Suppression of citrus leafminer, Phyllocnistis citrella, with an attract-and-kill formulation

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1 Suppression of citrus lefminer, Phyllocnistis citrell, with n ttrct-nd-kill formultion Luksz L. Stelinski 1 *&D.Czokjlo 2 1 Entomology nd Nemtology Deprtment, Citrus Reserch nd Eduction Center, University of Florid, 700 Experiment Sttion Rod, Lke Alfred, FL 33850, USA, nd 2 Alph Scents, 7676 Tuttle Rd., Bridgeport, NY 13030, USA Accepted: 8 Octoer 2009 DOI: /j x Key words: ttrcticide, ehviorl modifiction, citrus cnker, Grcillriide, Lepidopter, mting disruption, pheromone Astrct The citrus lefminer, Phyllocnistis citrell Stinton (Lepidopter: Grcillriide), is worldwide pest of citrus crops nd is responsile for prolifertion of citrus cteril cnker, Xnthomons xonopodis (Hsse) pv. citri (Gmm Proteocteri: Xnthomondcee). We developed nd evluted n ttrcticide formultion, termed MlEx, for control of P. citrell. MlEx is viscous pste with UV-protective properties tht is dispensed s 50-ll droplets using custom-mde clirted pumps. A formultion contining 0.016% P. citrell pheromone [3:1 lend of (Z,Z,E)-7,11,13-hexdectrienl nd (Z,Z)-7,11-hexdecdienl] nd 6% permethrin ws found to suppress mle response to pheromone in the field etter thn formultions contining 10 less pheromone. Although formultions without permethrin showed some suppression of mle ctivity ecuse of mting disruption, ddition of 6% permethrin to the formultion ws required for optiml efficcy. When MlEx, contining 0.016% pheromone nd 6% permethrin, ws pplied t point sources h )1, the ppliction height did not influence efficcy of mle P. citrell suppression within mture 4-m tll citrus trees. Decresing the rte of MlEx from to droplets h )1 reduced efficcy s mesured y oth mle P. citrell ctivity nd lrvl infesttion. Although droplets h )1 did not result in sttisticlly etter efficcy thn droplets h )1, there ws noticele trend for higher efficcy s droplet density incresed. Continuous tretment of 0.5-h locks of citrus with MlEx over the course of 112 dys reduced lrvl infesttion of new flush, s compred with those in untreted control plots, y depending on droplet ppliction density. In lortory ehviorl iossys, the ttrctiveness of MlEx droplets to mle P. citrell ws drsticlly reduced fter 21 dys of field ging. However, our lortory investigtion confirmed tht 100% of mles contcting MlEx droplets, ged up to 35 dys in the field, were killed within 24 h. Direct oservtion of mle P. citrell ehvior in the field confirmed tht ttrcted mles mde contct with droplets. Control of P. citrell with MlEx should reduce the numer of required rod spectrum sprys for lefminer mngement in oth field nd citrus nursery settings. Introduction The citrus lefminer, Phyllocnistis citrell Stinton (Lepidopter: Grcillriide), is mjor worldwide pest of citrus production (Heppner, 1993). Lrvl feeding within serpentine mines dmges leves, which cn result in yield loss (Peñ et l., 2000). Furthermore, lef wounds cused *Correspondence: Luksz L. Stelinski, Entomology nd Nemtology Deprtment, Citrus Reserch nd Eduction Center, University of Florid, 700 Experiment Sttion Rod, Lke Alfred, FL 33850, USA. E-mil: stelinski@ufl.edu y P. citrell lrvl feeding predisposes trees to infection y citrus cteril cnker, Xnthomons xonopodis (Hsse) pv. citri (Gmm Proteocteri: Xnthomondcee), responsile for lemished fruit, premture fruit drop, nd tree decline (Grhm et l., 2004). Feeding P. citrell lrve ter the lef cuticle, exposing mesophyll to direct infection. Wounds cused y P. citrell do not hel redily, which increses the exposure period to the cterium nd its spred y lrve moving throughout feeding glleries (Grhm et l., 2004). Feeding lrve within the lef mines re protected from folir pplictions of toxicnts rendering insecticidl control of the lrvl stge difficult or in Ó 2009 The Authors Entomologi Experimentlis et Applict 134: 69 77, 2010 Journl compiltion Ó 2009 The Netherlnds Entomologicl Society 69

