TurkJBiol 25(2001)419-425 ©TÜB‹TAK ViralControloftheEuropeanPineSawfly, Neodiprionsertifer (Geoffroy)inTurkey MustafaYAMAN,RemziyeNALÇACIO⁄LU,ZihniDEM‹RBA⁄ DepartmentofBiology,FacultyofArtsandSciences,KaradenizTechnicalUniversity, 61080,Trabzon-TURKEY Received:23.10.2000 Abstract: TheEuropeanpinesawfly, Neodiprionsertifer (Geoffr.)(Hymenoptera:Diprionidae),isa seriouspestofpineplantationsandornamentals. Chemicalpesticidesusedtocontrolthispesthavea detrimentaleffectontheenvironment.The Neodiprionsertifer nuclearpolyhedrosisvirus(NsNPV) frequentlycausesconsiderabledecreasesinthepopulationsbut dosesnotharmotheranimalsor plants. Inthisstudy,wetestedfivedifferentconcentrationsofNsNPVagainstthelarvaeofN.sertifer andshowed viralinfectionusingGiemsastaining.Whenaviralconcentrationof5x10 9 PIB/ml (PolyhedralInclusionBody)wasutilized,larvalmortalitystartedonthefourthdayandreached 90.4%ontheninthdayofthebioassay.However, lowerviralconcentrations,5x10 9,5x10 8 and 5x107 PIB/ml,showedthesamemortality,buttheinfectionperiodsoccurredondifferentdays.This studyshowsthatNsNPVcanbeeffectivelyusedagainst N.sertifer asasafebiologicalcontrolagent inTurkey. KeyWords: Neodiprionsertifer,NuclearPolyhedrosisVirus,BiologicalControl Neodiprionsertifer (Geoffr.)(Hymenoptera:Diprionidae)’in ViralKontrolü Özet: K›rm›z›mt›raksar›çal›antenliyaprakar›s› Neodiprionsertifer (Geoffr.)(Hymenoptera: Diprionidae)çamfidanl›klar›n›nönemlizararl›lar›ndanbiridir.Buzararl›y›kontroletmekiçinkullan›lan kimyasalpestisidlerçevredezararl›etkileresahiptir. N.sertifer nüklearpolihedrozisvirüsü(NsNPV) buzararl›n›npopulasyonundaönemlibirazalmayanedenolurken,çevredekidi¤erbitkivehayvanlar› etkilememektedir.Bumakalede, N.sertifer larvalar›nakarfl›NsNPV’nin farkl›konsantrasyonlar›test edildiveviralenfeksiyongiemsaboyamametoduylagösterildi.Larvalölümtestsüresinindördüncü günündebafllarken,5x10 9 PIB/mlkonsantrasyonunda9.günde %90.4’eulaflt›.5x10 9,5x108 and 5x107 PIB/mlkonsantrasyonlar›ayn›ölümoran›n›gösterirken,enfeksiyonsürelerifarkl›günlerde geliflti.Buçal›flmaNsNPV’nin N.sertifer’ekarfl›Türkiye’deetkilibirbiyolojikmücadeleajan›olarak kullan›labilece¤inigöstermektedir. AnahtarSözcükler: Neodiprionsertifer,NüklearPolihedrozisVirüs, BiyolojikKontrol 419 ViralControlofTheEuropeanPineSawfly, Neodiprionsertifer (Geoffroy)inTurkey Introduction TheEuropeanpinesawfly,Neodiprionsertifer (Geoffroy),isapestofmostspeciesoftwo- needledpinesthefollowingplacesinTurkey:‹stanbul,Bal›kesir-Dursunbey,Ankara-Beynam, Burdur-Kemer,Bucak,Mu¤la-Y›lanl›,Eskiflehir-Çatac›k,K›z›lcahamam-Çamkoru (1)and Trabzon.ItisparticularlyseriousinScotchpine,Pinussylvestris (L.),andredpine(P.resinosa (Ait.))plantations(2,3).Watershedareasareparticularlyvulnerabletoattack,becausetheuse ofchemicalinsecticidesisrestrictedtoavoidtheriskofwatercontamination.Asthechemical pesticidequestionisasocialissue,theobjectivesofnutrition,healthandenvironmentalquality canbeaddressedmoreefficientlybytheimplementationofintegratedpestmanagement techniques(IPM)ratherthanthroughcurrentcropprotectionpractices(4). BiologicalcontrolofthispestisespeciallydesirableinTurkey,becausemanyofthehost- treespeciesoccurinwatershedplantings.Fortunately,N.sertifer populationsarereducedbya