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GENETICCHARACTERIZATIONOFBEAUVERIABASSIANA ASSOCIATEDWITHFIREANTS,SOLENOPSISSPP.,ANDOTHERINSECTS BY YONGZENG ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT OFTHEREQUIREMENTSFORTHEDEGREEOF DOCTOROFPHILOSOPHY UNIVERSITYOFFLORIDA 1999 ACKNOWLEDGMENTS IwouldlikefirsttothankDr.J.L.Stimacforhisguidance,understanding, encouragementandsupportthroughallstagesofmygraduatestudies. Thisproject andIbenefitedimmenselyfromhistrust,perseverance,andhisemphasison objective-orientedfocusingandsystematicthinking. SpecialappreciationgoestoDr. H.C.Kistler,whogreatlyfacilitatedthisprojectthroughthekinduseofhislaboratory andhisexpertise. Ithankhimforhisunderstandingandconstantencouragement. I amverygratefiiltoDr.J.E.Maruniakforencouragementandthehelpheofferedat thestartofthisprojectandalsofortheinsightfiiladvicehehasgiventhroughout. I greatlywanttothankDr.J.W.KimbroughandDr.J.L.Nationfortheirvaluable inputintomygraduatestudy,inparticularthisproject. Also,Iwouldliketothankandacknowledgethefollowing:Dr.U.L.Rosewich forherenormousassistanceintheresearch;Dr.A.Jeyaprakashforhisgenerous technicalsupportandessentialsuggestions;Dr.D.G.Bouciasforhishelpfulinsights andsupportduringthework;L.A.Woodforhersubstantialhelponthisproject;and R.E.Pettwayforhisenthusiasmandvaluableskills. ManythanksmustalsogotoU. BennyandDr.A.Garcia-Maruniakfortheirti-emendoustechnicalsupport. Mythanks goouttoDr.H.G.Hall,Dr.R.M.Pereira,Dr.B.C.Larson-Vasquez,Dr.M.J. Parsons,H.L.KonsJr.andthefiiendsIhavemadeattheUniversityofFlorida. Finally,Ithankmyparents,mysisterYvonneandmywifeJenifferfortheirsupport, encouragement,andlove. ii TABLEOFCONTENTS Page ACKNOWLEDGEMENTS " LISTOFTABLES ^ LISTOFFIGURES ABSTRACT CHAPTERS 1 INTRODUCTION 1 BiologyofBeauveriabassiana 1 Classification ^ GeneticSystem ^ NaturalOccurrence ^ BeauveriabassianaasaBiologicalControlAgent 5 HistoryandStatus ^ ChallengesandPotentials 7 GeneticDiversityinBeauveriabassiana 8 ProteinandIsoenzymeAnalyses 8 GenomicDNAAnalyses 9 Objectives 11 2 DNAPOLYMORPHISMSINBEAUVERIABASSIANA:RFLPPROBE DEVELOPMENTANDSEQUENCEANALYSISOFTHEITSAND5.85 rRNAGENEREGION 13 Introduction 13 MaterialsandMethods 1^ FungalCulturesandB.bassianaIsolates 17 DNAExtraction 17 PCR-amplification,SequenceandRestrictionAnalysisoftheITSand5.85 rRNAGeneRegion 19 ConstructingandScreeningaGenomicLibrary 21 EnzymeDigestion,Electrophoresis,SouthernBlottingandProbeChoice 22 Results 23 SequencingandRestrictionAnalysisoftheITSand5.8sGeneregion 23 iii 1 IsolationandEvaluationofRecombinantClones 23 DNAPolymorphismsbyUsingHomologousRFLPProbes 29 DNAPolymorphismsbyUsingHeterologousDNAProbes 33 Discussion ^^ 3 AGSENSEOTCIICATDEIDVEWRISTIHTYFIIRNEBAENATUSV,ERSIOALEBNAOSPSSIIASNASPIP.S,OALANTDESOTHER HOSTSPECIES Introduction MaterialsandMethods ^-^ B.bassianaIsolates DNAExtraction,ChoiceofRestrictionEnzymesandProbes 43 DataAnalysis ResuGletnseticDiversityamongB.bassianaIsolatesAssociatedwi;thSolenopsi;s spp ComparisonofGeneticVariationbetweenIsolatesfromSolenopsisspp.and IsolatesfromOtherHostSpecies 5C3O Discussion •'^ 4 GENETICPOPULATIONSTRUCTUREINBEAUVERIABASSIANA ASSOCIATEDWITHFIREANTS,SOLENOPSISSPP 66 Introduction MaterialsandMethods 68 B.bassianaIsolates 68 DNAExtractionandChoiceofProbes 72 Results ; "72 GeneticVariationamongB.bassianaIsolatesAssociatedwithSolenopsis spp 72 B.bassianaIsolatesfromSolenopsisspp.intheUni;tedStates 78 Discussion 5 SUMMARYANDCONCLUSIONS 86 LISTOFREFERENCES 92 BIOGRAPHICALSKETCH 11 iv LISTOFTABLES Table Page 