2 70 Stelinski & Czokjlo some instnces ineffective. When spry progrms cn e implemented, they my require i-weekly pplictions given continul growth of new lef flush, which is highly detrimentl to nturl enemy popultions of P. citrell nd other citrus pests (Peñ et l., 2002). Development of effective control strtegies for P. citrell is of criticl importnce for the leding citrus producing countries, including Brzil nd the USA, where citrus cnker limits production (Leite & Mohn, 1990). Recently, the sex pheromone of P. citrell ws identified (Lel et l., 2006; Moreir et l., 2006). A 3:1 lend of (Z,Z,E)-7,11,13-hexdectrienl (Z7Z11E13-16Ald) nd (Z,Z)-7,11-hexdecdienl (Z7Z11-16Ald) is highly ttrctive to mles. Effective monitoring protocols using this pheromone hve een developed (Lpointe et l., 2006; Stelinski & Rogers, 2008) nd the potentil for mting disruption of P. citrell hs een investigted (Mfi et l., 2005; Stelinski et l., 2008). Pheromone-sed mting disruption is highly effective for P. citrell control (Stelinski et l., 2008). However, given the high cost of synthesis of the P. citrell pheromone components, it is uncler whether mting disruption will e vile commercil option for mngement of this pest. Given the species-specific mode of ction of mting disruption, high input costs trgeting P. citrell mngement lone in worldwide citrus production my e impossile given the emergence of the Asin citrus psyllid, Diphorin citri Kuwym, s nother worldwide pest limiting citrus production (Hlert & Mnjunth, 2004). Thus, ehviorl modifiction tools tht re oth effective nd economicl re needed for mngement of P. citrell. One potentil lterntive to mting disruption is the development of n ttrct-nd-kill method for P. citrell.there is lrge precedent for effective ttrcticides trgeting lepidoptern pests, which comine the use of very low dose of oth synthetic sex pheromone nd permethrin insecticide (Chrmillot et l., 1996, 2000; Suckling & Brockerhoff, 1999; Krupke et l., 2002; Evenden & McLughlin, 2004). Such formultions re typiclly pplied s smll droplets, which relese pheromone t rte highly ttrctive to mles. Responsive mles follow the plumes from ttrcticide droplets nd cn otin lethl dose of toxicnt upon contct with the source of ttrctnt. Although some investigtions hve shown tht formultions developed with the intent of controlling lepidoptern pest y ttrct-nd-kill ctully function y mting disruption (Evenden & McLughlin, 2004), others hve proven tht the ddition of toxicnt improved control over formultions contining only pheromone ctive ingredients (AI) (Chrmillot et l., 1996; Suckling & Brockerhoff, 1999). An dvntge of ttrct-nd-kill over mting disruption is tht killed mles re permnently removed from the reeding popultion. As competitive ttrction is popultion density dependent (Miller et l., 2006), ttrct-nd-kill should prove more effective thn mting disruption y flse plume following t high-pest densities. Furthermore, ttrct-nd-kill formultions use less pheromone AI per crop re thn mting disruption formultions. In the pst decde, gel mtrix with UV-protective properties ws developed s n ttrcticide for lepidoptern pests. This formultion ws registered in Switzerlnd under the trde nme Sirene (Chrmillot & Hofer, 1997) nd susequently s LstCll in the USA, Europe, nd South Afric (Evenden & McLughlin, 2004). The originl trget pest of this formultion ws the codling moth, Cydi pomonell L. (Chrmillot & Hofer, 1997); however, LstCll hs een dopted for multiple other pests since then (Brockerhoff & Suckling, 1999; Evenden & McLughlin, 2004; Nnsen & Phillips, 2004; Mxwell et l., 2006; Evenden et l., 2008). The gol of this investigtion ws to develop effective ttrct-nd-kill for P. citrell y dopting the controlled relese gel, termed MlEx, which is the currently licensed nme for formultion similr to LstCll. The specific ojectives were to determine the effect of (1) pheromone dosge; (2) presence of toxicnt; (3) droplet density per h of crop; (4) plcement height within the tree cnopy; nd (5) formultion ging on suppression of mle P. citrell ctivity, lef infesttion, s well s P. citrell mortlity in oth field nd lortory investigtions. In ddition, ehviorl oservtions were conducted to document ttrction of P. citrell mles to droplets of MlEx in the field. Mterils nd methods Attrcticide formultion The formultion used in oth field nd lortory experiments, termed MlEx (Alph Scents, Bridgeport, NY, USA), intended for control of P. citrell ws comprised of n inert viscous proprietry pste (94%), permethrin (6% y weight), nd P. citrell pheromone (0.016% y weight). The pheromone components of P. citrell, Z7Z11E13-16Ald nd Z7Z11-16Ald, were synthesized s descried previously (Lel et l., 2006; Moreir et l., 2006). Z7Z11E13-16Ald ws 94 nd 90% chemiclly nd isomericlly pure, respectively, nd Z7Z11-16Ald 86 nd 72%, respectively. A 3:1 lend of these components, which is optiml for ttrcting mles (Lel et l., 2006; Moreir et l., 2006), ws loded into the MlEx formultion. Ml- Ex ws dispensed from custom-mde pumps clirted to deposit 50 ll droplets. This se formultion ws slightly modified for one experiment descried elow to determine the effect of pheromone dosge nd presence of insecticide on efficcy ginst P. citrell.

