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PATHOGENICITYOFBeauveriabassianaTOADULTDiaprepesabhreviatus By JENNIFERLYNNGILLETT ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT OFTHEREQUIREMENTSFORTHEDEGREEOF DOCTOROFPHILOSOPHY UNIVERSITYOFFLORIDA 2003 IwouldliketodedicatethisworktothememoryofmylatementorDr.EverettRoyal Mitchell.Hissupportthroughmyundergradwork,timeinthePeaceCorpsandmaster's workwillalwaysberemembered.Hisworkininsectbiologicalcontrolwasagreat inspiration. ACKNOWLEDGMENTS IwouldliketoextendmythankstoDr.J.W.Kimbroughforpricelessguidancein mycology,goodadviceaboutprivatematters,andagoodjokeeverynowandagain.I wouldalsoliketothankhiswife,Jane,formanygoodhome-cookedmeals. Iwouldalso liketothankthemembersofmycommitteeDr.F.W.Zettler,Dr.C.W.McCoy,Dr.J.M. Sivinski,andDr.K.L.Buhr,fortheirenthusiasmforthisprojectaswellastheirinput intoitsproductionintoafinalproduct.IwouldliketothankDr.C.W.McCoyforhis initialinterestandinputinthisproject;withouthisinnovativeideasthisprojectwould nothavebeenasuccess.Dr.Zettler'sassistanceinacquiringanacademicfellowshipwill alwaysbeappreciatedaswellashissenseofhumorandnumerouslettersof recommendationforvariousawards.Dr.K.L.BuhrwasthereasonIjoinedthePeace CorpsafterreceivingmyB.S.;hisagreeingtoserveasmyexternalcommitteemember wasgreatlyappreciated. TheassistanceoftheDepartmentofPlantPathologystaffisgreatlyappreciated. GailHarris,DonnaPerryandLaurettaRahmeshavealwaysbeenwillingtodropwhat theyaredoingtorectifyaproblemorgiveahug. Manyoftheexperimentscouldnot havebeenperformedwithoutLuciousMitchell'sconstantgoodnature,coffeesupply, andabilitytofindanysupplyatamoment'snotice. GeneCrawford'shelpinthe greenhouseandwithrowdyfreshmengavemethetimetoperfectmymicroscopeskills andkeptmefi-omhavingtotalkovermystudents. iii Unfortunately,Dr.E.R.Mitchellpassedawaybeforethecompletionofthisproject. Hehadbeenmymentorininsectbiologicalcontrolsince1992andwithouthisguidance andleadershipitisdoubtfulthatIwouldhavebeencompelledtoresearchafungusthat caneatinsects. Dr.JohnSivinskimadespaceformeunderhiswingandpickedmeupat theUSDAafterthedeathofDr.Mitchell. John'sconstantenthusiasmformycologyand patiencehavebeengreatlyappreciated.MyfriendsandcolleaguesattheUSDArequire mentionfortheirloyalsupport. CharlieStuhldeserveshighestbillingasmyofficemate, andfriend,forhisabilitytotranscendthepilesofpapersaroundtheoffice,feedthefish, andfixmycomputer(andcar). IthankValerieMalcolm,ourunitsecretary,forkeeping meoutoftroublewiththeUSDApaperworkpolice.SpecialthanksgotoGinaPoseyand DelaineMillerforconstantemotionalsupportandlunchcompanionship. IamindebtedtotheFloridaDepartmentofAgricultureinGainesvilleforahealthy supplyofweevils.SuzanneFraserwasalwaysavailableatamoment'snoticetosend insectsandhelpfulhints.Theconstantandreliablesupplymademyresearchpossible. Itwouldberemisstoomitthesupportofmyfamilyfortheiracceptanceofmy ideasandsupportintimesofmyfailures,notonlyatthistimeinmylifebutforalifetime oflove. iv TABLEOFCONTENTS Page ACKNOWLEDGMENTS iii TABLEOFCONTENTS v ABSTRACT vii 1INTRODUCTION 1 2PROPENSITYOFBeauveriabassianaTOINFECTADULTDiaprepesabbreviatus, ESTABLISHINGATTACHMENTANDGERMINATION 7 Introduction 7 MaterialsandMethods 10 ModeofIngress 11 ConidialViabilityandWeevilGrooming 16 Results 16 Discussion 23 3BeauveriabassianaINTERNALGROWTHANDDEVELOPMENTINADULT Diaprepesabbreviatus 26 Introduction 26 MaterialsandMethods 27 Hematology 27 InternalDevelopmentandRamification 28 Results 29 Discussion 39 4HORIZONTALTRANSMISSIONOFBeauveriabassianaBYADULTDiaprepes abbreviatusANDSUBSEQUENTMORTALITYRATE 41 Introduction 41 MaterialsandMethods 42 MortalityRates 42 HorizontalTransmission 42 Results 43 Discussion 49 V 5 SUMMARYANDCONCLUSIONS LISTOFREFERENCES BIOGRAPHICALSKETCH AbstractofDissertationPresentedtotheGraduateSchool oftheUniversityofFloridainPartialFulfillmentofthe RequirementsfortheDegreeofDoctorofPhilosophy PATHOGENICITYOFBeauveriabassianaTOADULTDiaprepesabbreviatus By JenniferLynnGillett December2003 Chair: JamesW.Kimbrough MajorDepartment: PlantPathology TheDiaprepesrootweevil(DRW)isaseriousinsectpestinthestateofFlorida. Itcausesanestimated$75millionindamageseveryyeartoFlorida'scitrusindustry. Attemptsatchemicalcontrolofthispestarecostlywithlimitedeffectiveness.Theuseof