2-1. BeoanuvseerqiuaenbcaessainaanlaysiissoloaftetsheevIaTlSuaatnedd5f.o8rsDrNRANApogleynmeorrepghiiosnmosrbRaFseLdP probes 1^ 2-2. RestrictionenzymeanalysisoftheITSand5.8srRNAgeneregion 28 2-3. RFLPbandingpatternsamongisolatesfromfireants,GHAandNaturalis bysingleandlowcopyhomologousRFLPprobes 32 3-1. Bealuovceusriaandbarsespieatintaiviesoplraotebsesfromfireantsandnestsoilfortestingsingle 44 3-2. GeneticvariationofBeauveriabassianaisolatesfromSolenopsisspp. basedonrepetitiveprobe133andthetelomericprobeinconjunction withPstl 45 3-3. DNdAigbeasnteddinDgNpaAttoefrnBseaguevneerraitaebdabsysiraenpaetiistoilvaetpesrocboelslewcittedhHfrionmddIillf-ferent hosts 46 3-4. ComparisonofDNApatternbandingtypesgeneratedbysingle-locusand repetitiveprobeswithHinA///-digestedDNAofBeauveriabassiana isolatesfromfireantsandantnestsoil 47 3-5. GenetichomogeneityamongBeauveriabassianaisolatesfromSolenopsis speciesdigestedwithEcoRI,HindIIIandPst1 49 3-6. GeneticvariationofBeauveriabassianaisolatesfromSolenopsisspp. digestedwithBamHI,EcoRVandHaeIIIandhybridizedtorepetitive probe133andthetelomericprobe 50 4-1. GeneticvariationamongBeauveriabassianaisolatescollectedfrom SolenopsisinvictainCuiaba,MatoGrosso,Brazilin1986and1987 69 4-2. GeneticvariationamongBeauveriabassianaisolatesfromSolenopsis speciesornestsoilindifferentlocations 70 V 4-3. BeauveriabassianaisolatescollectedinGainesville,Florida 71 4-4. BeauveriabassianaisolatescollectedfromSolenopsisxyloniin Bakersfield,California vi LISTOFFIGURES Figure Va^s. 2-1. SequencealignmentoftheITSand5.85rRNAgeneregionsforBeauveria bassianaisolate447and14otherisolatesobtainedfromGenbank 24 2-2. DNAfingerprintingprofileofBeauveriabassianaisolatesbyusingprobe 133.GenomicDNAwasdigestedwithHin&III(A)andPstI(B) 30 2-3. DNAfingerprintingprofileofBeauveriabassianaisolatesbyusingprobe 205. GenomicDNAwasdigestedwithHindIII(A)andPst(B) 31 2-4. DNAfingerprintingprofileofBeauveriabassianaisolatesbyusinga telomericprobefromFusariumoxysporum.GenomicDNAwas digestedwithHindiIII(A)andPstI(B) 34 2-5. DNmiAtobcahnodnidnrgiaplatptreorbnseoffrBoemaFuuvsearriiaumbaosxsyipaonrauims.olGateensobmyiucsDinNgAawas digestedwithEcoRI(A)andHindIII(B) 35 3-1. DNSoAlefnionpgseirpsrsipnpt.inGgepnroofmiilecoDfNBeAauwvaesridaigbeasstseidawniatihsBolaatmesHcIolalnedctedfrom hybridizedtoatelomericprobefromFusariumoxysporum 52 3-2. DNAfingerprintingprofileofBeauveriabassianaisolatescollectedfrom Solenopsisspp.andotherinsects. GenomicDNAwasdigested withHindHIandhybridizedtoatelomericprobefromFusarium oxysporum 54 3-3. DendrogramshowingthegeneticrelationshipsamongBeauveriabassiana isolates.Clusteranalysiswasperformedbytheunweightedpair-group methodwitharithmeticmean(UPGAM).Similaritycoefficientswere basedon59bandsgeneratedbythetelomericprobehybridizedtoHind IIIdigestedDNA 55 3-4. DendrogramshowingthegeneticrelationshipsamongBeauveriabassiana isolates.Clusteranalysiswasperformedbytheunweightedpair-group methodwitharithmeticmean(UPGAM).Similaritycoefficientswere basedon45bandsgeneratedbyprobe133hybridizedtoHindIII digestedDNA 56 vii 3-5. DendrogramshowingthegeneticrelationshipsamongBeauveriabassiana isolates.Cladisticapproachwasperformedbyparsimonyanalysiswith datafromthetelomericprobe.Numbersbelowbranchesrepresent bootstrapvaluebasedon500replicates.Onlyvaluesgreaterthanor equalto50%areshown 56 3-6. DendrogramshowingthegeneticrelationshipsamongBeauveriabassiana isolates.Cladisticapproachwasperformedbyparsimonyanalysiswith datafromprobe133.Nimibersbelowbranchesrepresentbootstrap