Season-long mating disruption of citrus leafminer, Phyllocnistis citrella Stainton, with an emulsified wax formulation of pheromone

Transcription

1 J. Appl. Entomol. Seson-long mting disruption of citrus lefminer, Phyllocnistis citrell Stinton, with n emulsified wx formultion of pheromone L. L. Stelinski 1, S. L. Lpointe 2 & W. L. Meyer 1 1 Entomology nd Nemtology Deprtment, Citrus Reserch nd Eduction Center, University of Florid, Lke Alfred, FL, USA 2 U. S. Horticulturl Reserch Lortory, United Sttes Deprtment of Agriculture, Agriculture Reserch Service, Fort Pierce, FL, USA Keywords ehviourl modifiction, citrus cnker, emulsified wx, mechnisms of disruption, pheromone dispenser Correspondence Dr Luksz Stelinski (corresponding uthor), Entomology nd Nemtology Deprtment, Citrus Reserch nd Eduction Center, University of Florid, 7 Experiment Sttion Rod, Lke Alfred, FL 3385, USA. E-mil: stelinski@ufl.edu Received: July 3, 29; ccepted: Septemer 8, 29. doi: /j x Astrct The citrus lefminer, Phyllocnistis citrell Stinton (Lepidopter: Grcillriide), is mjor worldwide pest of citrus. Lrvl feeding y this insect fcilittes prolifertion of citrus cteril cnker, Xnthomons xonopodis pv. citri. Herein, we descrie seson-long disruption tril of P. citrell with newly developed, emulsified wx dispenser of pheromone (SPLAT- CLM TM ). A formultion contining 3 : 1 lend of (Z,Z,E)-7,11,13-hexdectrienl:(Z,Z)-7,11-hexdecdienl t.2% loding rte of ctive ingredient y weight nd deployed twice per seson (24 weeks totl) t 49 g of formultion/h cused seson-long disruption of mle moth ctch in pheromone trps s well s reduced lef infesttion. Anlysis of pheromone relese from dispensers y gs chromtogrphy reveled tht effective disruption of P. citrell occurred t deployment rte of 126 lg of (Z,Z,E)-7,11,13-hexdectrienl/h/h. Direct oservtion of moth ehviour in the field suggested tht disruption y this formultion occurred y non-competitive mechnism. A formultion of the 3 : 1 ttrctive lend t.2% pheromone loding rte cused only 2 6 weeks of disruption per deployment nd did not reduce lef infesttion during mid nd end of the seson evlutions. A formultion contining.2% of (Z,Z)-7,11-hexdecdienl lone nd deployed t 49 g/h cused 6 7 weeks of moth disruption to pheromone trps nd did not prevent lef infesttion, while n identicl formultion loded with.2% (w/w) of (Z,Z)-7,11-hexdecdienl lone hd no effect on P. citrell orienttion to pheromone trps. The SPLAT formultion evluted herein ppers to e n excellent relese device for (Z,Z,E)-7,11,13-hexdectrienl given tht pproximtely 1 dys of stedy relese occurred following n initil rief (c. 7 dys) urst of higher relese. The dvntges of SPLAT s formultion for P. citrell disruption include low cost of mnufcturing, iodegrdle nd wether resistnt chrcteristics, nd flowility llowing mchine ppliction. Mting disruption should e n effective lterntive to insecticides for mngement of P. citrell nd my reduce the incidence of citrus cnker. Introduction The citrus lefminer, Phyllocnistis citrell Stinton (Lepidopter: Grcillriide), is n importnt pest of commercil citrus production throughout the world (Heppner 1993). Lrve dmge leves y creting serpentine feeding mines, which hve een shown to reduce yield (Peñ et l. 2). More importntly, 512 J. Appl. Entomol. 134 (21) ª 29 Blckwell Verlg, GmH

2 L. L. Stelinski, S. L. Lpointe nd W. L. Meyer Citrus lefminer mting disruption lef wounds cused y lrvl feeding predisposes trees to infection y citrus cteril cnker, Xnthomons xonopodis pv. citri, which results in lemished fruit, premture fruit drop, lef drop, nd tree decline (Grhm et l. 24). Feeding P. citrell lrve wound the lef cuticle, which exposes lef mesophyll to direct infection. Wounds cused y P. citrell do not hel redily, which further increses the exposure period to the cterium nd its spred y moile lrve throughout feeding glleries (Grhm et l. 24). Phyllocnistis citrell is pest of ll citrus vrieties s well s other Rutcee nd certin ornmentls; however, grpefruit, tngerine, nd pumello re the most susceptile hosts (Legspi nd French 23). Control of lrvl P. citrell with insecticides is inconsistent nd t times ineffective ecuse lrve within the lef mines re protected from folir spry residues. Typicl spry progrms my require iweekly insecticide pplictions to protect emerging, highly susceptile lef flush. Such progrms lso reduce the popultions of nturl enemies of P. citrell nd other citrus pests (Peñ et l. 22). Development of effective lterntives to conventionl insecticides for P. citrell mngement is of criticl importnce for leding citrus-producing countries, including Brzil nd the USA, where citrus cnker is known to limit yield nd profit (Leite nd Mohn 199). The complete sex pheromone lend of P. citrell ws recently reported (Lel et l. 26; Moreir et l. 26). Of three totl components identified, 3 : 1 lend of (Z,Z,E)-7,11,13-hexdectrienl nd (Z,Z)-7,11-hexdecdienl is sufficient to optimlly ttrct mles to pheromone trps (Lel et l. 26; Moreir et l. 26); however, neither single component is ttrctive y itself (Lpointe et l. 29). Following identifiction of the pheromone, it ws confirmed tht it is highly ttrctive to mle P. citrell in Florid, USA (Lpointe et l. 26). Monitoring protocols with pheromone trps were optimized soon therefter (Stelinski nd Rogers 28). Furthermore, mting disruption of P. citrell hs een investigted in oth Jpn (Mfi et l. 25) nd the USA (Stelinski et l. 28). Control of P. citrell y mting disruption hs proven highly effective t remrkly low deployment rte of pheromone [1.5 g of ctive ingredient (AI)/h] (Stelinski et l. 28). However, the high cost of synthesis for the P. citrell pheromone components limits the commercil viility of mting disruption for this pest even though very little pheromone is needed to chieve efficcy. Given the species-specific mode of ction of mting disruption, P. citrell mngement my e constrined due to the emergence of Diphorin citri Kuwym s nother glol pest currently thretening the sustinility of citriculture (Hlert nd Mnjunth 24). Diphorin citri vectors phloem-restricted cteri in the genus Cndidtus Liericter tht cuse hunglonging disese, which kills citrus trees (Bové 26). Therefore, oth effective nd economicl ehviourl modifiction tools re needed for mngement of P. citrell. One potentil option for more economicl mting disruption for P. citrell is development of single pheromone component relese device. This option is fesile ecuse mting disruption of P. citrell occurs y non-competitive mechnism; i.e., cmouflge or desensitiztion (Stelinski et l. 28; Lpointe et l. 29), nd ecuse oth individul components cn disrupt mle orienttion to the complete two-component ttrctive lend (Lpointe et l. 29). Given tht (Z,Z)-7,11-hexdecdienl is much less expensive to synthesize thn (Z,Z,E)-7,11,13-hexdectrienl (Lpointe et l. 29), we sought to determine whether disruption of P. citrell could e chieved y deploying this single component in n effort to reduce cost of mting disruption for this pest. Furthermore, lthough disruption of P. citrell hs proven effective with hnd-pplied ruer devices (Stelinski et l. 28), flowle formultion mendle to mchine ppliction (Stelinski et l. 27) is required if mting disruption is to e dopted on lrge (>5 h) citrus growing opertions, which occur in the USA nd Brzil. Given tht commercilly produced citrus is grown on such lrge scles, hnd ppliction of pheromone dispensers would e economiclly prohiitive. The gol of this investigtion ws to develop flowle emulsified wx formultion for pheromone relese mendle to mechnicl ppliction for lrge scle mting disruption of P. citrell. The specific ojectives of this investigtion were to: (i) compre the effect of the ttrctive two-component P. citrell pheromone lend vs. single pheromone component when relesed from emulsified wx on disruption of mle moth orienttion nd crop injury; (ii) evlute two loding rtes of ech pheromone tretment; (iii) gin insights into the mechnism of disruption y direct oservtion of moth ehviour in the field; nd (iv) quntify seson-long pheromone relese rte y gs chromtogrphy in order to relte efficcy dt with sesonl pheromone relese. J. Appl. Entomol. 134 (21) ª 29 Blckwell Verlg, GmH 513

3 Citrus lefminer mting disruption L. L. Stelinski, S. L. Lpointe nd W. L. Meyer Mterils nd Methods Pheromone relese formultion A flowle formultion of emulsified wx designed to provide slow relese of semiochemicls (SPLAT TM ; ISCA Technologies, Riverside, CA) ws formulted with either (Z,Z,E)-7,11,13-hexdectrienl nd (Z,Z)-7,11-hexdecdienl (henceforth referred to s triene nd diene, respectively) or the diene lone, ech t two loding dosges. Both pheromone components were synthesized y ISCA Technologies Inc., s previously descried (Lel et l. 26; Moreir et l. 26). The triene ws 94% nd 9% chemiclly nd isomericlly pure, respectively, while the diene ws 86% nd 72% chemiclly nd isomericlly pure, respectively. The diene ws chosen s the single component for evlution ecuse P. citrell disruption occurs vi non-competitive mechnism (Stelinski et l. 28) nd cn e chieved with either the triene or diene (Lpointe et l. 29); however, the diene is pproximtely 1-fold less expensive to synthesize thn the triene (Lpointe et l. 29). Ech pheromone lend formultion ws incorported into SPLAT t.2% or.2% AI y weight. This resulted in totl of four pheromone tretments for field