3 Attrcticide for citrus lefminer 71 Effect of pheromone dosge nd insecticide The ojective of this experiment ws to investigte the effect of pheromone dosge nd presence of permethrin on efficcy of MlEx in suppressing ctch of mle P. citrell in pheromone-ited trps positioned in treted plots. A totl of four MlEx tretments were formulted. Two loding dosges of pheromone ( nd 0.016% y weight) were formulted with nd without 6% permethrin y weight. The experiment ws rrnged s rndomized complete lock design. Tretments were rndomly ssigned to five 0.5-h replicte plots in n 8-yer-old ornge orchrd [Citrus sinensis (L.) vr. Vlenci (Rutcee)] in Clermont, FL, USA(28 N, 81 W). Trees were plnted on m spcing with 4.0 m-verge cnopy height. Replicte plots were seprted y 60 m nd locks of tretments were seprted y 80 m. MlEx formultions were rndomly ssigned to replicte plots nd dispensed t rte of deposits h )1 (pproximtely 6 droplets per tree). Droplets were plced onto rk of tree rnches 2.0 m ove ground level. Tretments were pplied on 8 9 June 2008 nd the experiment ws conducted through 29 June 2008, when efficcy egn to noticely decrese s mesured y moth ctch in pheromone trps. Control plots were left completely untreted nd no dditionl insecticides were spryed in this orchrd during the course of the experiment. Mle P. citrell ctivity in experimentl plots ws quntified using two pheromone trps (LPD Scenturion Gurdpost; Suterr, Bend, OR, USA) deployed within ech replicte plot. Trps were plced six trees prt in the centrl row of ech plot. All trps were ited with single red ruer septum lure loded with 0.1 mg Z7Z11E13-16Ald nd 0.03 mg Z7Z11-16Ald, nd hung t lest 1.0 m from the nerest MlEx droplet, t c m ove ground level (Stelinski & Rogers, 2008). Moths cptured in trps were counted nd removed weekly. Effect of droplet density The ojective of this investigtion ws to determine the effect of MlEx droplet density on P. citrell control efficcy. The formultion used ws the se formultion descried ove contining 6% permethrin nd 0.016% pheromone, ecuse this ws the most effective tretment in the initil experiment (see Results). The tretments compred were 0, 1 500, 3 000, nd MlEx droplets h )1. Tretments were rndomly ssigned to 0.5-h replicte plots rrnged in rndomized complete lock design with five replictes in the citrus orchrd descried ove. Spcing etween tretments nd locks ws s descried ove. The experiment ws initited y pplying tretments on 6 7 July Tretments were re-pplied 7 8 August. The experiment ws terminted on 29 Septemer Tretments were evluted weekly using pheromone monitoring trps ccording to the procedure descried ove. Pheromone lures were replced every 7.5 weeks (Lpointe & Lel, 2007). In ddition, dmge to newly flushed leves ws ssessed hlf wy through (10 12 August) nd t the end (28 29 Septemer) of the tril. Dmge ws ssessed y inspecting lef flush smples chosen t rndom. For ech tree, 10 smples were inspected from mid-cnopy (2.5 m) nd 10 from lower cnopy (1.0 m) on 20 trees per replicte lock (2 000 flush smples per tretment). The numer of shoots per tree contining live mining P. citrell lrve ws recorded. Effect of droplet height within the tree cnopy The ojective of this experiment ws to determine the effect of height of MlEx droplet plcement on suppression of P. citrell mle flight s mesured y pheromone trpping. The formultion used ws the se formultion descried ove contining 6% permethrin nd 0.016% pheromone. All tretments were pplied t rte of MlEx droplets h )1 or pproximtely 6 droplets per tree. The ppliction heights compred reltive to the ground were 0.6, 2.0, nd 3.5 m within the citrus orchrd descried ove, which ws comprised of trees verging 4 m in height. Control plots were left untreted. Tretments were rndomly ssigned to 0.1-h replictes rrnged in rndomized complete lock design with five replictes. Replicte plots were seprted y 40 m nd locks of tretments were seprted y 50 m. Suppression of mle P. citrell flight ctivity ws mesured with weekly ssessments of pheromone trps. One pheromone trp ws deployed centrlly per replicte plot c m ove ground level ccording to the procedures descried ove. The experiment ws conducted 9 30 July Effect of droplet ging The ojective of this experiment ws to sses the ehviorl response of mle P. citrell to droplets of MlEx following vrious durtions of field ging. Four formultions of MlEx were compred (Tle 1) nd 40 droplets were evluted per formultion nd ging period comintion. Individul 50-ll droplets of MlEx were deposited onto 1 1 cm pieces of luminum foil. Aluminum foil pieces were ffixed using thum tcks to cm pieces of wood prticle ord. Prticle ords contining MlEx smples were ffixed with wire into the cnopies of eight citrus trees in the orchrd descried ove. Droplets were deployed on severl dtes 5 25 August 2008, to estlish stggered ging schedule for ech formultion. This llowed testing of ech formultion nd ging period tretment on given dy until sufficient replictes were ccrued for ech formultion nd dy of ging tretment