Beauveriabassiana,acommonnativeentomopathogenasabiologicalcontrolagentof Diaprepesabbreviatuscouldreducepesticideusageincontrollingthispest.A commerciallyproduceddrypowderformulationofBeauveriabassianawasusedinthis researchtodetermineitsaffectonweeviladults.ConidiaofBeauveriabassianawere foundtogerminateontheweevilexoskeletonatvariousratesdependingonthebody vii region.Conidialocatedininter-segmentalareasoftheabdomenandheadgerminatedat twelvehourspost-inoculation;onotherareasoftheabdomen,germinationwasfirst observedeighteenhourspost-inoculation.Germinationontheelytraoccurred30hours post-inoculationandeventhen,germinationwaslow.Internalramificationofhyphal bodiesbegantoappear72hourspost-inoculation.Thetimebetweencuticlepenetration andthedevelopmentofhyphalbodiesinthehemolymphappearedtobedelayedbyan immuneresponseofthehemocytes.Internalproliferationofhyphaerapidlyconsumed theinsectbodycavitycausingdeathafterasfewas6days.Sporulationonthecadaver usuallyoccurredtwotothreedaysafterdeathundermoistconditions.Diaprepesroot weevilsinteractbytouchinginthewild.Thistouchingisnotlimitedtosexualcontact, inter-sexualtouchingalsooccursandispossiblyaformofconmiunication.Weevils transmittedconidiathroughbothbisexualandsamesexcontact.Adultmortalityfromthis contactwasnotsignificantlydifferentbetweensexes.Thesedatasuggestthatadult weevilscanspreadfungalconidiawithinanativepopulation. viii CHAPTER 1 INTRODUCTION Theuseofthehyphomycete,Beauveriabassiana(Balsamo)Vuillemin(Fungi Imperfecti:Hyphomycetes:MoniUaceae),asamycoinsecticideinthebiologicalcontrol ofinsectshasalongandinterestinghistorybeginningwitharoleasapathogenofan economicallyimportantinsect.AccordingtoAinsworth(1956),AgostinoBassiwasone ofthefirstpeopletoexperimentwithinsectsinfectedwithBeauveria.Bassi(1835)bred silkworms{BombyxmoriL.)inanattempttosimulatewhitemuscardinedisease,acause ofeconomiclossesinthesilkindustry.Whitemuscardineisadiseasecharacterizedby insectdeathfollowedbyawhiteningoftheexoskeleton.Hedevisedmanyschemesto inducethedisease,includingseveralattemptsthatfailedatinducingdiseaseinhealthy insects. Bassi(1835)wasthefirsttopublishhisbeliefthatmuscardinediseasewascaused by"aplantofthecryptogamickind,aparasiticfungus."Inthelate18thcentury muscardinediseasewasreferredtoinFranceas"Muscardineduverasole"(Beauverie, 1911)andwasgiventheclassificationBeauveria(Vuillemin,1912).Beauveriabassiana wasdescribedbyBeauveriefirstin1911thenagainin1914asBotrytisbassiana. Beauverie(1911,1914)andVullemin(1912)originallydescribedBeauveriabassianaas follows:Hyphaeslender,1.5-2/xindiameter,hyaline,septate:coloniesflat,mealy,or finelypulverulentwithachalkyappearancesomewhatlikethesurfaceofanewlybroken pieceofchalk,whitetopalecreamonthesurface,doesnotcolortheundersurfaceof 1 2 potatodextroseagar;phialides,variableinshape,ventricosetofilamentous,developon themainhyphalbranchesoronshortbranchletsatrightanglestothemainaxis, branchingmayberepeated,forminggloboseheads;sporesborneonzigzagsterigmata thatextendfromthetipofthephialides,globose,2.4 indiameter(MacLeod,1954). BeauveriaisaclassicexampleofsympodialconidiogenesisintheHyphomycetes TeleomorphsareunknowninBeauveriabutarelikelytobelongtotheClavicipitaceae {Cordyceps,Torrubiellaandrelatedgenera)(Arx,1986;Kirketal.,2001). Beauveria bassianawasoneofthefirstentomopathogenstobestudiedandwasalsooneofthe oldestknownfossilizedentomopathogensfoundinDominicanamber(Poinarand Thomas,1984). ThisdescriptionoiBeauveriabassianaprovidedcharacteristicsusefulinthe identificationoftheorganism,helpedtoclarifymanytaxonomicerrors,andpulledmany synonymsintothegenusBeauveria(MacLeod,1954).ThegenusTolypocladiumhadat onetimebeenasynonymofBeauveriabasedonsimilarconidiogenousstructuresand similar,white,lanosecolonies(vonArx,1986).Thebiochemicaldatasupportingthis synonymyofthisgenuswithBeauveriahavebeenquestioned(Mugnaietal.,1989). AdditionalbiochemicalandgeneticworkusedrRNApolymorphism,isoenzyme variability,andmorphoontogeniccharactersconfirmingthatthesegeneraweredistinct (Rakotonirainyetal.,1991).Forthesereasons,Tolypocladiumwillnotbeconsidereda synonymofBeauveriainthiswork. SomesynonymsofBeauveriabassianaincludeBotrytisnecans,Botrytis stephanoderisandBeauverialaxa.AccordingtoFetch,(1926)confusionaboutsynonyms wasoftenbasedonquantitativeratherthanqualitativedata.Basedonmorphologicaland