testing. Field plots nd experimentl design The experiment ws conducted in 9-yer-old 4.5- h ornge orchrd [(Citrus sinensis [L.]) vr Vlenci. ] ner Orlndo, FL, USA. Trees were plnted on 3 6 m spcing nd verge cnopy height ws 4 m. The orchrd ws mnged y the University of Florid ccording to commercil pruning, irrigtion, hericide, nd fungicide mngement prctices ut without insecticides. Disruption trils were conducted compring five tretments: (i) untreted control; (ii) 3 : 1 triene:diene lend t.2% AI loding; (iii) 3 : 1 triene:diene lend t.2% AI loding; (iv) diene lone t.2% AI loding; nd (v) diene lone t.2% AI loding. The experiment ws rrnged s rndomized complete lock design with five replictes, ech replicte consisting of pproximtely 1 trees (c..25 h). Replicte plots were seprted y 4 m nd locks of tretments were seprted y 5 m. SPLAT tretments were pplied using custom mde hnd-held pplictors clirted to deliver 1. g dollops. Point source density nd concentrtion of pheromone tested were sed on previously optimized disruption reported with the 3 : 1 triene:diene lend relesed from ruer septum dispensers (Stelinski et l. 28). Ech tree within tretment plot received pproximtely 3 dollops of SPLAT resulting in pproximtely 49 g of ech formultion per h. SPLAT ws pplied to tree rnches within cnopies pproximtely 2. m ove ground level, which is the loction of gretest mle P. citrell ctivity within trees pproximtely 4 m tll (Stelinski nd Rogers 28). The experiment ws divided into two 12-week intervls comprising (i) spring/erly summer nd (ii) lte summer/fll. Tretment reppliction ws deemed necessry t 12 weeks into the tril sed on recent investigtion of P. citrell disruption with ruer sleeve dispenser of pheromone (Stelinski et l. 28) nd ecuse disruption of trp ctch in some tretments hd declined to levels comprle to controls (see Results). Tretments were pplied on 1 My 28 nd 23 July 28; disruption of P. citrell flight nd ssocited dmge y lrve were evluted over the course of n entire growing seson. Tretment evlution Disruption of mle P. citrell orienttion ws quntified using two pheromone trps (LPD Scenturion Gurdpost; Suterr, Bend, OR) deployed within ech replicte plot. One trp ws plced in the centrl tree of ech plot nd the second ws plced two rows from the plot edge. All trps were ited with single red ruer septum lure loded with.1 mg of triene nd.3 mg of diene s this hs een shown to e highly effective for trpping mle P. citrell in Florid (Lpointe et l. 26; Stelinski nd Rogers 28). Monitoring trps were hung t lest 1. m from the nerest mting disruption dispenser, t pproximtely m ove ground level in mid-cnopy (Stelinski nd Rogers 28). Pheromone lures were replced pproximtely every 6 weeks throughout the seson sed on known longevity of ttrctiveness (Lpointe nd Lel 27). Adhesive trp liners were replced nd counted weekly. In ddition to monitoring disruption y quntifying mle ctch in pheromone trps, dmge to newly flushed leves ws ssessed t the end of two 12-week tretment deployment intervls on 23 July nd 1 Octoer 28. Twenty shoots, 1 from midcnopy (2.5 m) nd 1 from lower cnopy (1. m), were inspected t rndom from 2 trees per replicte lock (2 flush smples per tretment) nd the numer of shoots per tree contining live mining P. citrell lrve ws recorded. 514 J. Appl. Entomol. 134 (21) ª 29 Blckwell Verlg, GmH

4 L. L. Stelinski, S. L. Lpointe nd W. L. Meyer Citrus lefminer mting disruption Behviourl oservtions in the field SPLAT droplets from ech of the four formultion tretments were directly oserved in the field to test the hypothesis tht mle P. citrell pproch pheromone dispensers. Previous investigtions hve confirmed tht mle moths of vrious species orient to, closely pproch, nd even contct pheromone dispensers of wide rnge of relese rtes in the field, which suggests tht competitive ttrction is n opertive mechnism of disruption for these species (Stelinski et l. 24; Epstein et l. 26). Oservtions of dispensers in tree cnopies were conducted for pproximtely 2 h ech night etween 21: nd 23: hours, the time when mle P. citrell re known to respond to pheromone lures in the field (Stelinski nd Rogers 28). Oservtions were conducted on 1 nights etween 5 My nd 1 Octoer 28 (5, 2 My; 6, 13 June; 8, 14 July; 15, 28 August; 12 Septemer; 1 Octoer). An oserver rotted mong plots conducting 2-min oservtionl outs per tretment such tht multiple tretments were oserved on given night. The order of oservtions cross tretments ws rndomized nightly nd different sets of dispensers were oserved on ech night. In ddition, mle moth orienttion to pheromone trps (s descried ove) ws oserved for 2-h periods during the sme intervl in control plots to verify tht