4 72 Stelinski & Czokjlo Tle 1 Behviors of Phyllocnistis citrelll in response to vrious formultions of MlEx following three durtions of droplet ging in the field Dys of ging % ttrction % contct Control % AI % AI % AI 0.016% AI % AI 0.016% AI % AI 0.016% AI No permethrin With permethrin Control No permethrin With permethrin 0 0.0A 89.4A 92.7A 90.2A 94.7A 0.0A 65.3A 72.6A 67.9A 75.3A 7 0.0Ac 74.5A 89.4A 67.2B 90.4A 0.0Ac 61.7A 68.7A 49.5B 68.3A Ac 67.3B 71.4B 54.7B 77.1B 0.0Ac 51.5B 56.4B 38.4B 54.6B Ac 19.0C 29.8C 12.1C 14.2C 0.0Ac 9.6C 16.1C 4.8C 6.3C Mens followed y the sme uppercse letter within column or the sme lowercse letter within row (seprtely for % ttrction nd % contct) re not significntly different (G 2 test of homogeneity: P>0.05). 1 Phyllocnistis citrell pheromone, 3:1 Z7Z11E13-16Ald nd Z7Z11-16Ald. comintion. Droplets were collected t rndom for ehviorl ssys fter 7, 21, nd 35 dys of field ging. In ddition, 40 fresh droplets were evluted to estlish the ehviorl response of mles to MlEx prior to field ging. Behviorl responses were evluted in Y-tue ssy ccording to the procedures descried in Wenninger et l. (2008). In rief, the Y-tue consisted of 14-cm-long stem nd two 10-cm-long rms, ech with 2-cm inner dimeter. A screened glss plug t the se of the stem ws used to introduce insects into the Y-tue. Odor sources were plced t the upwind end of one rm of the Y-tue such tht ttrcted mles were le to mke contct with MlEx droplets. Chrcol-filtered, humidified ir ws metered through the two rms of the Y-tue vi polytetrfluoroethylene tuing t 500 ml min )1. The ir exiting the wire screen plug t the se of the stem ws C nd 70 85% r.h.; light generted y two 95-W fluorescent uls (model F96T12; Philips, Eindhoven, the Netherlnds) ws mesured t c lux just ove the rnching point of the Y-tue. The position (left or right) of the test odor source ws rndomly selected for the first of ny set of oservtions nd lternted therefter. For the lnk control tretment, 1 1 cm piece of clen luminum foil without MlEx ws inserted into one of the rms of the Y-tue per replicte moth tested. Between ssys, glsswre ws rinsed thoroughly with cetone nd deionized wter, soked in hot sopy wter for min, nd kept in drying oven (150 C) for t lest 2 h (nd usully overnight) efore re-use. Phyllocnistis citrell were collected t the pupl stge from n infested greenhouse (Lke Alfred, FL, USA; 28 N, 81 W) mintined t 26 C nd60%r.h.pupe were sorted y sex (Jcs & Grrido, 1996) nd mles were llowed to emerge in 1-l plstic cges contining 5% (wt vol) sucrose in plstic cups with cotton dentl wicks protruding from their lids. Adult mles were mintined for 2 3 dys t 26 C, 60% r.h., nd n L12:D12 photocycle prior to testing. All tests were initited h prior to the end of scotophse to mtch the time when P. citrell re sexully ctive (Jcs & Peñ, 2002). Mle moths (n = 40 per tretment) were relesed individully into the glss plug t the se of the stem of the Y-tue, nd their ehvior ws oserved over 3-min period. The response of one mle P. citrell ws evluted per droplet. Ech MlEx formultion tretment ws presented to previously untested mle P. citrell on ech dy of testing until 40 replictes were ccrued per tretment comintion. The first choice of ech moth ws recorded s the rm in which the mle first entered c. 1 cm into the rm. In ddition, the numer of mles contcting MlEx droplets ws recorded. Following ech ssy, the mle ws crefully removed from the Y-tue nd plced individully into 240-ml plstic cup contining moistened dentl cotton wick tht ws seled with perforted lid. Ech cup ws mrked to indicte tretment nd whether or not mles mde contct with the droplet of MlEx. Cups were mintined in n environmentl chmer set t 26 C nd 60% r.h. for 24 h, fter which mortlity ws ssessed. All insects lying on their ck nd remining motionless fter prodding with fine proe were scored s ded. Behviorl oservtions in the field Mle P. citrell ehvior ws oserved in the field to determine whether mle moths pproched droplets of MlEx. Oservtions of MlEx droplets in tree cnopies were conducted for pproximtely 2 h ech night etween 21:00 nd 23:00 hours, period when mle P. citrell re known to exiit response towrd pheromone sources in the field in Florid (Stelinski & Rogers, 2008). Oservtions were conducted on eight nights etween 9 nd 29 June. Oservtions were conducted in the tretment plots descried

5 Attrcticide for citrus lefminer 73 ove designed to investigte the effect of pheromone loding nd presence of permethrin. An oserver rotted mong plots conducting 20-min oservtionl outs per tretment such tht multiple tretments were oserved on given night. The order of oservtions cross tretments ws rndomized nightly. During oservtions, dt were dictted into hnd-held microcssette udio recorder y n investigtor stnding c. 0.3 m from the MlEx droplet under oservtion. Oservtions fter dusk employed night-vision goggles (Model 3250; Rigel, DeWitt, IA, USA) s descried y Stelinski et l. (2004). Sttisticl nlysis Moth ctch dt were sujected to nlysis of vrince (ANOVA) fter trnsformtion to ln(x + 1), which normlized the distriutions nd homogenized vrinces. Becuse flush injury trends were nerly identicl t the mid- nd end-points of the droplet density study, injury dt from the two smpling dtes were comined nd rcsine trnsformed prior to ANOVA. Dt on the men numer of mles pproching vrious formultions of MlEx in the field were lso sujected to ANOVA. A logistic model ws used to mesure the proility tht mle P. citrell would