naturalnuclearpolyhedrosisvirus(NPV)(Baculovirus),firstreportedbyEscherich(5)andlater describedindetailbyBirdandWhalen(6).Thisvirusiscapableofcausingdiseaseinlarvaeof N.sertifer,butdoesnotharmotheranimalsorplants.Nuclearpolyhedrosisvirusesrepresent aneffective,selectiveandsafebiologicalcontrolagent(7).Thebestresultswithpolyhedrosis viruseshavebeenachievedagainstconifersawflies,especiallyinEurope.Manyfactorssuchas temperature,moisture,acidrainsanddosageinfluencetheeffectofthe Neodiprionsertifer nuclearpolyhedrosisvirus(NsNPV).Thisinsectcanhaveresistanceagainstthevirusindifferent geographicregions(8-10). Therefore,manyscientistshavestudiedtheeffectofNsNPVin severalcountries(8,11-13). Inpestcontrolstrategiesforenvironmentalprotection,theuseofvirusesisrelatively new inTurkey,althoughtherearerapiddevelopmentsinthebacterialcontrolofplantpests(14- 18). ThisstudywillencourageTurkishscientiststoinvestigateanduseinsectvirusesagainst plantpests. MaterialsandMethods Collectingandrearinglarvaeof N.sertifer LarvaeofN.sertifer werecollected onyoungpinefoliageinÇamburnu-Sürmene,Trabzon, andbroughttothelaboratoryinappropriateboxes.Thelarvaewererearedinspecially preparedjars(Figure1).Eachgroupwasfedwithanequalamountofpinefoliage. Viralsuspensionandbioassays Theviralsuspension(Virox)wasprovidedbyDr.Ziemnicka,InstituteofPlantProtection, DepartmentofBiologicalControlandQuarantine60-318,Poznan,Poland.Theconcentration of polyhedrainthesuspension wascountedinahemacytometer.Freshpinefoliagewasdipped infivedifferentconcentrationsof thevirussuspension:5x109,5x108,5x107,5x106 and5x105. 420 M.YAMAN,R.NALÇACIO⁄LU,Z.DEM‹RBAG Figure1. Generalviewofthe experiment. Thefoliagewasairdriedonfilterpaperandplacedinasmalljar containingwater,whichwas putintoalargejar(Figure1).Forthecontrol,20larvaereceivedleavesdippedinwater.The larvalmortalitieswererecordeddailyanddeadlarvaewereremoved.Alllarvaetestedwere keptat21±2ºCand60%RHona12:12hphotoperioduntiltheyspuncocoons(8).All bioassayswererepeated3times.DatawereevaluatedbyusingAbbott’sformula(19). Determinationofviralinfection Thedeadlarvaeweredissected,andtheinfectionwasobservedunderlightmicroscopy.In addition,occurrenceofviralinfectionwasprovedwithGiemsastaining.Forthis,athinsmear ofinfectedinsecttissueslidewaspreparedunderamicroscopeanddriedinair.Thesmearwas immersedfor1-2mininGiemsa’fixativeandrinsedunderrunningtapwaterfor5-10s.Then thesmearwasstainedfortwohoursin10%Giemsastainin0.02Mphosphatebuffer.After staining,thedyewasrinsedoffinrunningtapwaterfor5-10sandgentlyblottedwith absorbentpaper,andthenitwasdriedandexaminedwithanoilimmersionobjective(20). ResultsandDiscussion InallbioassayscarriedouttodeterminetheeffectofNsNPVonthelarvaeoftheEuropean pinesawfly,Neodiprionsertifer (Geoffroy),larvalmortalitieswere foundindifferentratiosand periodsfordifferentviralconcentrations(Figure2).AsseeninFigure2,theviral concentrations,5x109,5x108 and5x107 showedthesamemortality,buttheinfectionperiods occurredondifferentdays.Themortalperiodoflarvaedecreasedasthedosesincreased.At 421 ViralControlofTheEuropeanPineSawfly, Neodiprionsertifer (Geoffroy)inTurkey 100 — Figure2. EffectofNsNPVonthesecond 90 — 1 instarlarvaeofN.sertifer. 