valuebasedon500replicates.Onlyvaluesgreaterthanorequalto50% areshown 57 4-1. ExampleofDNAbandingpatternsof5eaMverm bassiana collectedfromSolenopsisinvictainCuiaba,MatoGrosso,Brazilin 1986(AandC)and1987(B).GenomicDNAwasdigestedwithHind IIIandhybridizedtoatelomericprobefromFusariumoxysporum (AandB)andprobe133(C) 74 4-2. ExampleofDNAbandingpatternsofBeauveriabassianaisolates collectedfromSolenopsisspp.indifferentlocations.GenomicDNA wasdigestedwithHindIIIandhybridizedtoatelomericprobefrom Fusariumoxysporum 76 4-3. ExampleofDNAbandingpatternsofBeauveriabassianaisolates collectedfromSolenopsisspp.indifferentlocations. GenomicDNA wasdigestedwithHindIIIandhybridizedtoprobe133 77 4-4. ExampleofDNAbandingpatternsofBeauveriabassianaisolates collectedfromSolenopsisquinquecuspis,S.saevissima and Solenopsisspp.GenomicDNAwasdigestedwithHindIIIand hybridizedtoatelomericprobefromFusariumoxysporum 79 4-5. ExampleofDNAbandingpatternsofBeauveriabassianaisolates collectedfromSolenopsisinvictaornestsoilinFlorida(A)andfrom S.xyloniinCalifornia(B).GenomicDNAwasdigestedwithHindHI andhybridizedtoatelomericprobefromFusariumoxysporum 80 vni AbstractofDissertationPresentedtotheGraduateSchool oftheUniversityofFloridainPartialFulfillmentofthe RequirementsfortheDegreeofDoctorofPhilosophy GENETICCHARACTERIZATIONOFBEAUVERIABASSIANA ASSOCIATEDWITHFIREANTS,SOLENOPSISSPP.,ANDOTHERINSECTS By YongZeng August1999 Chairman:JerryL.Stimac MajorDepartment:EntomologyandNematology Beauveriabassianaisolate447,obtainedfromSolenopsisinvictainBrazil,has beendevelopedasamicrobialcontrolagentagainstfireantsintheUnitedStates.To geneticallycharacterizeisolate447andotherB.bassianaisolates,including commercialproductisolates,variousmolecularmarkerswereevaluated. Thelimited DNAsequenceheterogeneityoftheribosomalinternaltranscribedspacers(ITS)and 5.85rRNAgeneregionwasconsideredinsufficienttoefficientlydiscriminateamong isolatesbyusingrestrictionanalysis. TwohomologousrepetitiveDNAsequences (probe133andprobe205)andtwoheterologousprobes(atelomericprobeanda mitochondrialprobefi"omFusariumoxysporum)wereabletodistinguishisolate447 fi-omtheothercommercialproductisolates,GHAandNatiiralis. Noneof17isolatesfi-om12non-fireantspeciesthatweretestedwithprobe133 andthetelomericprobeweresimilartoisolatesfromSolenopsisspp. SomeB. bassianagenotypesfromSolenopsisspp.appeartobelongtoaSolenopsisgenus- preferentialclonelineage.Genotypesimilarityinthelineagesuggestsaco- ix evolutionaryhistorybetweenthisB.bassianalineageandSolenopsisspp. Genetic diversityanalysiswithprobe133,thetelomericprobeandsinglelocusprobes indicatesthatasexualreproductionisapredominantreproductionmodeamongB. bassianaisolatesassociatedwithSolenopsisspp. ParsimonyanalysissuggeststhatB. bassianamaynotbeaspecieswithexclusivelyclonalreproduction. Probe133andthetelomericprobecanbeusedtoevaluatetheecological adaptabilityofisolate447asabiologicalcontrolagentagainstfireants.WhenB. bassianaisolatescollectedfromfourSolenopsisspeciesintenlocationswere examined,multiplegenotypeswerecommonlyfoundamongisolatescollectedfrom thesamespeciesatthesamelocation. Onegenotypewaspredominantregardlessof theSolenopsisspeciesorgeographiclocation.Thisindicatesthatthispredominant genotype,whichalsoincludesisolate447,cansurviveinawiderangeofecological conditions. Afterisolate447wasreleasedattreatmentsitesinFloridaandCaUfomia, isolateswiththesamegenotypeofisolate447wererecoveredoneyearlater, indicatingthesuccessfulcolonizationofanexoticisolatereleasedintheUnitedStates. x

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