pproch droplets of vrious formultions of MlEx using Proc GENMOD in SAS (SAS Institute, 2000). Susequently, nlyses of numers of mle moths responding were crried out using the G sttistic, testing the null hypothesis of no preference (Sokl & Rohlf, 1981). Proportions of moths responding were compred seprtely etween formultion tretments within ech ging period nd etween the ging periods tested within ech formultion tretment. In ll cses, the significnce level ws Results Effect of pheromone dosge nd insecticide Fewer mle P. citrell were cptured in plots treted with MlEx contining permethrin, t oth pheromone concentrtions tested, thn in control plots (F 4,16 =11.3, P<0.0001; Figure 1). Ctch of mle P. citrell ws sttisticlly equivlent etween the two pheromone concentrtions tested when MlEx formultions contined permethrin (P>0.05; Figure 1). More mle P. citrell were cught in plots treted with MlEx without permethrin, t oth pheromone concentrtions tested, thn in plots treted with MlEx contining permethrin (P<0.0001; Figure 1). Ctch of mle P. citrell ws reduced, compred with the control, in plots treted with MlEx not contining permethrin t the 0.016% pheromone concentrtion (P<0.0001), ut not t the % concentrtion (Figure 1). Effect of droplet density Fewer mle P. citrell were cptured in plots treted with ech density of MlEx ttrcticide tested thn in control plots (F 3,12 = 12.2, P<0.001; Figure 2A). Mle ctch ws lower in plots treted with nd droplets h )1 thn in plots treted with droplets h )1 (P<0.001; Figure 2A). Although there ws no sttisticl difference in mle ctch suppression etween the two highest density tretments tested (P>0.05; Figure 2A), nerly 15 fewer mle P. citrell were cught in plots treted with droplets h )1 thn in plots treted with droplets h )1. Infesttion of citrus leves y lrvl P. citrell ws lower in plots treted with nd MlEx droplets h )1 thn in control plots (F 3,12 = 6.8, P<0.025; Figure 2B). However, there ws no sttisticl difference in P. citrell infesttion etween plots treted with MlEx droplets h )1 nd control plots (P>0.05; Figure 2B). Although there ws no sttisticl difference (P>0.05) in lef infesttion etween the two highest density tretments tested (Figure 2B), the infesttion level oserved with droplets h )1 ws hlf of tht oserved with droplets h )1. Effect of droplet height within the tree cnopy Ctch of mle P. citrell ws lower in plots treted with MlEx ttrcticide, t ech cnopy height tested, thn in control plots (F 3,12 = 7.4, P<0.01; Figure 3). When pplied t density of droplets h )1, suppression of mle P. citrell ctch ws sttisticlly equivlent mong the three droplet ppliction heights compred (P>0.05; Figure 3). No. mles/trp Control % 0.016% % 0.016% pheromone pheromone pheromone pheromone no no with with pyrethroid pyrethroid pyrethroid pyrethroid Tretment Figure 1 Men (± SE) cptures of mle Phyllocnistis citrell in pheromone-ited trps s influenced y pheromone concentrtion s well s presence of permethrin in MlEx ttrcticide pplied s droplets h )1. Brs leled with the sme letter re not significntly different (ANOVA followed y LSD test, =0.05). c c

6 74 Stelinski & Czokjlo A No. mles/trp B % lrvl infesttion No. drops/h Figure 2 (A) Men (± SE) cptures of mle Phyllocnistis citrell in pheromone-ited trps nd (B) men percent lef flush infesttion y P. citrell lrve s influenced y droplet density of Ml- Ex contining 0.016% pheromone nd 6% permethrin. Brs within pnel leled with the sme letter re not significntly different (ANOVA followed y LSD test, = 0.05). Effect of droplet ging Following ech ging period, more mle P. citrell were ttrcted (G = 6.8, d.f. = 1, P<0.01) to nd contcted No. mles/trp Untreted control c Droplet height (m) Figure 3 Men (± SE) cptures of mle Phyllocnistis citrell in pheromone-ited trps s influenced y droplet ppliction height ove ground level of MlEx contining 0.016% pheromone, 6% permethrin, nd pplied s droplets h )1.Brs leled with the sme letter re not significntly different (ANOVA followed y LSD test, =0.05). c (G = 7.2, d.f. = 1, P<0.01) MlEx droplets, with or without permethrin nd regrdless of the pheromone concentrtion tested, thn lnk controls (Tle 1). Approximtely 90% of relesed mle P. citrell were ttrcted to fresh droplets of ech MlEx formultion nd over 65% of those exhiiting ttrction lso contcted droplets within the 3-min ssy period (Tle 1). There were no sttisticl differences etween P. citrell responses to the vrious MlEx formultions when mles were ssyed to fresh droplets (dy 0) (G = 0.9, d.f. = 1, P = 0.2; Tle 1). At 7 dys of field ging, fewer mle P. citrell were ttrcted to nd contcted MlEx droplets contining % pheromone nd permethrin thn on dy 0 (G = 5.9, d.f. = 1, P<0.01); however, mle responses to the other formultions tested were sttisticlly equivlent on dys 7 nd 0 (G = 1.2, d.f. = 1, P = 0.1; Tle 1). Behviorl responses of mle P. citrell to droplets of ech MlEx formultion were lower fter 21 dys of field ging thn on dy 0 (G = 6.3, d.f. = 1, P<0.01; Tle 1). On dy 21, fewer mle P. citrell were ttrcted to nd contcted MlEx droplets contining permethrin nd % pheromone thn to the other formultions tested (G = 4.8, d.f. = 1, P<0.01; Tle 1). Mle responses to droplets of ech MlEx formultion were lower on dy 35 thn on dy 