mle P. citrell could e oserved orienting to n ttrctive point source in the field under the sme conditions during which SPLAT dispensers were eing oserved. The numer of moths oserved orienting to trps nd the numer cught in trps under oservtion were ssessed. In oservtions of oth dispensers nd trps, dt were dictted into hnd-held microcssette udio recorder y n investigtor stnding.25.5 m from the pheromone source under oservtion. Oservtions employed night-vision goggles (Rigel, Model 325, DeWitt, IA) s descried y Stelinski et l. (24). Relese rte mesurements Smples of the 3 : 1 triene:diene lend formultion of SPLAT t the.2% pheromone loding rte were pplied s 1. g dollops to 2 3 cm cette film strips. The strips were stpled individully to five 5 45 cm wooden ords with wires ttched t ech corner. One smple ws tken from ech ord t test onset nd prior to field ging nd plced in pre-clened (cetonitrile rinse) 2-ml scintilltion vil (Fisher Scientific, Pittsurgh, PA). Five millilitres of n internl stndrd solution of 14 ng/ll of undecnl in cetonitrile (99.9% purity; Sigm-Aldrich, St. Louis, MO) ws dded to ech vil. These smples were plced t )1 C until use for susequent nlyses. The ords contining the remining smples were mintined t 24 C in the lortory for 24 h nd then plced in the field on 22 My 28. The ords were hung horizontlly within the cnopies of five 4-m-tll trees in mixed citrus plnting t the Citrus Reserch nd Eduction Center (Lke Alfred, FL, USA). The ords were hung pproximtely 1.5 m ove ground on the edges of trees in prtil shde creted y higher rnches. Smples were collected from ll ords 1, 2, 3, 4, nd 7 dys following deployment in the field nd then weekly for totl of 15 weeks. All smples were trnsported to the lortory individully within scintilltion vils with 5 ml of the internl stndrd solution descried ove. All smples were mintined frozen s descried ove prior to nlyses. Immeditely efore nlysis, ech smple ws removed from the freezer nd plced in wter th t 8 85 C for 3 5 min. Therefter, vils were shken for 1 min nd returned to the wter th for 3 min, then removed nd gitted gin. The solvent ws removed from ech smple with glss Psteur pipette nd filtered through second pipette, fitted with cm tringulr piece of Kimwipe (Kimerly-Clrke Corp., Roswell, GA) folded to form plug t the tpered end of the pipette, into preclened 8-ml vil with polytetrfluoroethylenelined cp (Chromtogrphy Reserch Supplies Inc., Louisville, KY). Pheromone within smples ws quntified using Vrin CP38 gs-chromtogrph (GC) (Vrin Inc., Plo Alto, CA) fitted with 3-m Rtx-5 cpillry column (.25 mm I.D.;.5 lm film) (Restek Corp., Bellefonte, PA) nd equipped with flmeioniztion detector. The initil GC temperture ws held t 5 C for 1 min nd then incresed t rte of 1 C/min to finl temperture of 26 C where it ws held for 1 min. The crrier gs ws He t flow rte of 1 ml/min. One microlitre of ech smple vil ws injected for nlysis. The pheromone content of the smples ws clculted using the internl stndrd method (McNir nd Miller 1998) nd ws corrected for the originl weight of ech smple. Sttisticl nlyses nd other clcultions Moth ctch dt were trnsformed to ln (x + 1) to normlize distriution nd homogenize vrince nd then sujected to nlysis of vrince (nov). Per J. Appl. Entomol. 134 (21) ª 29 Blckwell Verlg, GmH 515

5 Citrus lefminer mting disruption L. L. Stelinski, S. L. Lpointe nd W. L. Meyer cent injury dt from the two smpling dtes were rcsine trnsformed prior to nov. Mens were seprted y Tukey s test (SAS Institute 2) following significnt novs ( =.5). Percent orienttionl disruption of mle moth ctch in trps ws clculted s 1 ) (men moth ctch per trp in the pheromone-treted plot/men moth ctch per trp in the control plot) 1. Results Disruption of mle P. citrell ctch in pheromone trps During the 12-week period following the first SPLAT ppliction, disruption of mle P. citrell ctch in pheromone trps in plots treted with.2% 3 : 1 lend ws consistently round 9%, reltive to control plots (fig. 1). Moth ctch in trps within plots treted with.2% 3 : 1 lend ws reduced only during the first 2 weeks compred with untreted plots (fig. 1). During the initil 7 weeks of the erly seson tril, disruption of mle trp ctch in plots treted with.2% diene rnged from 5% to 9%; Men (±SEM) numer of P. citrell cptured per trp per week () Erly seson (7 My 23 July) Control Diene.2% Diene.2% 3 : 1 lend.2% 3 : 1 lend.2% 5/7 5/21 6/4 6/18 7/2 7/16 () Lte seson (3 July 15 Oct) Control Diene.2% Diene.2% 3 : 1 lend.2% 3 : 1 lend.2% 7/3 8/13 8/27 9/1 9/24 1/8 Dte Fig. 1 Men SEM cptures of Phyllocnistis citrell mles per week in the spring nd erly summer () nd lte summer nd fll () throughout the 28 seson in replicted.25-h pheromone-treted nd control plots. Pheromone tretments were pplied on 1 My nd 23 July. 3 : 1 Blend refers to 3 : 1 lend of (Z,Z,E)-7,11,13-hexdectrienl nd (Z,Z)-7,11-hexdecdienl. Diene refers to (Z,Z)-7,11-hexdecdienl. y the eighth week there ws no longer discernle effect of this tretment reltive to the control (fig. 1). Weekly mle ctch in plots treted with.2% diene ws pproximtely equivlent to tht oserved in control plots (fig. 1). Over the course of the initil 12 weeks of the investigtion, significntly (F = 12.3; d.f. = 4, 16; P <.1) fewer mle P. citrell were cptured in plots treted with.2% diene nd 3 : 1 lend thn in control plots (fig. 2). However, significntly (P <.1) fewer mles were cptured in plots treted with.2% 3 : 1 lend compred with.2% diene (fig. 2). At the.2% loding dosge, neither pheromone tretment formultion reduced moth ctch in trps compred with the control (P vlues >.5) (fig. 2). During the 12-week period following the second SPLAT ppliction (lte summer/fll), P. citrell popultion densities were generlly lower compred with the period following the first SPLAT ppliction (spring/erly summer) (fig. 1). Disruption of mle ctch in plots treted with.2% 3 : 1 lend verged pproximtely 9% ut tended to decrese ner the end of the tril (fig. 1). Disruption following the second SPLAT ppliction with.2% diene lsted 6 weeks, similr to tht oserved during spring nd erly summer (fig. 1). Disruption of 4 8% ws Cumultive no. mles per trp per trpping period () () Control Diene.2% Diene 3 : 1 lend.2%.2% Tretment c c 3 : 1 lend.2% Fig. 2 Men SEM cptures of mle Phyllocnistis citrell in pheromone-ited trps in plots treted with vrious formultions of SPLAT pheromone dispensers over the course of two 12-week periods during erly () nd lte () seson. 3 : 1 Blend refers to 3 : 1 lend of (Z,Z,E)-7,11,13-hexdectrienl nd (Z,Z)-7,11-hexdecdienl. Diene refers to (Z,Z)-7,11-hexdecdienl. Brs within ech pnel followed y the sme letter re not significntly different ( =.5, ANOVA followed y Tukey s test). 516 J. Appl. Entomol. 134 (21) ª 29 Blckwell Verlg, GmH

6 L. L. Stelinski, S. L. Lpointe nd W. L. Meyer Citrus lefminer mting disruption oserved with.2% 3 : 1 lend for the initil 4 weeks following the second SPLAT ppliction (fig. 1), which ws pproximtely twice s long s tht oserved during the erly seson intervl (fig. 1). Consistent with the first prt of the seson, there ws no evidence of disruption with.2% diene throughout this second deployment intervl (fig. 1). Over the course of the entire second deployment intervl, significntly (F = 8.9; d.f. = 4, 16; P <.1) fewer mle P. citrell were cptured in plots treted with.2% diene s well s in those treted with the 3 : 1 lend t oth loding dosges compred with control plots (fig. 2). However, significntly (P <.1) fewer mles were cptured in plots treted with.2% 3 : 1 lend thn in ny other tretment (fig. 2). Lef infesttion y lrvl P. citrell Leves infested y P. citrell (%) () () Control Diene.2% Diene.2% Tretment 3 : 1 lend 3 : 1 lend.2%.2% Fig. 3 Men SEM percent lef flush infesttion y Phyllocnistis citrell lrve during erly () nd lte () seson s influenced y vrious lend nd dosge tretments of SPLAT pheromone dispensers pplied to replicte.25-h plots of citrus. Evlutions were conducted t the end of two 12-week periods on 23 July nd 1 Octoer. On ech dte, 2 lef flushes were evluted per tretment. Brs within ech pnel followed y the sme letter re not significntly different ( =.5, ANOVA followed y Tukey s test). Following the initil 12 weeks of the study, significntly (F = 7.4; d.f. = 4, 16; P <.1) fewer flush shoots were infested with lrvl P. citrell in plots treted with.2% 3 : 1 lend compred with control plots (fig. 3). There ws no significnt (P vlues >.5) difference in injury etween ny of the other tretments nd the control (fig. 3). At the end of the study, significntly (F = 6.4; d.f. = 4, 16; P =.5) less lef injury ws recorded in plots treted with.2% 3 : 1 lend compred with control plots (fig. 3). There ws no difference (P vlues >.5) in injury etween ny of the other tretments tested compred with the control (fig. 3). Behviourl oservtions in the field No mle P. citrell were oserved orienting to SPLAT dollops in plots treted with ny of the pheromone formultions evluted during 2 h of oservtion over 1 dys of smpling. In contrst, (men SEM) mles/night were oserved orienting to monitoring trps ited with 3 : 1 lure (N =1 nights) in control plots. Of those moths oserved, mle P. citrell were cptured/trp per night on verge. Relese rte mesurements The relese profile of triene from SPLAT over the course of the seson, shown in fig. 4, fits third order polynomil decy curve t n R 2 =.98. During the initil 7 dys of mesurement, relese rte of triene occurred t pproximtely 7 lg/dy (fig. 4). However, for the remining 14 dys of mesurement, the relese rte ppered reltively liner t pproximtely 6.3 lg of triene/dy (fig. 4). During this ltter 14 dys of relese, pheromone loss per dy from SPLAT dollops remined t constnt decy of pproximtely.5 4.% (fig. 4). Discussion Of the SPLAT tretments evluted, the formultion contining 3 : 1 lend of the two-component ttrctive P. citrell lend ws the only one to cuse seson-long disruption s well s reduce lef infesttion. However, it required deploying 49 g/h of the SPLAT formultion contining.2% AI y weight twice per seson, which mounts to deploying 2 g of P. citrell pheromone per h per seson. Although reduced P. citrell lrvl injury should contriute to reduced incidence of citrus cnker infection (Grhm et l. 24), we were unle to document this hypothesis ecuse the disese did not pper to e present in the citrus orchrd where experiments were conducted (dt not shown). Recently, Lpointe et l. (29) demonstrted tht disruption J. Appl. Entomol. 134 (21) ª 29 Blckwell Verlg, GmH 517

7 Citrus lefminer mting disruption L. L. Stelinski, S. L. Lpointe nd W. L. Meyer () g of Triene (±SEM) remining per g of SPLAT () % loss of Triene per dy y =.2x x 2 9E + 6x + 1E + 11 R 2 = Dys following deployment Fig. 4 Weekly relese profile of (Z,Z,E)-7,11,13-hexdectrienl from 1. g dollops of SPLAT contining.2% of 3 : 1 lend of (Z,Z,E)- 7,11,13-hexdectrienl nd (Z,Z)-7,11-hexdecdienl y weight. of P. citrell is lrgely driven y the triene pheromone component; therefore, relese of this component from the two 3 : 1 lends proly medited disruption in this tril. Our relese rte quntifiction indicted tht effective disruption of P. citrell occurred when 1 g SPLAT dollops were relesing.26 lg of triene/h. Therefore, pproximtely 126 lg of triene/h/h re required to effectively disrupt P. citrell. Mle P. citrell did not pproch dollops of SPLAT of ny formultion tested in this investigtion, which further confirms tht disruption of this species occurs y non-competitive mechnism (Stelinski nd Rogers 28; Lpointe et l. 29). The mount of pheromone required for effective disruption of P. citrell is much lower thn wht is required for other species. For exmple, 5 2 mg AI/ h/h re required for effective disruption of the orientl fruit moth, Grpholit molest (Busck); 15 mg AI/h/h re required for lightrown pple moth, Epiphys postvittn (Wlker); nd 1 35 mg/h/h re required for grpe erry moth, Prloesi viten (Clemens); severl other exmples re in the sme rnge of tens of mg AI/h/h for effective disruption (reviewed y Crdé nd Minks 1995). Given tht disruption of P. citrell did not fll elow 9% with the 3 : 1 lend tretment t the.2% loding level, it is possile tht the threshold relese rte for effective disruption is elow 1 lg of triene/h/h. Therefore, the pheromone relese rte required to disrupt P. citrell is pproximtely 1-fold lower thn for other lepidoptern species where this hs een quntified. The SPLAT formultion ppers to e n excellent relese device for the P. citrell pheromone given tht pproximtely 1 dys of stedy relese occurred following n initil rief (c. 7 dys) urst of higher relese (fig. 4). These results re in contrst to wht hs een quntified with the mjor pheromone components of codling moth [Cydi pomonell (L.)], (E,E)-8,1-dodecdien-1-ol, (Epstein et l. 26); G. molest, (Z)-8-dodecen-1-yl-cette, (Stelinski et l. 25); nd P. viten, (Z)-9-dodecenyl cette, (Jenkins nd Iscs 28) from SPLAT emulsified wx formultions. In ech of those cses, the pheromones evported from emulsified wx ccording to first order exponentil functions. The proility of insect pheromone evportion from solid surfces decreses s chin length of the chemicl increses nd my e influenced y functionl groups (Gut et l. 24). Therefore, it is possile tht the seemingly liner nd much lower relese rte of the principl P. citrell pheromone component [(Z,Z,E)-7,11,13-hexdectrienl] from SPLAT reltive to the 12 cron tortricid pheromones discussed ove, is due its longer chin length. The remrkly low concentrtion of pheromone required for effective disruption of P. citrell comined with its fvourle relese chrcteristics from wx dispensers mkes it n excellent cndidte for control y mting disruption. Unfortuntely, our explortions to reduce cost of mting disruption formultions for P. citrell y either decresing the mount of AI per g of SPLAT or relesing the diene component lone were unsuccessful. The.2% 3 : 1 triene:diene lend disrupted mle P. citrell for only 2 5 weeks (fig. 1,) suggesting tht relese rte from this formultion rpidly dropped elow the threshold required for effective disruption. This durtion of efficcy would likely require t lest six deployments of the.2% 3 : 1 lend formultion to mintin seson-long disruption. The.2% diene formultion lso showed some promise cusing pproximtely 6 7 weeks of mesurle disruption, ut ws lso not sufficiently 518 J. Appl. Entomol. 134 (21) ª 29 Blckwell Verlg, GmH

8 L. L. Stelinski, S. L. Lpointe nd W. L. Meyer Citrus lefminer mting disruption effective to mintin seson-long disruption. The first investigtion of P. citrell mting disruption reported ner 1% effective orienttion disruption with the diene lone; lthough, the smll.8-h test plots did not resolve tretment effect with respect to lrvl infesttion (Mfi et l. 25). However, Mfi et l. (25) deployed the diene from 5 to 13 polyethylene tues per h t loding of 6 mg of diene per tue. Although they did not quntify relese rte from the polyethylene tues, such dispensers loded with tht quntity of pheromone re known to relese 1 3 mg of AI/h/h (Witzgll et l. 28); therefore, the relese rte of diene in their study my hve een t lest n order of mgnitude higher thn in the current investigtion. Furthermore, Lpointe et l. (29) recently provided evidence tht 2% diene only SPLAT formultion my e pproximtely equivlent to the.2% 3 : 1 lend SPLAT formultion tested in this study; however, their tril ws conducted for only 26 dys in lte seson (Octoer). The ove results suggest tht effective seson-long disruption of P. citrell should e possile with the diene component lone. In future studies, we pln on testing the efficcy of diene formultions t deployment rtes higher thn those tested herein with seson-long trils. The threshold relese rte for effective disruption of P. citrell with the diene component remins to e determined. Given tht the diene comprises only third of the 2-component ttrctive P. citrell lend, it is perhps not surprising tht more diene thn triene should e required for effective disruption of P. citrell y non-competitive mechnism such s desensitiztion or sensory imlnce (Brtell 1982). The emulsified wx dispenser for semiochemicl relese ws developed over decde go (Atterholt et l. 1998). Since then, effective formultions hve een developed, nd in some cses re now commercilly ville, for severl lepidoptern pests including C. pomonell (Epstein et l. 26), G. molest (Stelinski et l. 27), P. viten (Jenkins nd Iscs 28) the gypsy moth, Lymntri dispr (L.), (Tcheslvski et l. 28) mong others. Recently, n ttrct & kill version of n emulsified wx formultion ws lso developed for control of diptern, the orientl fruit fly [Bctrocer dorslis (Hendel)] (Vrgs et l. 29). Emulsified wx formultions, such s SPLAT, for relese of semiochemicls offer severl dvntges over more trditionl hnd-pplied reservoir dispensers. They re inexpensive to mnufcture nd store, iodegrdle, reltively wetherproof, nd mendle to mchine ppliction y ground or ir equipment. Mchine ppliction would e requirement for P. citrell mngement y mting disruption in the min citrus-producing countries such s the USA nd Brzil given tht citrus is grown s multi-thousnd h monocultures. Mechnized ppliction devices hve een developed for SPLAT (Stelinski et l. 27) nd should e esy to dopt for ppliction in commercil citrus. ISCA Technologies hs initited registrtion of n emulsified wx formultion for control of P. citrell y mting disruption (SPLAT-CLM TM ) in commercil citrus in the USA nd Brzil. In the interim, we continue to optimize the formultion nd re developing mechnized delivery system for use in commercil citriculture. Acknowledgements In Jckson nd Angel Hoyte re grtefully cknowledged for ssistnce with pheromone nd trp deployment nd susequent moth ctch dt collection. LLS thnks the IR-4 Project for prtilly funding this project. A previous version of the mnuscript ws improved y Drs Steve Futch (University of Florid), Wyne Hunter (United Sttes Deprtment of Agriculture-Agriculture Reserch Service), nd two nonymous referees. References Atterholt CA, Delwiche MJ, Rice RE, Krocht JM, Study of iopolymers nd prffin s potentil controlled-relese crriers for insect pheromones. J. Agric. Food. Chem. 46, Brtell RJ, Mechnisms of communiction disruption y pheromone in control of Lepidopter: review. Physiol. Entomol. 7, Bové JM, 26. Hunglonging: destructive, newlyemerging, century-old disese of citrus. J. Plnt Pthol. 88, Crdé RT, Minks AK, Control of moth pests y mting disruption: successes nd constrints. Annu. Rev. Entomol. 4, Epstein DL, Stelinski LL, Reed TP, Miller JR, Gut LJ, 26. Higher densities of distriuted pheromone sources provide disruption of codling moth (Lepidopter: Tortricide) superior to tht of lower densities of clumped sources. J. Econ. Entomol. 99, Grhm JH, Gottwld TR, Cuero J, Achor DS, 24. Xnthosmons xonopodis pv. citri: fctors ffecting successful erdiction of citrus cnker. Mol. Plnt Pthol. 5, Gut LJ, Stelinski LL, Thompson DR, Miller JR, 24. Behvior modifying chemicls: prospects nd J. Appl. Entomol. 134 (21) ª 29 Blckwell Verlg, GmH 519

9 Citrus lefminer mting disruption L. L. Stelinski, S. L. Lpointe nd W. L. Meyer constrints in IPM. In: Integrted pest mngement: potentil, constrints, nd chllenges. Ed. y Koul O, Dhliwl GS, Cuperus G, CABI Press, Wllingford, UK, Hlert SE, Mnjunth KL, 24. Asin citrus psyllids (Sternorrhynch: Psyllide) nd greening disese of citrus: literture review nd ssessment of risk in Florid. Fl. Entomol. 87, Heppner JB, Citrus lefminer, Phyllocnistis citrell, in Florid. Trop. Lepidopter. 4, Jenkins PE, Iscs R, 28. Mting disruption of Prloesi viten in vineyrds using pheromone deployed in SPLAT-GBM TM wx droplets. J. Chem. Ecol. 34, Lpointe SL, Lel WS, 27. Descriing sesonl phenology of the lefminer Phyllocnistis citrell (Lepidopter: Grcillriide) with pheromone lures: controlling for lure degrdtion. Fl. Entomol. 9, Lpointe SL, Hll DG, Murt Y, Prr-Pedrzzoli AL, Bento JMS, Vilel EF, Lel WS, 26. Field evlution of synthetic femle sex pheromone for the lefmining moth Phyllocnistis citrell (Lepidopter: Grcillriide) in Florid citrus. Fl. Entomol. 89, Lpointe SL, Stelinski LL, Niedz RP, Hll DG, Mfr-Neto A, 29. Sensory imlnce s mechnism of orienttion disruption in the lefminer Phyllocnistis citrell: elucidtion y multivrite geometric designs nd response surfce models. J. Chem. Ecol. (in press). Lel WS, Prr-Pedrzzoli AL, Cosse AA, Murt Y, Bento JMS, Vilel EF, 26. Identifiction, synthesis, nd field evlution of the sex pheromone from citrus lefminer, Phyllocnistis citrell. J. Chem. Ecol. 32, Legspi JC, French JV, 23. The citrus lefminer nd its nturl enemies [WWW document]. URL primr.tmu.edu/kcchome/pus/lefminer.htm. Leite RP Jr, Mohn SK, 199. Integrted mngement of the citrus cteril disese cused y Xnthomons xonopodis pv. citri in the stte of Prná, Brzil. Crop Prot. 9, 3 7. Mfi SA, Vng LV, Nkt Y, Ohyshi N, Ymmoto M, Ando T, 25. Identifiction of the sex pheromone of the citrus lefminer (Phyllocnistis citrell Stinton, Lepidopter: Grcillriide) with tril of control y the communiction disruption method. J. Pestic. Sci. 34, McNir HM, Miller JM, Bsic gs chromtogrphy. Wiley, New York. Moreir JA, McElfresh JS, Millr JG, 26. Identifiction, synthesis, nd field testing of the sex pheromone of the citrus lefminer, Phyllocnistis citrell. J. Chem. Ecol. 32, Peñ JE, Hunserger A, Schffer B, 2. Citrus lefminer (Lepidopter: Grcillriide) density: effect on yield of Thiti lime. J. Econ. Entomol. 93, Peñ JE, Shrp JL, Wysoki M, 22. Tropicl fruit pests nd pollintors: iology, economic importnce, nturl enemies, nd control. CABI Press, Wllingford, UK. SAS Institute, 2. SAS/STAT user s guide, version 6. 4th edition, Vol. 1. SAS Institute, Cry, NC. Stelinski LL, Rogers ME, 28. Fctors ffecting cptures of mle citrus lefminer, Phyllocnistis citrell, in pheromone-ited trps. J. Appl. Entomol. 132, Stelinski LL, Gut LJ, Pierzchl AV, Miller JR, 24. Field oservtions quntifying ttrction of four tortricid moth species to high-dosge, polyethylene-tue pheromone dispensers in untreted nd pheromone-treted orchrds. Entomol. Exp. Appl. 113, Stelinski LL, Gut LJ, Mllinger RE, Epstein D, Reed TP, Miller JR, 25. Smll plot trils documenting effective mting disruption of orientl fruit moth y using high densities of wx-drop pheromone dispensers. J. Econ. Entomol. 98, Stelinski LL, Miller JR, Ledeuhr R, Gut LJ, 27. Seson-long mting disruption of Grpholit molest y one mchine ppliction of pheromone in wx drops (SPLAT-OFM). J. Pest. Sci. 8, Stelinski LL, Miller JR, Rogers ME, 28. Mting disruption of citrus lefminer medited y non-competitive mechnism t remrkly low pheromone relese rte. J. Chem. Ecol. 34, Tcheslvski K, Thorpe K, Zeni D, Bernrdi C, Mfr-Neto A, Coler R, 28. The new pheromone delivery system for gypsy moth mting disruption. In: Proceedings, 18th U.S. Deprtment of Agriculture intergency reserch forum on gypsy moth nd other invsive species 27; 27 Jnury 9 12; Annpolis, MD. Gen. Tech. Rep. NRS-P-28. Ed. y Gottschlk KW, U.S. Deprtment of Agriculture, Forest Service, Northern Reserch Sttion, Newtown Squre, PA, 78 pp. Vrgs RI, Piñero JC, Mu RFL, Strk JD, Hertlein M, Mfr-Neto A, Coler R, Getchell A, 29. Attrction nd mortlity of orientl fruit flies to SPLAT-MATmethyl eugenol with spinosd. Entomol. Exp. Appl. 131, Witzgll P, Stelinski L, Gut L, Thomson D, 28. Codling moth mngement nd chemicl ecology. Annu. Rev. Entomol. 53, J. Appl. Entomol. 134 (21) ª 29 Blckwell Verlg, GmH