80±0.17a 4.85±0.51a 0.60±0.11a 5.00±0.40a G 0.65±0.13a E2 0.65±0.13a 4.80±0.42a E1 4.80±0.56a D 5.00±0.51a C Np G E2 E1 D C Np EPG variables

c c

days after application). b

riables for6-hmonitoringof Thiamethoxam FdfP Control Imidacloprid 4.10 ±0.51a not differ significantly (P > 0.05) using Fisher LSD test LSD Fisher using >0.05) (P significantly differ not a .5±04a .0±04a .50 7 0.7757 57 0.2550 4.40±0.49a 3.95±0.44a Diaphorina citri on Citrus sinensis nurserytreestreatedbydrenchwith eform duration per

This article isprotected bycopyright. Allrights reserved. Accepted Article event). neonicotinoid insecticides(35 Table 2.Means(±SE)ofEPGva c b a

WDE WDI NWEI NPI 61 .8 .6±N 45 17 ------0.972 0.811 0.004 61 4.54±1.75 --- 59 41 0.983 0.028 0.209 --- 2.66±NA 6.071 0.010 324 1.93±0.34a 0.73±0.07a 83.93±16.72a 286 0.016 --- 0.045 6.10±1.58 22.09± 11.32b 1.78±0.34a 0.264 7.026 20.09±2.66a 0.65±0.06a 36 15.41±1.92b 13.63±4.93 0.000 23.05±3.60b 36 1.61±0.35a 19.42± 3.29a 0.81±0.17a 3.359 34 1.382 54.38 ± 7.93a 2.66±NA 20.39±5.33a 0.125 0.032 4.22±0.72a 19.723 1.50±0.30a 56.27±7.76a 0.034 117.50±20.38a 57 57 0.010 0.012 6.10±1.58 22.09± 11.32b 2.59±0.70b 0.013 57 0.94±0.22a 2.149 3.655 0.018 57 57 16.69±3.11b G 140.61±18.05a 57 3.572 1.74±0.37b 0.75±0.43a 0.547 E2 0.88±0.18a 93.23 ± 17.80b 57 7.816 4.715 E1 4.643 1.05±0.17a 122.17±13.28b 92.80±22.66b 57 D 0.05±0.05a 4.268 1.75±0.40a 252.86±18.77a 1.65±0.33a C 253.21± 21.68a 0.50±0.11b 0.608 7.00±0.83a Np 0.20±0.09a 0.80±0.22b 0.80±0.22b 5.30±0.59a 0.65±0.13b 4.80±0.43b G 0.65±0.13b E2 0.65±0.13b 4.65±0.41a E1 4.55±0.49b D 4.50±0.47a C Np G E2 E1 D C Np EPG variables

c c

days after application). b

riables for6-hmonitoringof Thiamethoxam FdfP Control Imidacloprid 3.70 ±0.45a not differ significantly (P > 0.05) using Fisher LSD test LSD Fisher using >0.05) (P significantly differ not a .5±04a 49 .2 132 5 0.2693 57 1.3426 4.90±0.62a 3.95±0.46a Diaphorina citri on Citrus sinensis nurserytreestreatedbydrenchwith eform duration per

This article isprotected bycopyright. Allrights reserved. Accepted Article event). neonicotinoid insecticides(90 Table 3.Means(±SE)ofEPGva c b a

WDE WDI NWEI NPI 18 A .4±06 1.5±66 ------0.353 0.000 0.000 74 19.45±6.68 --- 74 0.005 49 1.053 3.24±0.61 10.947 295 --- 0.011 62.211 1.88±0.40a 213.62±25.80a 0.74±0.05a 5.238 268 0.427 1.87±NA --- 22.63± 8.97b 1.31±0.28a 19.78±4.28a 0.155 4.512 0.47±0.03b 43 19.68±2.22b 0.000 25.94±10.40 43 35.51±8.88b 9.59±1.10b 1.55±0.47a 0.49±0.08b 0.866 0.789 42 1.980 57.11 ± 8.10a 3.24±0.61 11.34±2.31b 0.483 2.88±0.70a 66.780 57 1.13±0.19a 52.03±7.46a 0.463 228.88±23.33a 57 0.153 1.87±NA 0.236 28.29± 10.99b 0.000 2.86±0.67a 0.153 57 1.02±0.27a 0.735 73.18±14.44a 0.016 57 22.30±2.64b G 57 57 0.778 1.96±0.60a 0.20±0.12a 0.007 E2 55.63± 7.44a 0.62±0.10a 57 1.937 16.595 E1 1.937 0.75±0.12a 118.96±28.37b 61.23±12.10a 57 D 0.15±0.08a 4.437 1.15±0.20a 277.00±14.88a 1.15±0.20a C 278.34± 11.83a 1.00±0.15a 5.313 3.70±0.67b Np 0.05±0.05a 1.75±0.40a 1.75±0.40a 3.35±0.60b 0.85±0.13a 5.80±0.62a G 0.95±0.15a E2 0.95±0.15a 4.85±0.43a E1 5.40±0.60a D 5.35±0.56a C Np G E2 E1 D C Np EPG variables

c c

days after application). b

riables for6-hmonitoringof Thiamethoxam FdfP Control Imidacloprid 4.40 ±0.56a not differ significantly (P > 0.05) using Fisher LSD test LSD Fisher using >0.05) (P significantly differ not a .5±04a .5±05a .02 7 0.1090 57 2.3042 3.05±0.56a 3.95±0.44a Diaphorina citri on Citrus sinensis nurserytreestreatedbydrenchwith eform duration per

This article isprotected bycopyright. Allrights reserved. Accepted Article trees treatedbydrenchwithneonicotinoi Table 4.Proportionofindividuals( a Days afterapplication Waveform Thiamethoxam Imidacloprid Control 90 DAA 35 DAA 15 DAA 2 1 1 1 053 0.765 --- 2 0.911 --- 0.533 0.262 --- 2 --- 2 14 0.186 0.214 --- 20 2.679 0.517 --- 2 15 --- 2 15 3.077 16 0.301 --- 20 1.318 20 2 16 --- 16 15 2.400 10 15 20 20 16 11 20 15 12 12 20 12 20 E2 12 D +E1 12 C 20 E2 12 D +E1 C E2 D +E1 C a

Diaphorina citri

d insecticides,during ) thatproducedaspecificwa a 6-htime period

veform type(PPW) (n=20)on χ 2 df P df Citrus sinensis nursery

This article isprotected bycopyright. Allrights reserved. Accepted Article by drenchwithneonicotinoidinsecticides,at Table 5.Mean(±SE)time toeventper b a Days afterapplication thesame sameletter, in the row, by followed Averages 90 DAA 35 DAA Thiamethoxam FdfP 15 DAA Control Imidacloprid EPG variables is 2fo tr frcrig 97 24a 22 .7 116 16a .1 4 0.496 0.338 42 0.341 43 0.711 43 1.112 121.65 ±21.69a 1.101 120.70 ±26.08a 82.21±9.47a 120.00±26.04a 71.81±9.11a 0.910 99.78± 12.40a 71.18±9.16a 0.807 97.72± 12.33a First E2fromstartofrecording 34 0.870 95.19±11.68a First E1fromstartofrecording 36 First Dfromstartofrecording 0.943 36 recording 0.214 124.95±15.77a First probefrom startof 0.139 115.79±20.62a 112.56±15.55a 0.876 132.58±23.72a 111.84±15.53a 107.15±19.74a 0.899 110.39±20.31a First E2fromstartofrecording 130.89±23.70a 0.899 36 130.04±23.74a 37 First E1fromstartofrecording 37 First Dfromstartofrecording 0.132 0.107 recording 110.52±20.46a 0.106 108.58±19.20a First probefrom startof 136.10±28.69a 108.14 ±19.22a 133.44±28.62a 100.41±16.25a 132.84±28.59a 99.10±16.23a First E2fromstartofrecording 98.67±16.21a First E1fromstartofrecording First Dfromstartofrecording recording First probefrom startof b

insect (minutes) for6-hmonitoring of 23.29 ±9.58a differenttime intervalsafterapplication. 53 .6 1.5±34a 17 74a .9 5 0.675 57 0.394 41.70±17.42a 0.843 12.85±3.49a 57 15.30 ±3.16a 0.170 26.98±8.03a 28.69±6.18a 27.22 ±8.13a do not differ significantly (P > 0.05) using Fisher LSD test LSD Fisher using >0.05) (P significantly differ do not a 95 .9 1.5±68a .8 5 0.835 57 0.188 17.45±6.82a 19.52±7.29a Diaphorina citri on Citrus sinensis nursery treestreated

This article isprotected bycopyright. Allrights reserved. Accepted Article plants ina given time interval afterre the observation chamber walls andceiling; or(D)died. Asterisk thiamethoxam or untreated); (B) moved from from th thiamethoxam (B)moved or untreated); Figure Legend Fig. 1 Mean (± SE) percentage ofmarked Diaphorina citri that: lease. Thiamethoxamapplicat

e original plantto the other plant; (C) moved from either thiamethoxam-treated or (A) remained on the original plant (Citrussinensis nursery tree indicates significant difference by ttest (P<0.05) between ion was done 30 days before to start the experiment. tostart the experiment. ion wasdone 30 daysbefore treated and untreated s treated with untreatedplantsto

This article isprotected bycopyright. Allrights reserved. Accepted Article in agiventime intervalafterrelease. observation chamber walls and ceiling; or (D imidacloprid or untreated); (B) movedfrom the original plant to Fig. 2 Mean (± SE) percentage ofmarked Diaphorina citri that:

Imidacloprid application was done 30daysbefore applicationwasdone Imidacloprid tostart the experiment. ) died.Asterisk indicates significant diff

(A) remained on the original plant (Citrussinensis nursery tree the other plant; (C) moved from plant; (C)moved other the erence by t test (P < 0.05) between trea (P<0.05) between byttest erence either imidacloprid-treated or

ted and untreated plants and untreated ted s treated with untreated plants tothe