21 (G = 8.4, d.f. = 1, P<0.01; Tle 1). After 5 weeks of field ging, <20% of the mle P. citrell ssyed contcted MlEx droplets in the Y-tue olfctometer. For the two formultions contining permethrin, ll mle P. citrell contcting MlEx droplets died within 24 h, irrespective of the durtion of field ging. However, mortlities of mle P. citrell contcting droplets of the two MlEx formultions without permethrin or the lnk control were <3% fter 24 h. Also, mortlity of mle P. citrell ttrcted to ut not contcting MlEx droplets with permethrin ws <3% fter 24 h. Behviorl oservtions in the field Mle P. citrell were oserved orienting to ech formultion of MlEx droplets (Tle 2). Fewer mle P. citrell were oserved pproching droplets of MlEx loded with % pheromone without permethrin compred with the other formultion tretments (F 3,12 = 8.5, P<0.001; Tle 2). Discussion Collectively, our results demonstrte tht n ttrcticide formultion comining pheromone nd permethrin suppressed flight ctivity of mle P. citrell to synthetic point sources of pheromone nd reduced lrvl infesttion of leves. Highly effective mting disruption of mle

7 Attrcticide for citrus lefminer 75 Tle 2 Men (± SE) numers of Phyllocnistis citrell oserved visiting droplets from vrious formultions of MlEx per night in the field Tretment No. mles % pheromone, no permethrin 12.3 ± % pheromone, no permethrin 27.0 ± % pheromone, with permethrin 23.1 ± % pheromone, with permethrin 19.5 ± 1.8 Mens followed y the sme letter re not significntly different (ANOVA followed y LSD test: P>0.05). P. citrell nd ssocited reduction of lef infesttion hs een documented for up to 221 dys with two deployments of 1.5 g pheromone AI h )1 (Stelinski et l., 2008). A single deployment of droplets of MlEx, contining 0.016% pheromone y weight, mounts to deploying pproximtely 24 mg pheromone h )1. To chieve 221 dys of comprle efficcy, pproximtely 10.5 deployments of this MlEx formultion would e required. This would require deploying 0.25 g pheromone AI h )1 over the 31.5-week intervl. Thus, the se MlEx formultion developed herein for P. citrell control could reduce cost of ppliction s compred with mting disruption y requiring nerly 12 less pheromone h )1 of crop per seson. The drwck, however, is tht MlEx would require pproximtely 5 s mny deployments thn mting disruption dispensers to chieve comprle durtion of efficcy. This discrepncy is likely ecuse of the difference in pheromone loding rte per relese source when compring mting disruption dispensers evluted for P. citrell (1.0 mg loding per dispenser; Stelinski et l., 2008) vs. single droplet of MlEx (8 lg loding per dispenser). Also, the loding rte of pheromone AI required for effective ttrct-nd-kill of P. citrell in the MlEx formultion is pproximtely 10 lower thn tht required for other lepidoptern species controlled y similr formultions (Chrmillot et l., 1996, 2000; Suckling & Brockerhoff, 1999; Krupke et l., 2002; Evenden & McLughlin, 2004). After 5 weeks of field ging, droplets of the se formultion of MlEx were still ttrctive to mle P. citrell. However, y this point they ttrcted pproximtely onefifth of the numer of mles tht were ttrcted y fresh droplets. Congruently, our field experiments proved tht MlEx droplets effectively suppressed mle P. citrell cptures in pheromone trps for c. 1 month. This durtion of ttrctiveness is lower thn tht oserved with ttrcticide formultions for other moth species. An ttrcticide formultion trgeting Epiphys postvittn (Wlker) remined highly ttrctive to mles over 3-month period (Brockerhoff & Suckling, 1999), wheres nother formultion trgeting Cloptili frxinell (Ely) remined ttrctive for t lest 5 weeks (Evenden et l., 2008). A mjor fctor limiting the utility of MlEx s prcticl control tool for mngement of P. citrell will e the durtion it remins ttrctive to mles in the field. The current formultion will require monthly repplictions to mintin efficcy. Extending this durtion of ttrctiveness is currently under investigtion. Reducing the pheromone loding rte in MlEx to % y weight decresed efficcy of mle P. citrell suppression s well s durtion of effectiveness y up to 1 week. This ws likely ecuse droplets contining only 0.8 lg of pheromone were not sufficiently competitive with femles throughout the deployment period s the relese rte dissipted elow n ttrctive threshold sooner thn the se formultion tested. The ddition of permethrin to the formultion lso proved necessry for efficcy given tht the formultions contining the insecticide suppressed mle ctivity 6 nd 10.6 more thn the formultions without permethrin (Figure 1). Both in our field nd lortory investigtions, there ws no evidence tht the 6% permethrin loding in the MlEx formultion inhiited mle P. citrell response to droplets contining pheromone. These results re congruent with severl other studies showing tht permethrin does not repel moths in pheromone-sed ttrcticide formultions t loding rtes rnging etween 1 nd 10% (Hynes et l., 1986; Evenden & McLughlin, 2004; Nnsen & Phillips, 2004; Curkovic & Brunner, 2006; Evenden et l., 2008). At lest one recent investigtion of this ttrcticide formultion showed tht the ddition of permethrin did not increse efficcy ginst G. molest, suggesting tht mting disruption ws the