80 — 2 (1=5x10 9;2=5x10 8;3= 70 — 3 5x10 7;4=5x10 6;5=5x — 4 60 105) 50 — 5 40 — — Mortality(%) 30 20 — 10 — 0 — — — — — — — — — — — — — — — 123456789101112131415 Infectionperiod(day) the5x109 PIBconcentration,larvalmortalitystartedonthefourthdayandreached90.4%on theninthdayofthebioassay.Thehighestdailymortalityduringthisperiodwasdeterminedto be40%onthesixthdayat5x10 9 PIBconcentration.Deadlarvaeshowedcommonsymptoms ofviralinfectionsuchashangingfromfoliage,liquefiedbodycontent,anddelicate,rupturing integument.Theinfectedlarvaelostappetite,werelessactiveanddied4to14daysafter infection.Thedeadlarvaehadafaint-greencolor(Figure3),andtheircuticulewaseasily broken,liberatingfluidinwhichalargenumberofpolyhedralbodieswaspresentunderthe microscope.Manydeadlarvaehungonpinefoliage,characteristicallyattachedbytheabdominal prolegs(Figure4).Someuninfectedorlowinfectedolderlarvaereachedthepupalstage.Inthe Giemsastain,performedtoshowviralinfection,stainedPIBsofNPVappearasclear,round objects(Figure5). Figure3. Healthy(left)andinfected (right)larvaeofN.sertifer. 422 M.YAMAN,R.NALÇACIO⁄LU,Z.DEM‹RBAG Figure4. NsNPV-Infected N.sertifer larvaehungingonpinefoliage. Figure5. PolyhedralInclusionBodies (PIBs)stainedwithGiemsa. Indifferentcountries,scientistshavefounddifferenteffectsofNsNPVonthelarvaeof N. sertifer atdifferentperiodsusingsimilarviraldosages.Glowackaetal.(13)foundthatthe larvalmortalityreached100%onthe9th dayof thelaboratorytestsatalldosesused(1x10 2- 1x10 6 PIB/ml).Podgwaiteetal.(11)determinedthatadoserateof2.5x10 9 PIB/haof NsNPVprovidedacceptablecontrol.Manyfactorssuchastemperature(8),dosage(9) moisture (10)andacidrains(21) influencetheeffectof Neodiprionsertifer nuclearpolyhedrosisvirus (NsNPV)and,also,thisinsectcanhaveresistanceagainstthevirusindifferentgeographic regions. 423 ViralControlofTheEuropeanPineSawfly, Neodiprionsertifer (Geoffroy)inTurkey ThespeedandhighrateofdeathsoflarvaeinfectedwithNsNPVindicatethatitisavery promisingagentforbiologicalcontroloftheEuropeanpinesawfly.Thenuclearpolyhedrosis virusofN.sertifer representsaneffective,selectiveandsafebiologicalcontrolagent.However, Greatheadetal.(22)statedthatparasitesandpredatorsactinthetransmissionofvirusand femalesinfectedwithsublethaldosessurviveandcarryoverthepathogen,viatheegg,tothe nextgeneration. TheresultsofourstudyshowthatNsNPVcanbeeffectivelyusedagainst N.sertifer asa safebiologicalcontrolagentinTurkey.Thisstudyisalsoagoodexampleforotherscientists whowanttouseinsectvirusagainstotherimportantplantpests. Acknowledgment TheauthorswishtoexpresstheirthankstoDr.JadwigaZiemnicka,InstituteofPlant Protection,DepartmentofBiologicalControlandQuarantine,60-318,Poznan,Poland,who providedtheviralsuspension(Virox). References 1. Çanakç›o¤lu,H.veMol,T.,OrmanEntomolojisi,Zararl›veYararl›Böcekler,‹stanbul,1998,‹.Ü.OrmanFakültesi, 541. 2. Lyons,L.A.,TheEuropeanpinesawfly, Neodiprionsertifer (Geoff.)(Hymenoptera:Diprionidae).Areviewwith emphasisonstudiesinOntario.Proc.Entomol.Soc.Ont.94:5-37,1964. 3. Wilson,L.F.,EffectsofdifferentpopulationlevelsoftheEuropeanpinesawflyonyoungScotchpinetrees.Ibid. 59:1043-1049,1966. 4. Norgard,R.B.,Integratingeconomicsandpestmanagement.In:Lawrence,J.A.&Smith,R.F.(Eds)Integrated PestManagement,PlenumPress,NewYork,1976,pp.63-76.