opertive mechnism of control (Evenden & McLughlin, 2004). Depending on the dosge of pheromone relesed per dispenser, mle orienttion my e disrupted y either non-competitive mechnism or competitive mechnism without source contct. Either of these scenrios likely occurred in the investigtion with G. molest (Evenden & McLughlin, 2004). However, in our investigtion, mle P. citrell definitely pproched nd contcted droplets of MlEx, s documented y direct ehviorl oservtions in the field. Some proportion of ttrcted mles ws suppressed y mting disruption without intoxiction y permethrin s trp cpture ws reduced in plots treted with the insecticide-free formultion (Figure 1). At deployment rte of MlEx droplets h )1,only8.7gpermethrin were deployed per h of crop with the 6% formultion. However, this low dose of insecticide likely plyed lrge role in suppression of P. citrell in the field s our lortory investigtion confirmed tht 100% of mles

8 76 Stelinski & Czokjlo contcting MlEx droplets, ged up to 35 dys in the field, were killed within 24 h. When the se formultion of MlEx ws pplied s point sources h )1, the height of ppliction did not influence efficcy of mle P. citrell suppression within mture 4-m tll citrus trees (Figure 3). These dt re congruent with recent finding showing tht mle P. citrell re ctive throughout the tree cnopy of mture citrus trees (Stelinski & Rogers, 2008) nd do not exhiit preferentil response to pheromone sources t specific height within the tree cnopy s is exhiited y certin other moths, such s C. pomonell (Howell et l., 1990). Similr findings hve lso een reported with n ttrcticide formultion developed for G. molest, where ppliction height of LstCll droplets did not influence efficcy; mting of sentinel virgin femles occurred only when plced in untreted portions of the tree cnopy (Evenden & McLughlin, 2004). Equivlent efficcy of MlEx ginst P. citrell t ech height tested within the cnopies of mture trees should simplify deployment of the formultion y either hnd or mechnized ground or eril ppliction methods. Decresing the rte of MlEx from to droplets h )1 reduced efficcy s mesured y oth mle P. citrell ctivity nd lrvl infesttion (Figure 1). These dt re in contrst to those otined with G. molest, where efficcy with nd droplets h )1 ws equivlent (Evenden & McLughlin, 2004). Although the highest rte tested (4 500 droplets h )1 ) did not result in sttisticlly etter efficcy thn droplets h )1, there ws noticele trend for improved efficcy s the numer of MlEx droplets per h ws incresed. The profile of mle P. citrell ctch in trps s function of point source density in Figure 2 is congruent with the conclusion tht competitive ttrction ws n opertive mechnism in this investigtion (Miller et l., 2006). Thus, lthough droplets h )1 my prove effective under certin popultion densities, efficcy will e popultion density dependent nd my require deployment of more droplets per h in proportion to the size of the P. citrell popultion. Continuous tretment of 0.5-h locks of citrus with MlEx ginst P. citrell over the course of 112 dys reduced lrvl infesttion of new lef flush y , depending on droplet ppliction density. A similr reduction of P. citrell infesttion ws otined with mting disruption, which deployed 3 g pheromone h )1 (Stelinski et l., 2008). Reduced lef infesttion should result in reduced spred of citrus cteril cnker (Grhm et l., 2004). Although this technology might enefit from compnion use of selective insecticides to decrese P. citrell popultion densities, it lso hs the potentil to reduce the numer of required rod spectrum sprys for P. citrell mngement in oth field nd citrus nursery settings. Trgeted deployment of insecticide with MlEx should decrese the hrmful impct of current P. citrell mngement prctices on the wide complex of nturl enemies tht re known to limit P. citrell popultion growth (Peñ et l.,2002).lrgescle commercil use of this formultion will likely mndte the development of specilized mechnicl pplictors (Stelinski et l., 2007) for rpid deployment of mteril in the field. Acknowledgements In Jckson is cknowledged for ssistnce with otining nd sexing P. citrell pupe. A previous version of the mnuscript ws improved y J. Ptt nd F.M. de Lme. LLS thnks the IR-4 Project for funding support. Comments from three nonymous reviewers improved n erlier version of the mnuscript. References Brockerhoff EG & Suckling DM (1999) Development of n ttrcticide ginst light rown pple moth (Lepidopter: Tortricide). Journl of Economic Entomology 92: Chrmillot PJ & Hofer D (1997) Control of codling moth, Cydi pomonell L., y n ttrct-nd-kill formultion. Technology trnsfer in mting disruption. IOBC WPRS Bulletin 20: Chrmillot PJ, Psquier D, Sclco A & Hofer D (1996) Essis de lutte le crpocpse Cydi pomonell L. pr un procédé ttrcticide. Mitteilungen der Schweizerischen Entomologischen Gesellschft 69: Chrmillot PJ, Hofer D & Psquier D (2000) Attrct-nd-kill: new method for control of the codling moth Cydi pomonell. Entomologi Experimentlis et Applict 94: Curkovic T & Brunner JF (2006) Evlution of permethrin for ttrcticide development ginst Choristoneur roscen nd Pndemis pyrusn (Lepidopter: Tortricide) mles. Crop Protection 25: Evenden ML & McLughlin JR (2004) Fctors influencing the effectiveness of n ttrcticide formultion ginst the Orientl fruit moth, Grpholit molest. Entomologi Experimentlis et Applict 112: Evenden ML & McLughlin JR (2004) Initil development of n ttrcticide formultion ginst the Orientl fruit moth, Grpholit molest (Lepidopter: Tortricide). Environmentl Entomology 33: Evenden ML, Gries R & Gries G (2008) Attrctiveness nd toxicity of n ttrcticide formultion on dult mles of sh lef cone roller, Cloptili frxinell. Entomologi Experimentlis et Applict 127:

9 Attrcticide for citrus lefminer 77 Grhm JH, Gottwld TR, Cuero J & Achor DS (2004) Xnthomons xonopodis pv. citri: fctors ffecting successful erdiction of citrus cnker. Moleculr Plnt Pthology 5: Hlert SE & Mnjunth KL (2004) Asin citrus psyllids (Sternorrhynch: Psyllide) nd greening disese of citrus: literture review nd ssessment of risk in Florid. Florid Entomologist 87: Hynes KF, Li WG & Bker TC (1986) Control of pink ollworm moth (Lepidopter: Gelechiide) with insecticides nd pheromones (ttrcticide): lethl nd sulethl effects. Journl of Economic Entomology 79: Heppner JB (1993) Citrus lefminer, Phyllocnistis citrell, in Florid. Tropicl Lepidopter 4: Howell JF, Schmidt RS, Horton DS, Khttck SUK & White LD (1990) Codling moth: mle ctivity to pheromone lures nd pheromone-ited trps t different elevtions within nd etween trees. Environmentl Entomology 19: Jcs JA & Grrido A (1996) Differences in the morphology of mle nd femle pupe of Phyllocnistis citrell (Lepidopter: Grcillriide). Florid Entomologist 79: Jcs JA & Peñ JE (2002) Clling ehvior of two different field popultions of Phyllocnistis citrell (Lepidopter: Grcillriide): effect of ge nd photoperiod. Florid Entomologist 85: Krupke CH, Roiterg BD & Judd GJR (2002) Field nd lortory responses of mle codling moth (Lepidopter: Tortricide) to pheromone-sed ttrct-nd-kill strtegy. Environmentl Entomology 31: Lpointe SL & Lel WS (2007) Descriing sesonl phenology of the lefminer Phyllocnistis citrell (Lepidopter: Grcillriide) with pheromone lures: controlling for lure degrdtion. Florid Entomologist 90: Lpointe SL, Hll DG, Murt Y, Prr-Pedrzzoli AL, Bento JMS et l. (2006) Field evlution of synthetic femle sex pheromone for the lefmining moth Phyllocnistis citrell (Lepidopter: Grcillriide) in Florid citrus. Florid Entomologist 89: Lel WS, Prr-Pedrzzoli AL, Cosse AA, Murt Y, Bento JMS & Vilel EF (2006) Identifiction, synthesis, nd field evlution of the sex pheromone from citrus lefminer, Phyllocnistis citrell. Journl of Chemicl Ecology 32: Leite RP, Jr & Mohn SK (1990) Integrted mngement of the citrus cteril disese cused y Xnthomons xonopodis pv. citri in the stte of Prná, Brzil. Crop Protection 9: 3 7. Mfi SA, Vng LV, Nkt Y, Ohyshi N, Ymmoto M & Ando T (2005) Identifiction of the sex pheromone of the citrus lefminer (Phyllocnistis citrell Stinton, Lepidopter: Grcillriide) with tril of control y the communiction disruption method. Journl of Pesticide Science 34: Mxwell EM, Fdmiro HY & McLughlin JR (2006) Suppression of Plutell xylostell nd Trichoplusi ni in cole crops with ttrcticide formultions. Journl of Economic Entomology 99: Miller JR, Gut LJ, De Lme FM & Stelinski LL (2006) Differentition of competitive vs. non-competitive mechnisms mediting disruption of moth sexul communiction y point sources of sex pheromone: (Prt I) theory. Journl of Chemicl Ecology 32: Moreir JA, McElfresh JS & Millr JG (2006) Identifiction, synthesis, nd field testing of the sex pheromone of the citrus lefminer, Phyllocnistis citrell. Journl of Chemicl Ecology 32: Nnsen C & Phillips TW (2004) Attrctncy nd toxicity of n ttrcticide for Indinmel moth, Plodi interpunctell (Lepidopter: Pyrlide). Journl of Economic Entomology 97: Peñ JE, Hunserger A & Schffer B (2000) Citrus lefminer (Lepidopter: Grcillriide) density: effect on yield of Thiti lime. Journl of Economic Entomology 93: Peñ JE, Shrp JL & Wysoki M (2002) Tropicl Fruit Pests nd Pollintors: Biology, Economic Importnce, Nturl Enemies, nd Control. CABI Press, Wllingford, UK. SAS Institute (2000) SAS STAT User s Guide, Version 6, 4th edn, Vol. 1. SAS Institute, Cry, NC, USA. Sokl RR & Rohlf FJ (1981) Biometry. WH Freemn, New York, NY, USA. Stelinski LL & Rogers ME (2008) Fctors ffecting cptures of mle citrus lefminer, Phyllocnistis citrell, in pheromoneited trps. Journl of Applied Entomology 132: Stelinski LL, Gut LJ, Pierzchl AV & Miller JR (2004) Field oservtions quntifying ttrction of four tortricid moth species to high-dosge, polyethylene-tue pheromone dispensers in untreted nd pheromone-treted orchrds. Entomologi Experimentlis et Applict 113: Stelinski LL, Miller JR, Ledeuhr R & Gut LJ (2007) Seson-long mting disruption of Grpholit molest y one mchine ppliction of pheromone in wx drops (SPLAT-OFM). Journl of Pest Science 80: Stelinski LL, Miller JR & Rogers ME (2008) Mting disruption of citrus lefminer medited y non-competitive mechnism t remrkly low pheromone relese rte. Journl of Chemicl Ecology 34: Suckling DM & Brockerhoff EG (1999) Control of light rown pple moth (Lepidopter: Tortricide) using n ttrcticide. Journl of Economic Entomology 92: Wenninger EJ, Stelinski LL & Hll DG (2008) Behviorl evidence for femle-produced sex ttrctnt in Diphorin citri. Entomologi Experimentlis et Applict 128: