ABSTRACT WALLACE, EMMA CATHERINE. Population Structure of ...

ABSTRACTWALLACE,EMMACATHERINE.PopulationStructureofPseudoperonosporacubensisonCommercialandNon-CommercialCucurbitsinNorthCarolinaDeterminedbySimpleSequenceRepeats(SSRs)Pseudoperonosporacubensisisanairborneoomyceteplantpathogenthatcauses

downymildew,afoliardiseaseaffectingallcommercialcucurbitcrops.IntheUnited

States(US),thispathogenwaseasilymanagedwithhostresistanceincucumber

untilitsresurgencein2004.Sincethen,P.cubensishascausedmajorlossesin

cucumbereveryyearintheUS,andintensivechemicaltreatmentsareneededto

managethisdisease.Inadditiontoothercommercialcucurbitcropssuchas

cantaloupe,pumpkin,squash,andwatermelon,P.cubensiscaninfectapproximately

60Cucurbitaceaespeciessomeofwhicharenotgrownextensivelyforcommercial

productionintheUS.ManyofthesespeciesaregrownonlyonasmallscaleinNorth

Carolina(NC)oroccurinthewildasweedyplants.Littleisknownaboutthe

contributionofwildandnon-commercialcucurbitstotheyearlydownymildew

epidemic.SinceP.cubensishasarelativelyhighgeneticdiversityinNC,andgenetic

differentiationisrelatedtocucurbithost,agoalofthisstudywastouseexamine

non-commercialandwildcucurbithostsforP.cubensisinNorthCarolinaandalsoto

developandutilizemolecularmarkerstodeterminethegeneticstructureofP.

cubensisoncommercialandnon-commercialcucurbitsinNC.Sixnon-commercial

andwildcucurbitswereplacedinsentinelplots,partoftheCDM-IPM-PIPE

forecastingsystem,inthreegeographicallydistinctregionsinNC.Theywere

monitoredfordownymildewinfectionsbynaturalinoculumthroughoutthe

cucumbergrowingseason.Koch’spostulateswerecompletedforP.cubensison

Momordicacharantia,M.balsamina,andCucurbitafoetidissima.Next,insilico

approacheswereusedtodevelopmolecularmarkerstostudythepopulation

geneticsofthepathogen.TheMicrosatelliteIdentificationTool(MISA)wasusedto

identifythefrequencyandlocationofmicrosatellites,orSimpleSequenceRepeats

(SSRs)intheP.cubensispredictedtranscriptome.Comparisonsweremadeto

microsatellitefrequencyandabundancetrendsinanotherdownymildewpathogen,

Hyaloperonosporaarabidopsidis,withgenomicresources.Althoughthepercentage

ofSSRsinexaminedsequenceswassignificantlydifferentbetweenP.cubensis(12%

of23,522transcripts)andH.arabidopsidis(15%of14,548transcripts),theSSR

motiftypesshowedsimilarfrequencies,abundances,anddensitiesinthe

transcriptomes.Primer3wasusedtoidentify2,088SSRs.When100markerswere

evaluatedinalabsetting,elevenmarkerswereidentifiedasreproducibleand

detectedpolymorphismacrossP.cubensisisolates.Amajorityofscreenedmarkers

(84%)weretransferabletoP.humuli,acloselyrelateddownymildewpathogenon

Humuluslupulus(hop).Theeleveninformativemarkerswereusedtogenotype38P.

cubensisisolatesand22P.humuliisolatesfromdiversehostsandlocationsand

greatgeneticdistance(D=0.97)wasdeterminedbetweenthetwo

Pseudoperonosporaspecies.Finally,theinformativemarkerswereusedtogenotype

344P.cubensisisolatescollectedfromsixcommercialhosts,threenon-commercial

hosts,andthreegeographicallydistinctregionsofNC.Pairwisepopulationmatrices

weregeneratedtocompareisolateswhengroupedbylocation,timeandlocation,

hostgenera,hostspecies,andhostspeciesandlocation.Analysesrevealedthe

greatestgeneticdifferentiationwasseenwhenisolatesweregroupedbyhost

speciesandhostspeciesandlocation,whichsupportstheconceptofhost-driven

differentiation.

©Copyright2016byEmmaCatherineWallaceAllRightsReserved

PopulationStructureofPseudoperonosporacubensisonCommercialandNon-CommercialCucurbitsinNorthCarolinaDeterminedby

SimpleSequenceRepeats(SSRs)By

EmmaCatherineWallace

AthesissubmittedtotheGraduateFacultyofNorthCarolinaStateUniversityInpartialfulfillmentofthe

RequirementsforthedegreeofMasterofScience

PlantPathology

Raleigh,NorthCarolina

2016

APPROVEDBY:

____________________________________________________________________________________Dr.LinaM.Quesada-OcampoDr.DavidShewCommitteeChair

__________________________________________Dr.IgnazioCarbone

ii

DEDICATIONTomyparents,JamesandMaureenWallace,andmygrandparents,PeggyWallaceandKennyandCatherineHughes.

iii

BIOGRAPHY

EmmaCatherineWallacewasbornJanuary28,1991inHalifax,NovaScotia.

ShegrewupinBrampton,OntarioandCuyahogaFalls,Ohio.Throughouther

schooling,Emmafoundinterestinfartoomanysubjectsandwhenshewasn’t

studying,shedevotedmuchofherpersonaltimeinhighschooltotheater,soccer,

andvolunteeringatspecial-needssummercamps.Appreciatingabalancebetween

thehumanitiesandsciencesinhighereducation,EmmaenrolledinMarymount

Universityin2009andchosetomajorinBiology,

ThroughoutherundergraduatedegreeEmmagotinvolvedwithseveral

studentgroupsandresearch.SheheldseveralleadershippositionsintheScience

ClubatMarymountandhelpedwithcoordinatingfieldtripstovariousmuseums,

labs,andseminars,bringingspeakerstotalkaboutcareersinscience,and

organizingoutreacheventsinthecommunity.Shewasalsoapeertutorandwriting

consultant,volunteeredinDr.ToddRimkus’turtlelab,andheldaninternshipatthe

USFishandWildlifeService.Herfirstintroductiontoconductingscientificresearch,

however,waswithDr.BarbaraKreutzerwholedresearchonbioremediationtools

insuburbansettings.Emmaworkedonseveralprojectsevaluatingtheeffectiveness

ofraingardensindecreasingpollutantsinrainwaterrunoff,whichculminatedin

herUndergraduateHonorsThesis.However,shewantedtolearnmoreaboutthe

microbesatplay,andwasfortunatetogetaninternshipattheSystematicMycology

andMicrobiologyLabatUSDA-ARSinBeltsville,Maryland.Thereshewas

introducedtoPlantPathologyandmolecularidentificationtools.Heradvisor,Dr.

JoAnnCrouch,encouragedEmmatoapplytoPlantPathologygraduateprograms

andhighlysuggestedgettingincontactwithDr.LinaQuesada-Ocampo,whowas

juststartingalabatNorthCarolinaStateUniversity.ThestarsalignedandEmma

beganherMaster’sDegreeAugust2013.

Emma’sworkonPseudoperonosporacubensis,herexperienceinNCSU’sPlant

PathologyDepartment,andherparticipationinDr.Quesada-Ocampo’sVegetable

PathologyLabhelpedherdevelopasascientistandshehopestocontinueasa

researcherinthefieldofplantpathology.

iv

ACKNOWLEDGEMENTS

IwouldliketothankmycommitteemembersDrs.LinaQuesada-Ocampo,

DavidShew,andIgnazioCarbonefortheirfeedbackandassistance.Dr.Lina

Quesada-Ocampoespeciallydeservesmygratitudeforherexcellenceinmentoring.

Hersupportandencouragementthroughoutthisprocesshashelpedmeinmy

developmentasascientist.Herdedicationtoherstudentsisevidentasshemakes

herselfaccessibleforfeedbackandgoesoutofherwaytoprovideuswithdiverse

learningopportunities.Iappreciatetheopportunitiesshehasgivenmetopresent

myworkandtheworkofthelabacrossseveralplatforms.Iwouldliketothank

severalsourcesoffundingthatmademyresearchpossible,includingUSDANational

InstituteofFoodandAgriculture,USDASpecialtyCropsBlockGrant,USDAAnimal

andPlantHealthInspectionService,andPicklePackersInternationalInc.

Ihavethoroughlyenjoyedworkingwithallpastandcurrentmembersofthe

VegetablePathologyLabincludingMikeAdams,Dr.LilianaCano,Dr.ElsaGongora-

Castillo,SauniaWithers,Dr.ThippeswamyBasaiha,Dr.AlamgirRahman,Dr.Edwin

Palencia,AndrewScruggs,NicolasNoel,CamiloParada,NathanMiller,LyndeRing,

andAbelWalker.IespeciallywouldliketothankSauniaWitherswhohelpedme

withpreliminarystepsoffragmentanalysis,NicolasNoelwhoassistedwithDNA

extractions,andMikeAdamswhohelpedincollectingdownymildewsamples.I

wouldalsoliketothankDr.CubetaforhismentoringwhenIwastheteaching

assistantforhiscourse,PP222:KingdomofFungi.

MyfriendsandfamilyprovidedmewiththesupportsystemIneededto

succeedingraduateschool.IamgratefulforthefriendsImadehereinRaleighas

myfellowstudentswelcomedmeandmademefeellikepartofthecommunity.I

appreciatetheadviceandlifeconversationsheldwithmywonderfulofficemates

EmilyMeyersandAndrewScruggs,andIamparticularlyindebtedtoMeganMiller,

NathanMiller,andLilianaCanofortheirfriendshipandkindness.Iwouldliketo

thankmyfriendsandfamilywhomadethetrektovisitmeinNorthCarolina,

especiallyAshleySen,KelseyWallace,andUditBhatnagar,whowerefrequently

ropedintospendinglatenightsinthelabwithmeandparticipatingindepartment

v

events.Finally,Ithankmyparentsandsiblingswhohavealwaysbeenmybiggest

sourceofencouragement,humor,andpop-cultureupdates.

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TABLEOFCONTENTSLISTOFTABLES…………………………………………………………………………………………..…..viiiLISTOFFIGURES…………………………………………………………………………………………….….x

CHAPTERI:LiteratureReview

TheHost:FamilyCucurbitaceae…………………………………………………………………1ThePathogen:Pseudoperonosporacubensis………………………………………………..2Epidemiology…………………………………………………………………………………………....3CDMIPM-PIPE……………………………………………………………………………………..……5VariabilityinPathogenVirulenceinDifferentHosts………………………….……..…6PseudoperonosporacubensisandP.humuli…………………………………………………9GeographicDispersal/GeneticDiversityofPseudoperonosporacubensis…….11Microsatellites…………………………………………………………………………………………13WildHosts……………………………………………………………………………………………....14

References…………………………………………………………………………………………………...…..16

CHAPTERII:DownyMildewPathogensInfectingWildandNon-CommercialCucurbitsinNorthCarolinaAbstract……………………………………………………………………………………………………..……..21FirstReportofPseudoperonosporacubensisCausingDownyMildewonMomordicabalsaminaandM.charantiainNorthCarolina.……………………...…....23

FirstReportofDownyMildewonBuffaloGourd(Cucurbitafoetidissima)CausedbyPseudoperonosporacubensisinNorthCarolina……………………………..26

FirstReportofPlasmoparaaff.australisonLuffacylindricaintheUnitedStates………………………………………………………………………………………………………………..29

CHAPTERIII:IdentificationandDevelopmentofMicrosatelliteMarkersinthePseudoperonosporacubensisTranscriptomeAbstract……………………………………………………………………………………………………….…..32Introduction………………………………………………………………………………………………….....33MaterialsandMethods…………………………………………………………………………………….37Results……………………………………………………………………………………………………….....….42Discussion……………………………………………………………………………………………………..…47Acknowledgements………………………………………………………………………………………....55TablesandFigures…………………………………………………………………………………………..56References……………………………………………………………………………………………….………70

vii

CHAPTERIV:PopulationStructureandDiversityofPseudoperonosporacubensisonCommercialandNon-CommercialCucurbitsinNorthCarolinaAbstract………………………………………………………………………………………………………..…..77Introduction………………………………………………………………………………………………….....78MaterialsandMethods………………………………………………………………………………….....81Results…………………………………………………………………………………………………………......84Discussion…………………………………………………………………………………………………….…..86TablesandFigures………………………………………………………………………………………..….92References……………………………………………………………………………………………..……….101

viii

LISTOFTABLES

ChapterIII:IdentificationandDevelopmentofMicrosatelliteMarkersinthePseudoperonosporacubensisTranscriptomeTable1.1.IsolatesusedininitialSSRscreen……………………………………………………….56Table1.2.PseudoperonosporaisolatesusedforSSRscreeningwithfragmentanalysis…………………………………………………………………………………………………………...…57Table1.3.NumberanddistributionofSSRsintranscriptsequencesaccordingtoMISA……………………………………………………………………………………………………………….…61Table1.4.Percentage,relativeabundance,andrelativedensityofSSRsindownymildewtranscripts………………………………………………………………………………………….….62Table1.5.Mostcommonrepeatmotifsidentifiedfromperfectandcompoundmicrosatellitesintwodownymildewtranscriptomes……………………………………….....63Table1.6.Resultsofmarkervalidationviagelelectrophoresis……………………………65Table1.7.PolymorphicSSRsinP.cubensis…………………………………………………………66Table1.8.StatisticsofpolymorphicprimersacrossP.cubensisisolates………………67Table1.9.PairwisepopulationmatrixofNei’sunbiasedgeneticdistance……………69ChapterIV:PopulationStructureandDiversityofPseudoperonosporacubensisonCommercialandNon-CommercialCucurbitsTable2.1.Polymorphicsimplesequencerepeatmarkers…………………………………..92Table2.2.Pseudoperonosporacubensisisolatesevaluated…………………………………93Table2.3.PairwisepopulationmatrixofNei’sunbiasedgeneticdistanceofP.cubensisisolatesgroupedbygeographicregion………………………………………………..…94Table2.4.PairwisepopulationmatrixofNei’sunbiasedgeneticdistanceofP.cubensisisolatesgroupedbytimeandlocation………………………………………….……...…95Table2.5.ExpectedheterozygosityofP.cubensisisolatesovertimeandlocation……………………………………………………………………………………………………………...96Table2.6.PairwisepopulationmatrixofNei’sunbiasedgeneticdistanceofP.cubensisisolatesgroupedbyhostgenera…………………………………………………………….97

ix

Table2.7.PairwisepopulationmatrixofNei’sunbiasedgeneticdistanceofP.cubensisisolatesgroupedbyhostspecies………………………………………………………...….98Table2.8.PairwisepopulationmatrixofNei’sunbiasedgeneticdistanceofP.cubensisisolatesgroupedbycommercialhostspeciesandlocation……………………...99

x

LISTOFFIGURES

CHAPTERIII:IdentificationanddevelopmentofmicrosatellitemarkersinthePseudoperonosporacubensistranscriptomeFigure1.1.Frequencyandnumberofrepeatsbymotif-typeandsourcetranscriptome…………………………………………………………………………………………………....64Figure1.2.Allelefrequencyperlocus………………………………………………………..……….68

1

CHAPTERI

LiteratureReview

TheHost:FamilyCucurbitaceae

ThemembersoftheCucurbitaceaefamilyarediverseandcontain118genera

thataregrownallovertheworldinareasconsideredsubtropicalandtropical.In

temperateregions,themaincucurbitcropsarecultivatedandincludecucumbers

(CucumissativusL.),watermelon(Citrullusspp.),musk-melon(CucumismeloL.),

squashes,pumpkins,andgourds(Cucurbitaspp.)(Whitaker,T.W.andDavis1962;

Robinson,R.W.andDecker-Walters1997)Althoughdiverse,membersofthe

Cucurbitaceaehavecommonalitiesincluding:adenserootsystemthatremains

relativelyclosetothesoilsurface;simple,threetofivelobedleaves;frost-sensitive;

andplantsproducefruitontendril-bearingvines(Whitaker,T.W.andDavis1962;

Robinson,R.W.andDecker-Walters1997).Adventitiousrootscanbefoundin

variousCucurbitaspecies(Whitaker,T.W.andDavis1962).

Ofthecucurbits,cucumberisofgreatesteconomicimportance.In2012,the

UnitedStates(US)planted47,330acresofcucumber,withavalueof$248million

(USDA2013).NorthCarolina(NC)isthethirdlargestproducerofbothfreshmarket

andprocessingcucumbersintheUS.TheNorthCarolinaDepartmentofAgriculture

(NCDA)reportedthat6,100acresoffreshmarketcucumberand9,700acresof

processingcucumberswereharvestedin2010withacombinedvalueof

$22,419,000.NCalsoranksinthetop10intheUSforwatermelonandsquash

production.In2012,7,100acresofwatermelonand3,400acresofsquashwere

harvested(NCDA&CS2013).

2

ThePathogen:Pseudoperonosporacubensis

PseudoperonosporacubensisbelongstotheStramenopilekingdom,meaning

itisnotatruefungi,rather,itismorecloselyrelatedtoalgaeanddiatoms.This

organismbelongstothePeronosporaceaefamilyalongwithPhytophthoraspp.P.

cubensisisanobligatebiotrophicparasite,meaning,itmustgrowwithinaliving

hosttosurvive.HostleavescanbeinfectedbyP.cubensisatanyageafterthey

unfurl.Thepathogencanalsoinfectthecotyledon(Palti&Cohen1980).Itssexual

structure,theoospore,cantheoreticallysurviveoutsideofthehost,buttheyhave

notbeenobservedintheUS(Lebeda&Cohen2011;Gökeretal.2007).

Dichotomouslybranchedsporangiophoresemergeoutofstomataingroups

ofonetofive.Eachsporangiophoretipholdsanalmond-shapedsporangiawitha

papilla.Sporangiophorescanbe180-400µmlong,andsporangiaare20-40x14-25

µm(Palti&Cohen1980).Eachsporangiumcontains2-15zoospores,measuring10-

13µm,thathavetwoflagella,oneofwhichhaslateralhairs.Thezoosporesinfect

susceptibleleaftissuebyproducingagermtubeandenteringthroughthestomata.

Theorganismcolonizestheleafashyphaegrowsintercellularlyandcreates

haustoriathattakenutrientsfromhostcellsandsecreteeffectorproteinsthat

contributetodevelopmentofacompatibleinteractionanddisease(Savoryetal.

2011).

Althoughtherehavebeenreportsofoosporesinlabandfieldsettings

(Rostowzew1903;Hiura&Kawada1933;Chenetal.1959),thesestructureshad

notbeenstudiedindepthuntilrecently.CohenandRuben(2012)recently

conductedanin-depthexperimenttostudythesexualreproductionofP.cubensis.

3

Underlabsettings,theyfound“thatP.cubensisisheterothallic,havingA1andA2

matingtypeswhichcancrossandenablesexualreproductionincucurbits”(Cohen

&Ruben2012).In2013,Thomasetalreportedtheproductionofoosporesin

laboratorysettings.TheyalsoidentifiedtheA2matingtypeintheUnitedStates

(Thomasetal2013).

LeafwetnessisaveryimportantfactorinthelifecycleofP.cubensis.Thomas

showedthateveninthepresenceofinoculum,dewmustalsobepresentontheleaf

surfacefor5-6hoursforP.cubensistoinfectcantaloupeleaves(1977).The

optimuminfectiontemperatureandwetnessdurationcombinationsare20Cfor2

hours,15-20Cfor6hours,10-20Cfor12hours,and5-20Cfor24hours.Itwas

foundthatthebestinoculumconcentrationforinfectiontooccurwas1000

sporangia/cm2(Cohen1977).

Epidemiology

IntheUS,downymildewhasbeenobservedoncucurbitsalongtheeast

coast,andinmid-westernstates,suchasMichigan(Coluccietal.2006).This

pathogen,beinganobligateparasite,canonlyliveinthepresenceofcompatible

hosttissue.Cucurbitcropsdonotsurvivethewinterinfieldsettingsabove30°

latitude(Palti&Cohen1980)andP.cubensishasnotyetbeenshowntoover-winter

intheseareas.Theseobservationshaveleadmanytobelievethepathogensurvives

yearroundinsouthernFloridaoringreenhouses.Itcansurviveoncucurbits

presentyearroundinFloridabecauseitdoesnotfrost,andconditionsremain

4

favorable.Eachsummer,sporangiafromFloridaarecarriedviawindcurrentsup

theeastcoast(Nusbaum1944).

Thefirststudytotracktheoccurrenceofcucurbitdownymildewand

weatherpatternsoverseveralstatesintheUSwasconductedbyNusbaumin1941

to1944.Throughcorrespondencewithworkersinstatesallalongtheeastcoast,he

wasabletoidentifysometrendsinthespreadofdownymildewthroughoutthe

growingseason.Hefoundthatthefirstappearanceofthediseaseintheyearwasin

Florida,aroundlateApriltoearlyMay.BytheendofJuneitreachedNC,then

DelawarebythebeginningofJuly,andfinallyMassachusettsbytheendofAugustto

thebeginningofSeptember.Inthesereports,Nusbaumnotedthatnewoutbreaks

anddiseaseprogressionofdownymildewalsowereaffectedbyenvironmental

conditions.Afewdaysofrainyweatherwouldbefollowedbyincreaseddisease

intensityoranewoutbreak.Thediseasewouldnotprogressasquicklywhenthere

wasadryperiod(Nusbaum1944).

However,thestateofcucurbitdownymildewepidemicsintheUShas

changedsignificantlyovertheyears.Theintroductionoftheresistantcucumber

cultivar,Poinsett,inthe1960s,andsubsequentcultivarsbredforresistancekept

cucurbitdownymildewincheckintheUS.In2004,cucurbitdownymildew

suddenlybecameaseriousproblemasresistantcultivarsnolongerwereeffectivein

controllingthedisease(Coluccietal.2006).InNC,Holmes(2006)notedthatdowny

mildewappearedearlierthanithadinthepast,anditwasmuchmoreaggressiveon

cucumber.Inthefollowingyears,thediseasehadspreadtonorthernstatesandthe

mid-west(Coluccietal.2006).There-emergenceofcucurbitdownymildewon

5

cucumbersintheUSandincreaseddiseaseseverityobservedincommercialcrops

maybeduetoanew,morevirulentstrain(Savoryetal.2011).

CDMIPM-PIPE

TheCDMipm-PIPE(CucurbitDownyMildewIntegratedPestInformation

PlatformforExtensionandEducation)isanonlinetoolthatmakesaccessible

informationregardingdiseaseoutbreaks,diseaseforecasts,andanalysisofthe

currentepidemic.Thissystemalertsregisteredmemberswhendownymildewis

likelytoarriveintheircountysogrowerscanplanaheadforappropriateprevention

ormanagementpractices.Previousdownymildewreportsandmeteorologicaldata

areusedtogeneratethesealerts.Havingacalculatedtimeofthearrivalofdowny

mildewcansavegrowersalotofmoneybecausetheycanbeginfungicidesprays

onlywhenthereisariskofinfection(Ojiamboetal.2011).

Severalusefulconclusionshavebeenmadethroughtheuseofthistool.The

datashowsitislikelythatoutbreaksinGeorgia,SouthCarolina,andNCserveasa

sourceofinoculumforcasesofcucurbitdownymildewintheMidwestandmid-

Atlantic,ratherthandirectlyfromsouthernFlorida.OjiamboandHolmes(2011)

alsopointedtothelikelihoodofthecontributionofothersourcesofinoculumto

epidemicsintheGreatLakesandmid-Atlanticregion.Forexample,growersin

MichiganandOntario,Canadaproducecucumbersyear-roundingreenhouses,and

itispossibleforcucumbersfromthesegreenhousestocontributeinoculumtofield

epidemics(Ojiambo&Holmes2011).Resultsfromgeneticstructurestudiessupport

thispossibilityasOntario,Michigan,andOhioisolateshadhighclustersimilarity

6

(Quesada-Ocampoetal.2012).Greenhousecucumberproductionmakesitpossible

tohaveyear-roundavailabilityofsusceptiblecucumbers,allowingP.cubensisto

survivethewinterabove30°latitude.Ifnotcontrolledingreenhousesettings,

infectedgreenhousecucumberscanprovideinoculumforoutbreaksofdowny

mildewatanypointthroughoutthecucurbit-growingseason(Ojiambo&Holmes

2011).

VariabilityinPathogenVirulencefromDifferentHosts

Thepathogen,Pseudoperonosporacubensis,ishost-specific,onlyinfectingthe

leavesoftheCucurbitaceaefamily.TheCucurbitaceaecontainsadiversegroupof

bothcultivatedandwildgenera.Betweenfieldobservationsandlab-based

experiments,P.cubensishasbeenreportedon60speciesin20generaofcucurbits

(Palti&Cohen1980).Intermsofagriculturalsignificanceandeconomicimpact,

onlyninecultivatedspeciesfromthisfamilyaresusceptibletodownymildew

(Cohen1981).IntheUS,downymildewprimarilyinfectssquash,pumpkin,melons,

watermelonstoalesserextent,andasof2004,cucumbers(Coluccietal.2006).

Incucumbersandsquash,chloroticlesionsareangular,asthepathogenis

boundbytheprominentleafveins.Onthebottomsideoftheleaf,thelesionswill

haveafuzzyappearancethatcanbebrown,gray,orevenpurple.This“downy”

aspectofthediseaseisthesporangiophoresandsporangia,anasexualsporeeofthe

oomycetes.Thisisthemostdiagnosticfeatureofthedisease.WhenP.cubensis

infectscantaloupeandwatermelonleaves,thelesionsareusuallysmallandcircular

toirregular-shaped.Theyarebrowninthecenterandhaveachlorotichalo.Itisless

7

commontoseethecharacteristicdownyappearanceontheundersideoftheleaf

(Cohen1981).

Comparedtootherdownymildews,P.cubensishasalargehostrange.

However,isolatesofP.cubensisvaryintheirabilitytoinfectdifferenthosts.This

variabilityinpathogenicityhaspromptedresearcherstodefinepathotypesand

assignP.cubensisisolatesaccordingly.Pathotypesaregroupscontainingpathogen

isolatesthatareabletoinfectthesamecombinationofhosts,downtoagenus,

species,orevencultivarlevel(Lebeda&Cohen2011).In1987,isolatesfromJapan,

Israel,andtheUSweregroupedintofiveP.cubensispathotypes.Afterinoculating

twenty-sixcultivars(spanningsevengenera)ofcucurbitswithmultiplepathogen

isolates,diseaseincidenceandseverityshowedthatthereweredistinct

pathogenicitydifferencesamongisolates(Thomasetal.1987).Thesetofhost

differentialswasalteredyearslater,andnowtherearetwelveCucurbitaceaetaxa

thatrepresentthemostimportantgenera:Cucumis,Cucurbita,Citrullus,Benincasa,

Luffa,andLagenaria.Inthisnewsystem,isolatesaregivenanumericalcodefor

pathotypeidentification.Researcherssuggestedthatstandardizationofthehost-

differentialstestandmakinginformationaboutthehosttaxaeasilyaccessible

wouldleadtobetterresearchonhost-pathogeninteractions,pathogengenetics,and

populationstructure(Lebeda&Widrlechner2003).

Toanalyzehost-rangevariabilityinP.cubensisisolatesfromtheUS,Colucci

(2008)performedthehost-differentialtestonAmericanisolatesofP.cubensis

collectedbeforeandafterthere-emergenceofcucurbitdownymildewintheUSin

2004.Shefoundthatalltheisolatesproducedhighlevelsofinfectiononcucumber

8

andcantaloupe.IsolatesinoculatedonotherCucumisspp.hadsuccessfulinfection

ratesaswell.Ofthethreesquashdifferentialsused,Cucurbitapepovar.pepo

showedthelowestlevelofinfection.IsolatesofP.cubensisfromCucumiscaused

onlylowlevelsofinfectiononCucurbitaspp.Ultimately,Colucci(2008)determined

thatitwasnotusefultocategorizeUSisolatesintopathotypesduetothegreat

variabilityinpathogenicityfoundinUSisolates.Overall,althoughCucumisspp.may

bemoresusceptibletodownymildewinfection,itshouldbenotedthat“all

commerciallygrowncucurbitcropsareatriskfordownymildewinfectionin

naturalpopulations”(Colucci2008).

Usingapopulationgeneticsapproach,Quesada-Ocampoetal.(2012)found

thatP.cubensisisolatesfromnon-cucumberhoststendedtobefromaparticular

geneticclustermoresothanisolatesfromC.sativus.Thesefindingssuggesteda

levelofhostspecificity,specifically,thatisolatesfromcertaingeneticclustersmay

differinabilitytoinfectdifferentcucurbithosts(Quesada-Ocampoetal.2012).

Usingamulti-locusapproach,Rungeetal.(2011)determined“cryptic

speciesorsubspecies”werepresentwithinP.cubensisandbelongedtotwodifferent

phylogeneticlineages.Isolatesfromclade1aredistributedworldwidewithhigh

geneticvariationinNorthAmerica,possiblyindicatingthisregionastheoriginofP.

cubensisoracenterofdiversity.IsolatesfromEastAsiatendtobelongtoclade2.

However,someisolatesfromtheCzechRepublic,Germany,andtheUSthatalso

belongtothisclade.Interestingly,theUSisolatesdesignatedtoclade2arestrains

thatoriginatedfromtherecentaggressiveoutbreaks.Rungeetal.explainsthattheir

phylogeneticstudiescoupledwithhistoricreportsofP.cubensisintheUSandJapan

9

seemtoconcludethatclade2ofP.cubensisfromJapanwasintroducedtotheUSand

causedthe2004resistance-breakingepidemic(Rungeetal.2011).

Morerecently,sequencingapproacheshavebeenimplementedtoanswer

questionsofhostspecialization.Theexistenceof“twodistinctevolutionary

lineages”wasdeterminedthroughacomparativeanalysisofwhole-genome

sequencesofP.cubensisisolatescollectedfromeachofthemajorcommercial

cucurbithosts.Whole-genomesequencedatageneratedbyIllumina’sHiSeq

platformshowedisolatesfromCucurbitapepo,C.moschata,andCitrulluslanatus

belongtoonelineage,andisolatesfromCucumisspp.andCucurbitamaximabelong

toaseparatelineage(Thomasetal.2014).

PseudoperonosporacubensisandP.humuli

OtherspeciesofPseudoperonosporaincludeP.humuli,P.cannabina,P.

celtidis,whichinfectplantsintheUrticaceae,Cannabinaceae,andUlmaceae,

respectively(Runge&Thines2012).Severalstudieshaveinvestigatedthe

phylogeneticrelationshipsbetweenP.cubensisandP.humuli.AstudybyChoietal.

(2005)claimedtherewerenorealdifferencesinmorphologybetweenP.cubensis

andP.humuli.TheyalsoconcludedthatbasedonthegeneticsimilarityofnrITS

sequences,P.humuliisthesamespeciesasP.cubensis.Theseconclusionshave

receivedcriticismandopposingdatafromseveralprojects.

ResultsfromRungeetal.(2011)supportthespeciesdistinctionbetweenP.

humuliandP.cubensisusingnrITS,coxII,andypt1.Thisstudyalsofoundthat

isolatesfromHumulusjaponicusfromKoreamightbeacrypticspecies,astheywere

10

foundtobebasaltothestudiedP.cubensisisolates.Theseresultsillustratethatthe

downymildewhostjumptoCucurbitaceaemostlikelycamefromHumulusspp.

(Rungeetal.2011).AstudybyMitchelletal.(2011)alsosupportedtheideaoftwo

distinctspeciesthroughphylogeneticanalysisofnrITS,B-tub,andcox2.However,

resultsfromthisstudyledauthorstoconclude,“P.humulidescendedfromP.

cubensis”.Thisstudyalsofoundthatinlaboratorysettings,P.cubensiscaninfecthop

andhavelightsporulation,whereasP.humulionlyproducedonesporangiophore

wheninoculatedoncucurbitplants.Thisfindingprovidesevidenceforthe

possibilityofahostjumpaswell,althoughinoculationstudiescannotdetermine

directionality(Mitchelletal.2011).Runge&Thinesrevisitedthisissuein2012,

claimingMitchell’sphylogenetictreesdonotsuggestP.humulidescendsfromP.

cubensis.Theyalsoconductedlab-basedinfectionexperimentsandfoundthatP.

cubensisinfectshopwithlimitedsuccessandsporulation,andP.humuliwasableto

infectandasexuallyreproduceonCucumissativus(Runge&Thines2012).The

increaseinmolecularapplicationstogeneticsstudiesmayassistinclarifyingthe

taxonomicstatusofP.cubensisandP.humuli.

Otherthanphylogeneticrelationships,thereareseveraldifferencesbetween

P.cubensisandP.humuli.IntheUStherearenoreportsofP.cubensisoverwintering

throughoosporesinfieldsettings.Commercialcucurbithostsarenotperennial,and

itisunknownwhetherP.cubensiscansurviveyear-roundincoolerregions.P.

humuli,however,regularlyproducesoosporesinseveralpartsoftheperennialhop

plantincludingleaves,cone,stem,andcrown(Mahaffeeetal.2009).P.cubensiswas

knowntosolelyinfectandsporulateonhostleaftissue.Arecentstudyhasmadethe

11

firstreportthatP.cubensiscanbeseed-transmitted(Cohenetal.2014).Itis

possibletomorphologicallydistinguishP.cubensisfromP.humuliinalabsetting,

butbecausedownymildewmorphologyisinfluencedbyenvironmentalandhost

factors,onewouldnotbeabletoreliablymorphologicallydifferentiatefieldsamples

ofthesespecies(Runge&Thines2012).

GeographicDispersal/GeneticDiversityofP.cubensis

ThepathogenP.cubensishasbeenreportedin70countries,rangingfromsub

tropicaltotemperatetosemi-aridenvironments(Palti&Cohen1980).Inrecent

years,strideshavebeenmadeinresearchrelatingtopopulationgeneticsofP.

cubensis.Asdiscussedearlier,thehostpathogenicityrangewithinP.cubensisasa

speciesishighlyvariable.However,Choietal.(2005)concludedP.cubensistobea

homogeneoustaxonduetotheextremesimilarityofITSrDNAsequences.Itwas

clearmoremolecularstudieswerenecessarytogaininsightongeneticdiversityof

P.cubensisanditspopulations(Choietal.2005).Studiesweredevelopedto

examineP.cubensiswithinpopulationstolearnmoreabouthost-parasite

interactions,andbetweenpopulationstofindconnectionsbetweenstructureand

geographicdispersalofthepathogen(Sarrisetal.2009;Quesada-Ocampoetal.

2012;Polatetal.2014).

AcombinationofAFLP(AmplifiedFragmentLengthPolymorphism)andITS

rDNAanalysiswereusedtocompareP.cubensispopulationsfromEuropean

locations.ThroughtheuseofaclusteringalgorithmontheAFLPdata,itwas

determinedthattheisolatesweredividedintotwomaingroups:ContinentalEurope

12

(WesternEuropeisolatesandCzechRepublic)andMediterraneanEurope(Crete,

Greece).AFLPanalysiswasabletoestablishgeneticisolationbetweenthesetwo

groups.Inaddition,therewasenoughdifferenceamongITS2sequencesforcorrect

groupingofAsianvs.Europeanisolates(Sarrisetal.2009).

Inlaterstudies,evenmoregeneticsiteswereusedtogroupP.cubensis

isolates.UsingtwomitochondrialandfivenuclearSNPmarkers,andBayesian

clustering,Quesada-Ocampoetal.(2012)foundsixgeneticclustersinacollectionof

P.cubensisisolatesfromaroundtheworld.Althoughallsixofthegeneticclusters

werepresentineachcontinent,structurewasdeterminedinvariousgeographic

regions.ThisstudyalsofoundthatisolatesfromNorthCarolinashowedhighgenetic

diversityandwere“moderatelytohighlydifferentiated”fromFloridaisolates.Itis

currentlybelievedP.cubensispersistsyear-roundasactivemyceliuminsouthern

Florida,thendispersesabovethe30°latitudelineasthecucurbitseasonprogresses.

ThegeneticdifferentiationofP.cubensisisolatesfromNorthCarolinaandFlorida

suggestinoculumfromothersourcessuchasMexicoandgulfcoaststates,couldbe

contributingtothegeneticdiversityinNorthCarolina.Researcherssequencedtwo

mitochondrialregionsandfivenuclearregionsthatcontained50informativeSNPs,

(singlenucleotidepolymorphism)andusedBayesianclusteringtogroupisolates

intogeneticclusters.Whenisolatesclassifiedintogeneticclustersweresortedby

geographicregions,ageneticstructurewasobserved,showingdifferentiation

betweenisolatesfromtheUSandisolatesfromEurope(Quesada-Ocampoetal.

2012).

13

Microsatellites

TolearnmoreaboutgeneticpatternswithinandbetweenP.cubensis

populations,severalmoleculartoolscanbeused.Erhardt(2009)suggeststheuse

ofmolecularmarkerscouldshedlightonthegeneticrelatednessofP.cubensis

populationsintheMidwestandP.cubensispopulationsfoundinSouthernstates

earlyinthediseasecycle(Erhardt2009).

Variousmoleculartechniques(RFLPs,mtDNA,SSRs,AFLPs,SNPs)canbe

usedtogaininformationonpopulationdiversityandpopulationgenetics(Cooke&

Lees2004).Microsatellites,orSimpleSequenceRepeats(SSRs)arerepeating

patternsofnucleotidesthatoccurthroughoutgenomes.Commonmotifsincludedi-,

tri-,tetra,penta-,andhexa-nucleotideunitsthatoccurinarepeating,consecutiveor

interruptedsequence.Duetotheirfrequencyandtheirhighmutationrate,theycan

beusedasgeneticmarkers,whereindividualsofapopulationmayhavevarying

numbersofarepeatingmotifatagivenlocus(Guichouxetal.2011).Molecular

markers,ingeneral,canbeusedinpopulationgeneticsstudiestodefine

populations,assignindividualstopopulations,andmeasurevariationwithinand

amongpopulations(Pritchardetal.2000;McDonald&Linde2002).Inferences

aboutapopulation’sevolutionarypastareshapedbygeneticstructuredata.Within

thefieldofplantpathology,researcherspredictpossibleevolutionarymechanisms

thatmayshapecurrentandfuturepopulations.Forplantpathologists,this

informationisvaluable,asitguidesinformeddecisionsaboutdiseasemanagement

strategiesspecifictocertainpathogenpopulationsandhowtheyareexpectedto

changeovertime(McDonald&Linde2002).

14

Withtheavailabilityofwhole-genomesequencesofP.cubensis,identifying

SSRsanddesigningprimerstoamplifythesesequencesisnowaprocessthatcanbe

completedinsilico.

WildHosts

SeveralstudieshaveshownthatitislikelyP.cubensisoverwintersasactive

myceliaoncommercialcucurbitsinsouthernFlorida,andthenaeriallydispersesup

theeastcoasteachyear(Ojiambo&Holmes2011;Nusbaum1944).Resultsfrom

Quesada-Ocampoetal.(2012)showedthatP.cubensisisolatesfromFloridawere

geneticallydifferentfromP.cubensisisolatesfromGeorgia,NorthCarolina,Texas,

SouthCarolina,andIndiana.Theseresultssuggestothersourcesofinoculum

contributetoP.cubensisdiversityotherthanorinadditiontoinoculumfrom

isolatesabletooverwinterinsouthernFlorida(Quesada-Ocampoetal.2012).

Researchersoftencitewildhostsasapossiblesourceforthisdiversity(Lebeda&

Cohen2011;Quesada-Ocampoetal.2012;Ojiamboetal.2015;Ojiamboetal.2011).

Severalstudiescalledforaninvestigationofwildhostsandperennialhosts

ofP.cubensisintheUS(Ojiambo&Holmes2011).P.cubensiscanoverwinteron

Bryoniadioicainlaboratorysettings,givingsupporttothenotionofwildhosts

contributingtothedownymildewepidemic.AlthoughB.dioicagrowsinthewildin

Europe,itisnotfoundintheUnitedStates(Runge&Thines2009).However,

severalwildspeciesofCucurbitaceaeoftenusedinhost-rangestudies(Lebeda

1992;Lebeda&Widrlechner2003)arecommonlyfoundinmanystatesthroughout

theUS(USDAPLANTSDatabase).AsianmelonsaregainingpopularityintheUSfor

15

bothsmall-scaleproductionandforcucurbitbreedingprograms(McCreightetal.

2012),andMomordicabalsaminaandM.charantiaaresporulatinghostsforP.

cubensisinfieldsettings(Wallaceetal.2014).Finally,manyspeciesof

Cucurbitaceaegrowinthewildandareconsideredweeds.SpeciessuchasSicyos

angulatus,Echinocystislobata,andCucurbitafoetidissimahavebeenreportedas

hoststoPseudoperonosporacubensisinnaturalsettingsintheUnitedStates

(PLANTS,Wallaceetal.2015).Itremainstobeseenifthesewildandnon-

commercialcucurbitscontributetothedownymildewepidemicintheUS.

16

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cubensisandP.humulibasedonmolecularandmorphologicaldata.Mycological

Research,109(7),pp.841–848.

Cohen,Y.etal.2014.SeedTransmissionofPseudoperonosporacubensis.PLOSONE,

9(10),e109766.

Cohen,Y.1977.Thecombinedeffectsoftemperature,leafwetness,andinoculum

concentrationoninfectionofcucumberswithPseudoperonosporacubensis.

CanadianJournalofBotany,55,pp.1478–1487.

Colucci,S.J.2008.HostRange,FungicideResistanceandManagementof

Pseudoperonosporacubensis,CausalAgentofCucurbitDownyMildew.MS

Thesis,NorthCarolinaStateUniversity.RetrievedfromNCSUInstitutional

Repository.

Colucci,S.J.,Wehner,T.C.&Holmes,G.J.2006.Thedownymildewepidemicof2004

and2005intheEasternUnitedStates.InG.J.Holmes,ed.Cucurbitaceae2006.

Asheville,NorthCarolina:UniversityPress,pp.403–411.

Cooke,D.E.L.&Lees,A.K.2004.Markers,oldandnew,forexaminingPhytophthora

infestansdiversity.PlantPathology,53(6),pp.692–704.

Erhardt,C.2009.DevelopingaHigh-Throughput,SNP-BasedPopulationScreening

MethodforPseudoperonosporacubensis.MSThesis,MichiganSateUniversity.

RetrievedfromProQuestDissertaions&ThesesGlobal,304931218.

Göker,M.etal.2007.Howdoobligateparasitesevolve?Amulti-genephylogenetic

analysisofdownymildews.FungalGeneticsandBiology,44(2),pp.105–22.

17

Guichoux,E.etal.2011.Currenttrendsinmicrosatellitegenotyping.Molecular

EcologyResources,11(4),pp.591–611.

Lebeda,A.1992.ScreeningofwildCucumisspeciesagainstdownymildew

(Pseudperonosporacubensis)isolatesfromcucumbers.Phytoparasitica,20(3),

pp.203–210.

Lebeda,A.&Cohen,Y.2011.Cucurbitdownymildew(Pseudoperonospora

cubensis)—biology,ecology,epidemiology,host-pathogeninteractionand

control.EuropeanJournalofPlantPathology,129(2),pp.157–192.

Lebeda,A.&Widrlechner,M.P.2003.AsetofCucurbitaceaetaxafordifferentiation

ofPseudoperonosporacubensispathotypes.JournalofPlantDiseaseand

Protection,110(4),pp.337–349.

Mahaffee,W.,Pethybridge,S.&Gent,D.H.2009.CompendiumofHopDiseasesand

Pests,AmericanPhytopathologicalSociety,Minneapolis.

Mccreight,J.D.,Staub,J.E.&Wehner,T.C.2012.GoneGlobal :FamiliarandExotic

CucurbitsHaveAsianOrigins.HortScience,48(9),pp.1078–1089.

McDonald,B.A.&Linde,C.2002.Pathogenpopulationgenetics,evolutionary

potential,anddurableresistance.AnnualReviewofPhytopathology,40,pp.349–

79.

Mitchell,M.N.etal.2011.GeneticandpathogenicrelatednessofPseudoperonospora

cubensisandP.humuli.Phytopathology,101(7),pp.805–18.

NCDA&CS,2013.AgriculturalStatistics-2012AnnualStatisticsBook.Raleigh.

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Nusbaum,C.J.1944.NoTitle.PlantDiseaseReporter,28,pp.82–85.

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Ojiambo,P.S.etal.2015.EpidemiologyandPopulationBiologyof

Pseudoperonosporacubensis :AModelSystemforManagementofDowny

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Ojiambo,P.S.&Holmes,G.J.2011.Spatiotemporalspreadofcucurbitdownymildew

intheeasternUnitedStates.Phytopathology,101(4),pp.451–61.

Ojiambo,P.S.,Holmes,G.J.&Adams,M.L.2011.CucurbitDownyMildewipmPIPE :A

NextGenerationWeb-basedInteractiveToolforDiseaseManagementand

ExtensionOutreach.PlantHealthProgress,(January).

Palti,J.&Cohen,Y.1980.DownyMildewofCucurbits(Pseudoperonosporacubensis):

TheFungusanditsHosts,Distribution,EpidemiologyandControl.

Phytoparasitica,8(196),pp.109–147.

Polat,İ.etal.2014.CharacterizationofPseudoperonosporacubensisisolatesfrom

EuropeandAsiausingISSRandSRAPmolecularmarkers.EuropeanJournalof

PlantPathology,139(3),pp.641–653.

Pritchard,J.K.,Stephens,M.&Donnelly,P.2000.Inferenceofpopulationstructure

usingmultilocusgenotypedata.Genetics,155(2),pp.945–59.

Quesada-Ocampo,L.M.,Granke,L.L.&Olsen,J.2012.TheGeneticStructureof

PseudoperonosporacubensisPopulations.PlantDisease,96(10),pp.1459–1470.

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York.

Runge,F.,Choi,Y.-J.&Thines,M.2011.Phylogeneticinvestigationsinthegenus

PseudoperonosporarevealoverlookedspeciesandcrypticdiversityintheP.

cubensisspeciescluster.EuropeanJournalofPlantPathology,129(2),pp.135–

19

146.

Runge,F.&Thines,M.2009.ApotentialperennialhostforPseudoperonospora

cubensisintemperateregions.EuropeanJournalofPlantPathology,123(4),

pp.483–486.

Runge,F.&Thines,M.2012.ReevaluationofHostSpecificityoftheCloselyRelated

SpeciesPseudoperonosporahumuliandP.cubensis.PlantDisease,(96),pp.55–

61.

Sarris,P.etal.2009.Molecularpolymorphismsbetweenpopulationsof

PseudoperonosporacubensisfromGreeceandtheCzechRepublicandthe

phytopathologicalandphylogeneticimplications.PlantPathology,58(5),

pp.933–943.

Savory,E.A.etal.2011.Pathogenprofile:Thecucurbitdownymildewpathogen

PseudoperonosporacubensisSUMMARY.MolecularPlantPathology,12(3),

pp.217–226.

Thomas,A.,Carbone,I.&Ojiambo,P.2014.Comparativegenomicanalysisof

Pseudoperonosporacubensistoelucidatethegeneticbasisofhostspecialization.

Phytopathology,104(S3)pp.118.

Thomas,C.,Inaba,T.&Cohen,Y.1987.PhysiologicalSpecializationin

Pseudoperonosporacubensis.Phytopathology,77,pp.1621–1624.

USDA.2013.Cucumbers:BackgroundStatistics.EconomicResearchService.

Retrievedfromhttp://www.ers.usda.gov/topics/in-the-news/cucumbers-

background-statistics.aspx.

Wallace,E.C.etal.2014.FirstReportofPseudoperonosporacubensiscausingdowny

20

mildewonMomordicabalsaminaandM.charantiainNorthCarolina.Plant

Disease,98(9),pp.1279.

Wallace,E.C.,Adams,M.&Quesada-Ocampo,L.M.2015.FirstReportofDowny

MildewonBuffaloGourd(Cucurbitafoetidissima)causedbyPseudoperonospora

cubensisinNorthCarolina.PlantDisease,99(12),pp.1861.

Whitaker,T.W.andDavis,G.N.1962.Cucurbits:Botany,CultivationandUtilization.

IntersciencePublishersInc.,NewYork.

21

CHAPTERII

DownyMildewPathogensInfectingWildandNon-Commercial

CucurbitsinNorthCarolina

ThischapterwaspublishedasthreeDiseaseNotesinPlantDiseaseAbstractTheoomycetepathogensmakingupthedownymildewsallhaveanobligate

biotrophiclifestyleinwhichtheyrequirealivinghosttosurvive.Thesepathogens

includingPlasmoparaspp.,Peronosporaspp.,andPseudoperonosporaspp.alsotend

tohavenarrowhostranges,onlyinfectingasmallnumberofspecies.Thepathogen

Pseudoperonosporacubensis,thecausalagentofcucurbitdownymildew,however,

infectsapproximately60hostsintheCucurbitaceaefamily.Differentcombinations

ofhostsandpathogenstrainsproducevariablelevelsofinfectionandpathogen

sporulation.However,moststudiesonwildcucurbithostshavebeenperformedin

laboratorysettingswithartificialinoculationmethods.IntheUnitedStates,several

wildandnon-commercialCucurbithostshavebeenreportedtobehostsofP.

cubensis,butmanyofthesereportspre-datetheresurgenceofthepathogenin2004.

Itisunclearifthepathogenfrequentlyoccursonwildandnon-commercial

cucurbitsinnaturalconditions.Tobetterunderstandthecapabilityofnon-

commercialcucurbitstoserveashostsforP.cubensisinnaturalsettings,fiveplants

ofsixdifferentnon-commercialcucurbitswereplacedinsentinelplotsinLenoir,

Haywood,andRowancountiesinNorthCarolina.Thenon-commercialcucurbits

wereplantedalongsidecommercialcucurbits.Theplotsweremonitoredfordisease

throughoutthesummerandfallgrowingseasons.Symptomaticlesionswerefurther

inspectedwithadissectingmicroscopeinalaboratorysetting.Ifsporulationwas

observedKoch’spostulateswereperformed.Between2013and2014,P.cubensis

wasfoundsporulatingonMomordicacharantia,Momordicabalsamina,and

Cucurbitafoetidissima,andKoch’spostulateswerecompleted.Plasmoparaaustralis

wasalsofoundonLuffacylindrica,whichwasthefirstreportofthishost-pathogen

22

interactionintheUnitedStates.Identifyingnon-commercialandwildcucurbitsthat

hostthepathogeninnatureisanimportantstepinunderstandingtheroleofthese

cucurbitsintheyearlycucurbitdownymildewepidemic.

23

FirstReportofPseudoperonosporacubensisCausingDownyMildewonMomordicabalsaminaandM.charantiainNorthCarolina.

E.Wallace,M.Adams,K.Ivors,P.S.Ojiambo,andL.M.Quesada-Ocampo,DepartmentofPlantPathology,NorthCarolinaStateUniversity,Raleigh,NC27695.

Publishedmanuscript:

Wallace,E.etal.,2014.FirstreportofPseudoperonosporacubensiscausingdownymildewonMomordicabalsaminaandM.charantiainNorthCarolina.PlantDisease,98(9),P.1279.Availableat:http://dx.doi.org/10.1094/PDIS-03-14-0305-PDN.

Momordicabalsamina(balsamapple)andM.charantiaL.(bittermelon/bitter

gourd/balsampear)commonlygrowinthewildinAfricaandAsia;bittermelonis

alsocultivatedforfoodandmedicinalpurposesinAsia(Bharathi&John2013).In

theUnitedStates,thesecucurbitsgrowasweedsorornamentals.Bothspeciesare

foundinsouthernstatesandbittermelonisalsofoundinPennsylvaniaand

Connecticut(PLANTS).Cucurbitdownymildew(CDM),causedbytheoomycete

Pseudoperonosporacubensis,wasobservedonbittermelonandbalsamapple

betweenAugustandOctoberof2013insixNorthCarolinasentinelplotsbelonging

totheCDMipmPIPEprogram(Ojiamboetal.2011).Plotswerelocatedatresearch

stationsinJohnston,Sampson,Lenoir,Henderson,Rowan,andHaywoodcounties,

andcontainedsixdifferentcommercialcucurbitspeciesincludingcucumbers,

melons,andsquashesinadditiontotheMomordicaspp.Leaveswithsymptoms

typicalofCDMwerecollectedfromtheMomordicaspp.andsymptomsvariedfrom

irregularchloroticlesionstocircularlesionswithchlorotichalosontheadaxialleaf

surface.Sporulationontheabaxialsideoftheleaveswasobservedandacompound

microscoperevealedsporangiophores(180to200μmheight)bearinglemon-

shaped,darksporangia(20to35×10to20μmdiameter)withpapillaononeend.

24

GenomicDNAwasextractedfromlesionsandregionsoftheNADHdehydrogynase

subunit1(Nad1),NADHdehydrogynasesubunit5(Nad5),andinternaltranscribed

spacer(ITS)ribosomalRNAgeneswereamplifiedandsequenced(Quesada-Ocampo

etal.2012).BLASTanalysisrevealed100%identitytoP.cubensisNad1

(HQ636552.1,HQ636551.1),Nad5(HQ636556.1),andITS(HQ636491.1)sequences

inGenBank.SequencesfromadownymildewisolatefromeachMomordicaspp.

weredepositedinGenBankasaccessionnos.KJ496339through44.Tofurther

confirmhostsusceptibility,veinjunctionsontheabaxialleafsurfaceoffive

detachedleavesoflab-grownbalsamappleandbittermelonwereeitherinoculated

withasporangiasuspension(10μl,104sporangia/ml)ofaP.cubensisisolatefrom

Cucumissativus(‘Vlaspik’cucumber),orwithwaterasacontrol.Inoculatedleaves

wereplacedinhumiditychamberstopromoteinfectionandincubatedusinga12-h

light(21°C)anddark(18°C)cycle.Sevendayspostinoculation,CDMsymptomsand

sporulationwereobservedoninoculatedbalsamappleandbittermelonleaves.P.

cubensishasbeenreportedasapathogenofbothhostsinIowa(USDA1960).Toour

knowledge,thisisthefirstreportofP.cubensisinfectingtheseMomordicaspp.inNC

inthefield.IdentifyingtheseMomordicaspp.ashostsforP.cubensisisimportant

sincethesecucurbitsmayserveasasourceofCDMinoculumandpotentiallyan

overwinteringmechanismforP.cubensis.Furtherresearchisneededtoestablish

theroleofnon-commercialcucurbitsintheyearlyCDMepidemic,whichwillaidthe

effortsoftheCDMipmPIPEtopredictdiseaseoutbreaks.

25

References:

L.K.BharathiandK.J.John.MomordicaGenusinAsia-AnOverview.Springer,NewDelhi,India,2013.

P.S.Ojiamboetal.PlantHealthProg.doi:10.1094/PHP-2011-0411-01-RV,2011.

PLANTSDatabase.NaturalResourcesConservationService,USDA.Retrievedfromhttp://plants.usda.gov/,7February2014.

L.M.Quesada-Ocampoetal.PlantDis.96:1459,2012.

USDA.IndexofPlantDiseaseintheUnitedStates.AgriculturalHandbook165,1960.

26

FirstReportofDownyMildewonBuffaloGourd(Cucurbitafoetidissima)CausedbyPseudoperonosporacubensisinNorthCarolina.

E.Wallace,M.Adams,L.M.Quesada-Ocampo,DepartmentofPlantPathology,NorthCarolinaStateUniversity,Raleigh,NC27695

PublishedManuscript:

Wallace,E.etal.,2015.FirstreportofdownymildewonBuffaloGourd(Cucurbitafoetidissima)causedbyPseudoperonosporacubensisinNorthCarolina.PlantDisease,99(12),P.1861.Availableat:http://dx.doi.org/10.1094/PDIS-03-15-0348-PDN.

Cucurbitafoetidissima(buffalogourd)isavinyperennialcropnativetotheUnited

States(US).Thisplant,characterizedbylongtriangulargrey-greenleavesandfruits

thatare5to10cmindiameterandyellowatmaturity,isferalandconsidereda

weedtomany.However,C.foetidissimaservesseveralpurposesinNativeAmerican

culturesanditsoil-richseedshavepotentialeconomicprofitability(Bemisetal.

1978;Clowneyetal.2013).C.foetidissimagrowswellindryaridregions,butis

widelydistributedthroughouttheUS,growingin22statesandMexico(Bemisetal.

1978;PLANTS).InAugustthroughOctoberof2014,Pseudoperonosporacubensis

wasobservedonC.foetidissimaplantsinLenoir,Rowan,andHaywoodcountiesin

NorthCarolina(NC).TheseplantsweregrowninsentinelplotsaspartoftheCDM-

IPMPIPE,thecucurbitdownymildewdisease-forecastingsystem.Thediseasewas

characterizedbyirregularbrownlesionswithchlorotichalosandsporulationonthe

abaxialleafsurface.Theoomycetewascollectedfrominfectedleavesandobserved

withamicroscope,revealingcharacteristicP.cubensisstructures.Anaverage

sporangiophoretrunklengthof307.6μmandpigmented,papillated,lemon-shaped

sporangiaof23.9x17.9μmwereobserved.SporangiacollectedfromLenoirCounty

fieldsampleswereusedtoinoculatedetachedlab-grownC.foetidissimaleaves.A

27

104/mLsuspensionofsporangiawasappliedtotheabaxialsurfaceoftheleaves

usingaPrevalsprayer.Inoculatedleaveswerekeptinacrylicclearboxesandplaced

inincubatorssetforacycleof12-hoflightat21̊Cthen12-hofdarkat18̊C.

Humiditywasmaintainedbytheadditionofadamppapertowelinthebox.Sixdays

postinoculation,sporulationofthepathogenwasobservedontheabaxialsideof

theleaf.Speciesconfirmationwasalsocarriedoutmolecularly.DNAwasextracted

fromsporulatinglesionsandPCRwasusedtoamplifynuclearandmitochondrial

regions.PCRproductsweresequencedandBLASTsearchesshowedBetaTubulin

(Btub),NADHdehydrogenasesubunit1(Nad1),andNADHdehydrogenasesubunit

5(Nad5),had100%identitytoP.cubensissequencesinNCBI(AccessionNo.

JF304706.1,KJ141003.1,andHQ636556.1respectively).SequencesofP.cubensis

isolatesfromC.foetidissimawereaddedtoGenbank(AccessionNo.KP970684,

KP970682,KP970683).Littleisknownabouttheimpactofwildcucurbitsinthe

yearlycucurbitdownymildewepidemicandwarrantsfurtherresearch.The

perennialnatureofC.foetidissimamakesthishostofparticularimportanceasitis

believedP.cubensismayoverwinteronwildcucurbits(Ojiamboetal.2011).Thisis

thefirstreportofP.cubensisinfectingC.foetidissimainfieldsettingsintheUS.

Identifyingnon-commercialandwildcucurbitsthathostthedownymildew

pathogenisanimportantfactortoidentifyaswelearnmoreaboutthepathogenand

detailsofthediseasecycle.

28

References

W.Bemisetal.Econ.Bot.32,87,1978.

F.G.Clowneyetal.2013.Climbers:Cucurbitafoetidissima.UniversityofMichigan.Retrieved11August2014fromhttp://climbers.lsa.umich.edu/?p=258

P.S.Ojiamboetal.2011.CucurbitDownyMildewipmPIPE:ANextGenerationGenerationWeb-basedInteractiveToolforDiseaseManagementandExtensionOutreach.PlantHealthProg.OnlinePublication.doi:10.1094/PHP-2011-0411-01-RV.

PLANTSDatabase.NaturalResourcesConservationService,USDA.Retrieved22August2014fromhttp://plants.usda.gov/

29

FirstreportofPlasmoparaaff.australisonLuffacylindricaintheUnitedStates

E.Wallace1,Y.J.Choi2,,M.Thines3,4,L.M.Quesada-Ocampo1,1DepartmentofPlantPathology,NorthCarolinaStateUniversity,Raleigh,NC27695,2DepartmentofBiologicalScience,CollegeofNaturalSciences,KunsanNationalUniversity,Gunsan54150,Korea,3BiodiversityandClimateResearchCentre(BiK-F),D-60325Frankfurt(Main),Germany,SenckenbergGesellschaftfurNaturforschung,Senkenberganlage25,D-60325Frankfurt(Main),Germany,4JohannWolfgangGoetheUniversity,DepartmentofBiologicalSciences,InstituteofEcology,EvolutionandDiversity,D-60325Frankfurt(Main),Germany

PublishedManuscript:

Wallace,E.etal.,2016.FirstreportofPlasmoparaaff.australisonLuffacylindricaintheUnitedStates.PlantDisease,100(2),P.537.Availableat:http://dx.doi.org/10.1094/PDIS-06-15-0684-PDN.

Luffa,orthespongegourd,isasub-tropicalmemberoftheCucurbitaceae

family.Thefibrousnatureofthefruit’svascularsystemcreatesafunctionalsponge

andismainlycultivatedforthispurpose.AsiaandCentralAmericaleadin

commercialluffaproduction,buttheUnitedStates(US)hasrecognizedthedemand

andeconomicpotentialforluffaandeffortshavebeenmadetoimplementluffaasa

specialtycrop(Davis2008).InAugust2014,luffaleavesexhibitingirregularto

angularchloroticlesionswerecollectedfromHaywoodCountyinNorthCarolina.All

fiveluffaplants,locatedinasentinelplotpartoftheCDMIPMpipe,wereinfected

with20%diseaseseverity.Theabaxialsurfacerevealedwater-soakedlesionsand

whitesporulationconsistentwithdownymildewsymptoms.Sporangiophoresand

sporangiawerecollectedfromfieldsamplesbypipettingupanddownonthelesion

withwater,thenobservedandmeasuredusingalightmicroscope.Papillate,hyaline

sporangiawereprimarilyovoid-sphericalinshape,havingdimensionsof(13–)14–

15–17(-18)×(11–)12–13–13(–15)(n=54).Thesporangiophorelengthhad

30

dimensionsof(348–)438–550–661(–771)(n=54).Thetrunk’swidthwas(8-)10-

12-14(-16)(n=46),withthebaseslightlywiderandbecomingnarrowjustbelow

thefirstbranch.Branchingpatternwasmonopodialwithsecondarybranchesand

branchletsoccurringat90°anglesfromprimarybranchesandsecondarybranches,

respectively.MorphologywasconsistentwithPlasmoparaaustralisaccordingto

Constantinescu(2002).DNAwasextractedfromsporulatinglesionsonluffaleaves.

PCRwasusedtoamplifythenucleargeneD1-D3regionof28Slargesubunit

ribosomalRNA(LSU),andthemitochondrialgenecytochromecoxidasesubunit2

(cox2)(Choietal.2009).ProductsweresequencedandsubmittedtoGenBank

(KT159460-KT159463).Thereisnoprevioussequencedataavailablefor

PlasmoparaaustralisinGenBankforBLASTanalysesasoccurswithmanydowny

mildewpathogens;however,theD1-D3nrLSUsequenceswereusedtogeneratea

phylogenetictreeasseeninChoietal.2009.Cox2sequenceswerenotusedin

phylogeneticanalysis.Thisdata,alongwithmorphologicalcharacteristics,is

sufficienttodesignatethispathogenP.aff.australis.Remainingtissuewas

preservedandsubmittedtotheLarryF.GrandMycologicalHerbarium(CatalogNo.

20864).In2006,PlasmoparaaustraliswasfirstreportedonLuffacylindricainBrazil

(Soaresetal.2006).IntheUS,P.australisreportedlyoccursonEchinocystislobata

andSicyosangulatus,twowildcucurbitspeciescommonlyfoundacrossthecountry

(Farr&Rossman2015).ItisunclearhowL.cylindricacametobeahostforP.

australisintheUS.However,thisnewdiseaseisofconcernforsmall-scaleluffa

growers.Certainly,moreinvestigationmayshedlightonthediseasecycle,

epidemiology,andhost-specificityofthepathogen.

31

References

Y.J.Choietal.2009.Mycol.Res.113:Pt10.10.1016/j.mycres.2009.07.010

O.Constantinescu,2002.Sydowia.54:129.

J.Davis,2008.HorticulturalInformationLeaflet.Retrieved24March,2015,from

http://content.ces.ncsu.edu/commercial-luffa-sponge-gourd-production/

D.F.Farr&A.Y.Rossman,FungalDatabases,Syst.Mycol.andMicrobiol.Lab.ARS,

USDA.Retrieved24March,2015,fromhttp://nt.ars-grin.gov/fungaldatabases/

D.J.Soaresetal.2006.PlantPathol.55:2.10.1111/j.1365-3059.2006.01328.x

32

CHAPTERIII

IdentificationandDevelopmentofMicrosatelliteMarkersinthe

PseudoperonosporacubensisTranscriptome

EmmaC.Wallace,andLinaM.Quesada-Ocampo,DepartmentofPlantPathology,

NorthCarolinaStateUniversity,Raleigh,NorthCarolina,27695

Wallace,E.&Quesada-Ocampo,L.M.2016.IdentificationandDevelopmentof

MicrosatelliteMarkersinthePseudoperonosporacubensistranscriptome.PLOSONE,

Inpreparation.

Abstract

TenyearsafterthereemergenceofPseudoperonosporacubensisintheUnitedStates

(US),thecausalagentofcucurbitdownymildewcontinuestocausesevere

economiclossesinthecucurbitindustry.TherecentlypublishedP.cubensisdraft

genomeprovidedtheopportunitytodevelopmarkerstostudypopulationdynamics

andoptimizemanagementstrategies.Microsatellites,shorttandemlyrepeating

sequencesofDNA,occurfrequentlythroughoutgenomes,havehighmutationrates,

andserveasgoodmolecularmarkers.Insilicotechniqueswereusedtoidentify

2,738microsatellitesintheP.cubensispredictedtranscriptome,96%ofwhichare

perfectrepeats.Fromtheidentifiedmicrosatellites,2,088flankingprimerswere

designedtodevelopmarkers.Evaluationsof100primerpairsrevealedthat~10%

oftheprimerswerepolymorphicamongP.cubensisisolates.Transferability

33

betweenspecieswasdemonstrated,as84%ofthetotalprimersscreenedamplified

aproductinPseudoperonosporahumuliisolates,asisterspeciesofP.cubensis.

InformativemarkershadanaveragePolymorphismInformationContent(PIC)of

0.53andaverageheterozygocityof0.44.TheseSSRprimersmaybeinformativeina

studytobetterunderstandP.cubensisonapopulationlevel.

Introduction

Microsatellites,orSimpleSequenceRepeats(SSRs)arerepeatingbasepair

unitsconsistingof1-6nucleotidesequences.Theyoccurfrequentlythroughoutthe

genomeandhavehighmutationrates(10-3to10-4perlocuspergeneration)

resultingindifferencesinthenumberofrepeatedmotifsatparticularlocibetween

certainindividuals.AsSSRsizedifferencescanbeobservedandcomparedamong

individualsandpopulations,microsatellitesareadesirablemolecularmarker

(Schlötterer2000).Microsatelliteshaveproventobeusefulinseveralapplications

fromassociationstudiestopaternitytesting(Guichouxetal.2011).Beforethe

prevalenceandaccessibilityofgenomicresourcesandhigh-throughputsequencing

technology,microsatelliteidentificationwasanexpensiveandlaboriousprocess.De

novoidentificationinvolvedcreatingagenomiclibrary,identifyingSSR-containing

clones,sequencing,primerdesign,andlaboratoryvalidation,whichwasalabor-

intensiveandexpensiveprocess(Abdelkrimetal.2009;Zaneetal.2002).NowSSR

identificationandmarkerdevelopmentcanbestreamlinedwithnextgeneration

sequencingandbioinformaticstools.Severalgenomesandtranscriptomesare

publicallyavailableandmanyofthesoftwareusedtoidentifySSRsandtodesign

34

primerstoamplifyidentifiedSSRsarefreeofcost(Abdelkrimetal.2009).Many

studieshaveutilizedtheseresourcesandtechnologiestoreportthefrequencyand

locationofSSRsinvariousplantgenomes,expressedsequencetags(ESTs),and

transcriptomesinatime-efficientandcost-effectivemanner,mainlyforthe

purposesofbreeding,taxonomystudies,conservation,andgermplasm

characterization(Scaglioneetal.2009;Zhaoetal.2015;Caietal.2013;Yueetal.

2014).ObservationsandconclusionsaboutSSRtrendshavehelpedusunderstand

moreabouthowmicrosatellitesoperateinthecontextofagenome,howthey

functioningenomeevolution,andhowtheycontributetogeneticvariation(Tóthet

al.2000).

InthecaseofPseudoperonosporacubensis,thecausalagentofcucurbit

downymildew,methodstoeasilydeterminepopulationstructurearecrucialtoour

understandingoftheepidemiologyofthediseaseitcausesandtodevelopmentof

effectiveanddurablemanagementstrategies(Quesada-Ocampoetal.2012).This

needismoreapparentthaneverasdownymildewposesaseriousthreattocucurbit

productionintheUnitedStates(Holmesetal.2015;Wallaceetal.2014).Priorto

2004,lossesincucumberduetoP.cubensiswereminimalandweremanaged

entirelybyhostresistance.Aftertheemergenceofthehighlyvirulentstrainin2004,

fungicidesbecameanecessityinthecontrolofthispathogen(Coluccietal.2006;

Holmesetal.2015).Theprolificasexualsporulationofthepathogenontheleaf

surfaceandtheeasethroughwhichitisaeriallydispersedfromfieldtofieldalso

contributetothepathogen’sdamagingeffects.Althoughtheroleofsexual

reproductionandwildcucurbithostshavenotbeenthoroughlydetermined,

35

epidemiologicalstudieshavehelpedwithcontrolstrategies(Lebeda&Cohen2011;

Cohenetal.2015;Holmesetal.2015;Ojiamboetal.2015).Thecreationand

implementationofthecucurbitdownymildewintegratedpestmanagementpest

informationplatformforextensionandeducation(CDM-ipm-PIPE)forecasting

system,forexample,hasallowedgrowerstooptimizesprayprogramsthatcoincide

withtheanticipatedarrivalofthepathogenintheircountyorstate(Ojiamboetal.

2011).Evenwiththeuseoffungicidesandthehelpofdiseaseforecastingfromthe

CDMipm-PIPE,P.cubensiscontinuestocausemajorlossesasvitalfungicideslose

efficacyandenvironmentalconditionsremainfavorabletothepathogen(Ojiamboet

al.2015;Holmesetal.2015).

TherehavebeenfewattemptsatdevelopingSSRsforP.cubensisdenovoand

themarkersthatweredevelopedhavenotbeenusedinapopulationstudytodate

(Almanyetal.2009;Hadziabdicetal.2013).FewpopulationgeneticsstudiesonP.

cubensishavebeenconductedandexistingstudieshaveusedothergeneticmarkers

suchasAmplifiedFragmentLengthPolymorphisms,SingleNucleotide

Polymorphisms,Sequence-RelatedAmplifiedPolymorphisms,andInterSimple

SequenceRepeats(AFLPs,SNPsSRAP,ISSRs)(Sarrisetal.2009;Quesada-Ocampo

etal.2012;Polatetal.2014).Moreover,mostmarkersusedhavenotbeen

associatedtoparticulargenes.Markerswithingenesareusefulastheyprovide

functionalinformationaboutthemarker,tendtobeconserved,andaretransferable

betweenspecies(Liuetal.2013).SomeP.cubensispopulationanalyseshaveused

SNPsderivedfromgenes;however,thesestudiesonlyexaminedpopulationchanges

duringlongtimeframes(i.e.years)becausepolymorphismwasnotenoughto

36

examinepopulationchangeswithinagrowingseason(Quesada-Ocampoetal.

2012).TheavailabilityofoptimizedpolymorphicSSRprimerswouldallowresearch

aimedatunderstandingthepopulationdynamicsofdownymildewpathogensin

shorttimeframes(i.e.withinagrowingseason),whichcouldprovideimportant

epidemiologicalinformationtoimprovediseasemanagementstrategies(Naegeleet

al.2016).SSRsderivedfromtranscriptomesarepredictedtobelocatedingenesand

thereforearemorelikelytobetransferabletorelatedspecies.Thisisofparticular

interestbecausethereareseveralgeneraofeconomicallyimportantdownymildew

pathogenswithfewornogenomicresourcesavailablesuchasPseudoperonospora

humuli,Plasmoparaobducens,Peronosporabelbahrii,andPlasmoparaviticola,which

causedownymildewofhop,impatiens,basil,andgraperespectively(Lebeda&

Cohen2011;Gentetal.2009;Risticetal.2011;Kanetisetal.2013;Gessleretal.

2011).

InlightoftheeconomicimportanceofP.cubensisandthepotentialinsight

offeredthroughtheuseofinformativemarkers,wesoughtto:1.Analyzeand

comparethedistributionofSSRsthroughouttheP.cubensisandHyaloperonospora

arabidopsidis(causalagentofArabidopsisdownymildew)predicted

transcriptomes;2.Determinespeciestransferabilityofmarkerswiththeclosely

relatedhopdownymildewpathogenP.humuli;and3.EvaluateSSRsfor

polymorphismacrossP.cubensisisolates.Overall,inthisstudywedemonstrated

thatinsilicoidentificationofSSRsinpredictedgenesfromP.cubensisnext

generationsequencingdataprovidesasubstantialamountofpolymorphicmarkers

37

thatcanbetransferredtocloselyrelateddownymildewpathogensandusedin

populationstructureanalyses.

MaterialsandMethods

InsilicoidentificationandanalysesofSSRsinpredicteddownymildewtranscriptomes

Transcriptomespredictedfromgenomeassembliesfortwodownymildew

pathogens,PseudoperonosporacubensisandHyaloperonosporaarabidopsidisthat

arepubliclyavailablewereusedforSSRidentification.FASTAfilesfortheP.cubensis

andH.arabidopsidistranscriptomeswereobtainedfromSavoryetal.(E.aSavoryet

al.2012)andtheEMBL/Genbank/DDBJdatabasesunderaccession

GCA_000173235.2(Baxteretal.2010),respectively.

TheMicrosatelliteIdentificationTool(MISA)(Thieletal.2003)wasusedto

searchtheP.cubensisandH.arabidopsidistranscriptomesforthepresenceofSSRs.

MISAreportedperfectandcompoundSSRsrangingfromonetosixbasepairunits

withaspecifiedminimumnumberofrepeatsforeachmotif,specifically,1/20,2/5,

3/4,4/3,5/3,6/3(unitsize/minimumnumberofrepeats).Comparisonsbetween

microsatelliteabundance,frequencyandmotiftypesinthetwodownymildew

transcriptomeswerecalculatedwithaproportiontestasconductedinGarnicaetal.

2006(Garnicaetal.2006).

PrimerDesign

AcustomBioperlscriptwasusedtoparsetheMISAoutputfilesothe

programPrimer3(Rozen&Skaletsky2000)couldusesequencecoordinates

reportedbyMISAtodesignprimersflankingtheidentifiedSSRs.Theprogram

designedprimersthatwouldamplifyproductsbetween100-300bp.Theprimers

38

weretobebetween18and27bpwithoptimumlengthof20bp,withGCcontent

between20%and80%withoptimumGCcontentof50%,andwithamelting

temperaturebetween57and63°Cwiththeoptimummeltingtemperatureof60°C.

Primersrepresentativeofthedifferentmotifgroups(100total)wereorderedfrom

andmanufacturedbyIntegratedDNATechnologies,Inc.(Coralville,IA).Forward

primersweredesignedtoincludeanM13tailforfluorescentlabelingofproducts

andlaterfragmentanalyses(Schuelke2000).

TissuecollectionandDNAextraction

Atotalof13downymildewsampleswereusedtoevaluate100Primer3-

generatedSSRprimersforamplification,transferability,andpolymorphismviagel

electrophoresis.Cucurbitandhopleavesinfectedwithdownymildewwere

collectedfromseverallocationsthroughoutNorthCarolina(NC)(Lenoir,Haywood,

Johnston,andRowancounties).OneCucumissativusleafinfectedwithP.cubensis

wascollectedinMichigan(MI)andoneP.humuliisolatewascollectedinOregon

(OR).TheisolateusedinthesequencingoftheP.cubensisgenome(MSU-1)was

includedasapositivecontrol(E.A.Savoryetal.2012).DNAfromoneNCisolate

fromCucumissativus,theMIisolatefromcucumber,theP.humulifromOR,andthe

genome-sequencedisolatewereextractedfromsporangia.AllotherDNAwas

extractedfromasinglelesionininfectedfoliartissue.ThepresenceofP.cubensison

leaveswasconfirmedbytheobservationofsporulationusingadissecting

microscope.Leaflesionswereexcisedbysterilescalpel,placedinamicrofugetube,

andstoredat-80°CuntilthetimeofDNAextraction.

39

TissuewasdisruptedandDNAwasextractedandpurifiedviaphenol-

chloroformextractionsadaptedfrompreviouswork(Ahmedetal.2009).DNAwas

purifiedwithethanolwashesthensuspendedin1xTEbufferandquantifiedusinga

NanoDropND1000spectrophotometerandNanoDrop2.4.7csoftware(NanoDrop

TechnologiesInc.,Wilmington,DE).IntegrityoftheDNAwasconfirmedbygel

electrophoresiswiththepresenceofa>12,000bpband.Eachisolatewasamplified

inthenrITSandmitochondrialNad1andNad5(Quesada-Ocampoetal.2012)

wherepresenceofabandofappropriatesize(700bp,500bp,300bp,respectively)

confirmedthepresenceofP.cubensisorP.humuliDNAinlesiontissueandalackof

abandconfirmednegativecontrols(uninfectedcucumberleaftissueandwater

control).

PrimerEvaluation

TheSSRprimersreportfromthePrimer3outputwasdividedbySSRmotif

types(tri-,tetra-,penta-,hexa-nucleotiderepeats)andarrangedfromhighest

numberofrepeatingunitsindescendingorder,asmicrosatelliteswithmorerepeats

tendtobemorepolymorphic(Guichouxetal.2011;Ellegren2000).Thetopfifteen

primersofeachcategorywereselectedtobevalidatedforamplificationandtested

forconsistencyandtransferabilitytoP.humuliinascreenagainsttheelevenP.

cubensisisolatesfromdifferentcucurbithosts(Cucumissativus,Cucumismelo,

Cucurbitapepo,Cucurbitamaxima,Cucurbitamoschata,Citrulluslanatus,Momordica

balsamina,andMomordicacharantia)andtwoP.humuliisolates(Table1.1).

PreliminaryresultssuggestedSSRswithcertainmotifsweremorelikelytobe

polymorphic,somoremarkerswithtri-andhexa-nucleotiderepeatswere

40

evaluated.PCRreactionswereperformedwith10µLof2xGoTaq®HotStartGreen

MasterMix(Promega,Madison,WI),1µLof10µMforwardprimer,1µLof10µM

reverseprimer,10ngofDNA,andsterilewater.Thethermalcycler(Bio-Rad,

Hercules,CA)program,CDMSSR1,wassettohaveaninitialdenatureof94°Cfor3

m,and35repeatingcyclesconsistingofdenaturingat94°C,annealingat53°C,and

extensionat72°C,eachstephavingadurationof30s.Thisprogramconcluded

witha5mfinalextensionat72°C.

P.cubensisisolatesfromwatermelon,bittermelon,andbalsamapple,andthe

positiveandnegativecontrolswereamplifiedbytouchdownPCR(TDSSR1)because

infectedleaveswithlowlevelsofsporulationdidnotamplifyreliablywithstandard

PCRsettings.TheseisolatesunderwentPCRwiththermalcyclersettingswithan

initialdenaturingstepof94°Cfor5m,then20cyclesofa30sdenaturingstepat94

°C,a45satanannealingtemperaturestartingat62°C,andanextensionstepat72

°Cfor2m.Ateachcycle,theannealingtemperaturewoulddecreaseby0.5°C.Then

thereactioncontinuedwithanothertwentycyclesofa30seconddenaturestepat

94°C,a45sannealingstepat55°C,anda2mextensionstepat72°C.Thereaction

endedwitha5mfinalextensionat72°C(Korbie&Mattick2008).Theseisolates

werescreenedagainstatotalof100SSRmarkersandevaluatedviagel

electrophoresis.PCRproductswererunon4%ultrapureagarosegelsat40voltsfor

approximatelyfivehourstoevaluateamplificationanddetermineproductsize.

FragmentAnalysisofPolymorphicSSRPrimers

Fromtheinitialscreeningof100SSRmarkerswithagarosegel

electrophoresis,asubsetof17primerswithconsistentamplificationacrosssamples

41

andappearanceofpolymorphismacrossP.cubensisisolateswerefurtheranalyzed

withmorePseudoperonosporaisolatesforpolymorphismviafragmentanalysis

(Table1.2).PCRproductsofdownymildewisolatesamplifiedwiththepolymorphic

SSRprimersweresubjectedtoasecondroundofPCR,CDMSSR2,inordertoattach

fluorescentdyetotheamplifiedproducts.Reactionswerecarriedoutin10µL

volumeconsistingofthesamematerialsasabovewiththeexceptionofanM13

primerlabeledwithfluorescentdyeinplaceofthesite-specificforwardprimer

(Schuelke2000).ThermalcyclersettingsfortheprogramCDMSSR2werethesame

astheCDMSSR1program,butwith15repeatingcyclesofdenaturing,annealing,and

extensionsteps.ProductsfromtheCDMSSR2weredilutedfiftytotwenty-five-fold

and“pool-plexed”,combiningmultiplePCRproductsofdifferentSSRprimers

labeledwithdifferentfluorescentdyes(VICand6FAM)(AppliedBiosystems,Foster

City,CA).AgenotypingreactionwasperformedwhereHiDiFormamide,LIZ600

sizestandard,andthediluted,pool-plexedsamplewerecombinedthensubmitted

totheNCSUGenomicScienceLaboratory(GSL,Raleigh,NC)forgenotypingwitha

3730xlDNAAnalyzer(AppliedBiosystems,FosterCity,CA).

ResultswereindividuallyanalyzedinPeakScanner(AppliedBiosystems,

FosterCity,CA).Inanygivensetofsamples,peakswithsignalslowerthan5%of

thesignalofthetallestpeakwereautomaticallyremovedfromanalysis.Onagiven

isolateread,peakslessthan15%oftheheightofthelargerpeakwereremovedto

decreasetheriskofgenotypingstutterpeaks.Twoalleleswereassumedtobe

presentateachlocibecausePseudoperonospora,belongingtoOomycota,arediploid

organisms.Ifonepeakwasobservedatanygivenlocus,homozygositywasassumed.

42

Tandemwasthenusedtoassignalleles(Matschiner&Salzburger2009).Sixofthe

17makersevaluatedwithfragmentanalysisprovedtobemonomorphicacrossthe

evaluatedP.cubensisisolatesandwereremovedfromfurtheranalysis.Basic

summarystatisticswerecalculatedfor11reliablepolymorphicprimersacross38P.

cubensisisolatesfromdiversehosts,years,andlocations.Powermarkerv3.25was

usedtocalculatebasicsummarystatisticsincludingHeterozygosityand

PolymorphismInformationContent(PIC)usingdefaultparameters(Liu&Muse

2005).

Twenty-twoP.humuliisolatesalsoweregenotypedwiththe11informative

markersusingtheproceduredescribedabove.GenAlExwasusedtoexaminealleles

andgenerateapairwisepopulationmatrixofNei’sUnbiasedGeneticDistance

betweentheP.cubensisandP.humuliisolates.

Results

MISAanalysisrevealedthatofthe23,522P.cubensissequencesexamined,

2,398sequencescontainedSSRs,withatotalof2,738SSRsidentified.InH.

arabidopsidis,14,548sequenceswereexaminedand1,691oftheexamined

sequencescontainedSSRs.Atotalof2,119SSRswereidentifiedintheH.

arabidopsidistranscriptome.ThenumberofSSR-containingsequencesoutofthe

totalnumberofsequencesexaminedwassignificantlydifferentbetweenspecies

(p<0.0002).Asignificantdifferencealsowasfoundbetweenthepercentageoftotal

SSRsidentifiedoutofthetotalnumberofsequencesexaminedinP.cubensis

(11.6%)andH.arabidopsidis(14.5%)(P<0.0002).Itshouldbenotedthatthetotal

43

sizeofthesequencesexaminedforP.cubensis(26.9Mb)isalmosttwicethetotal

sizeofthesequencesexaminedforH.arabidopsidis(13.9Mb).Also,theN50score

forP.cubensisassembly(3.96kbp)isrelativelylowerthanthatofH.arabidopsidis

(41.56kbp)(Table1.3).

Relativeabundance(SSR/Mb)andrelativedensity(bp/Mb)valuesmaybe

moreinformativebecausethesevaluesaccountforthedifferencesinlengthof

examinedsequencesbetweenspecies.RelativeabundanceisthenumberofSSRs

observedperMegabaseofsequenceexamined,andrelativedensityisthetotal

lengthofsequencecontributedbySSRsperMegabaseofsequenceexamined.The

totalrelativeabundancesofSSRsinP.cubensisandH.arabidopsidis(101.79and

152.01,respectively),showagreaterdifferencebetweenorganismscomparedto

thepercentageofSSR-containingsequencesofthetotalnumberofsequences

examined(10.2%and11.6%,respectively).ThetotalrelativeabundanceofSSRsin

P.cubensisandH.arabidopsidisrevealedlessofadifferencecomparedtothetotal

relativedensitiesbetweenthetwospecies.H.arabidopsidishasamuchhigher

densityofSSRsinthepredictedtranscriptome(2,322.89)comparedtoP.cubensis

(1,421.66)(Table1.4).

AmajorityoftheidentifiedSSRsinbothP.cubensisandH.arabidopsidis

sequenceswereperfect(96%and93%,respectively),meaningthereareno

interruptingsequenceswithinthechainofrepeatingunits.Also,1.2%ofthe

sequencesexaminedfromtheP.cubensistranscriptomecontainedmorethanone

SSR,whereas2.2%oftheH.arabidopsidissequencesexaminedcontainedmorethan

oneSSR.

44

MISAanalysisindicatedthattri-nucleotiderepeatswerethemostfrequently

occurringmotiffoundinbothtranscriptomes(Table1.4).Thismotif-typemadeup

61%ofthetotalSSRsfromtheP.cubensistranscriptomeand71%ofthetotalSSRs

fromtheH.arabidopsidistranscriptome(Table1.4).Inbothspecies,di-nucleotide

repeatswerethesecondmostfrequentlyoccurringmotif,followedbytetra-,hexa-,

andpenta-nucleotiderepeats.Thereweresignificantdifferencesfoundbetweenthe

percentageofdi-andtri-nucleotiderepeatsbetweenspecies(P<0.0002),butthere

werenosignificantdifferencesfoundbetweenthepercentageoftetra-,penta-,and

hexa-nucleotideSSRs(P>0.1).Intermsofrelativeabundance,thesametrendheld

withtri-nucleotiderepeatshavingthehighestvalueinbothspecies,followedbydi-,

tetra-,hexa-,andthenpenta-nucleotiderepeats.Althoughtherelativedensity

valuesforeachmotifgroupinP.cubensisalsokeptthistrend,tetra-nucleotide

repeatshadahigherdensitythandi-,hexa-,andpenta-nucleotiderepeatsinH.

arabidopsidis.Also,inH.arabidopsidishigherrelativeabundanceandrelative

densityvalueswereseenforeachmotifgroupexceptdi-nucleotiderepeats,in

whichP.cubensisheldhighervalues(Table1.4).Itshouldalsobenotedthatinthe

H.arabidopsidistranscriptome,therewasonlyonemonomeridentifiedthatmetthe

specificationstatedintheMISAscriptof20repeatingunits.Therewereno

monomersidentifiedintheP.cubensistranscriptome.

Bothtranscriptomeshadthesamerepeatingsequencesoccurwithhigh

frequency.Forexample,themotifAGC/CTGwasthemostcommonlyoccurring

motifinbothtranscriptomes,beingtherepeatingsequencein451oftheSSRs

identifiedintheintheP.cubensistranscriptomeand495oftheSSRsidentifiedin

45

theH.arabidopsidistranscriptome.Elevenoutofthefifteenmostcommonmotif

sequenceswerethesameforbothP.cubensisandH.arabidopsidis(Table1.5).A

majorityoftheSSRsidentifiedineachmotif-typegroupfelltowardthelowerbound

oftherepeatrangesetforSSRidentificationusingMISA(Fig1.1).MISAwassetto

identifyrepeatingsequencesthatexceeded5repeatingmotifsfordi-nucleotide

repeats,4repeatingmotifsfortri-nucleotiderepeats,and3repeatingunitsfortetra-

,penta-,andhexa-nucleotiderepeats.Inbothspecies,over90%oftetra-,penta-,

andhexa-nucleotiderepeatSSRsweremadeupofthreerepeatingunits.Ahigher

percentageoflongerchainsofrepeatingunitsoccurredindi-andtri-nucleotide

repeats.Forexample,tri-nucleotiderepeatSSRswithmorethanfourrepeatingunits

madeup19%ofthetri-nucleotiderepeatSSRsinP.cubensisandapproximately

23%ofthetri-nucleotiderepeatSSRsinH.arabidopsidis(Fig1.1).

PrimersweresuccessfullydesignedbyPrimer3toamplify2,088SSRsoutof

the2,738SSRsidentifiedbyMISAinthetranscriptomeofP.cubensis.Amajorityof

theseprimers(97%)wereforamplificationofperfectmicrosatellites,meaning

therearenosequencesinterruptingtherepeatingmotif,however,3%ofthe

primersarepredictedtoamplifycompoundSSRs,wherenon-SSRbasepairsmaybe

foundwithintherepeatingmotifsequence.Ofthe2,088primersdesigned,417were

di-nucleotiderepeats,1,125weretri-nucleotiderepeats,248weretetra-nucleotide

repeats,40werepenta-nucleotiderepeats,and91werehexa-nucleotiderepeats.

Experimentalvalidationof100primersetswithgelelectrophoresis(S1

Table)revealedthat92%oftheselectedmarkersproducedaproductacrosstheP.

cubensisisolatesused,and90%ofthe92primerpairsthatproducedaproductwere

46

thesizepredictedbyPrimer3(83total)(Table1.6).Ofthe92primersthat

producedaPCRproductofthePrimer3prediction,sevenprimersonlyamplifiedthe

isolatethatwasusedtosequencetheP.cubensisgenome(MSU-1)(E.aSavoryetal.

2012).

Oftheinitial100primersetsselectedforexperimentalvalidationwithgel

electrophoresis,17primersetshadconsistentamplificationandappearedtobe

polymorphicacrossP.cubensisisolates.Whenthe17primerswereappliedtoa

largerpanelofP.cubensisisolates,only11primersetswereidentifiedtoreliably

amplifylociwithpolymorphicalleleswithinP.cubensis(Table1.7).Fiveofthese11

polymorphicprimersweretri-nucleotiderepeatsandfourwerehexa-nucleotide

repeats,initiallyselectedbasedonpredictednumberofrepeats.Thefinaltwo

polymorphicprimerswereselectedbecausetheywerepredictedtobelocatedin

function-associatedgenes.Thesetwomarkers,SSR97andSSR92,wereidentifiedin

Crinklerfamilyproteins.Whenthese11primerswereappliedtoadiversepanelof

P.cubensisisolatesfromallmajorcommercialhostsandthreenon-commercial

cucurbits(n=38)(Table1.2),within-populationstatisticscouldbedetermined.Over

elevenloci,fiftyalleleswerefoundintheisolatesevaluated.Thenumberofalleles

perlocirangedfromtwotoeight,withanaverageallelediversityof4.5allelesper

locus.Heterozygocityhadameanvalueof0.44andrangedfrom0.16to0.79.The

PICrangedfrom0.27to0.73andhadameanvalueof0.53(Table1.8).Fig1.2,

generatedbyGenAlEx,demonstratesthefrequencyofeachalleleinthispanel.A

majority(eight)ofthelocihaveadominantlyoccurringallele,withthemost

frequentallelebeing50%orgreater.ThisisparticularlyevidentinSSR34and

47

SSR57wherethemajoralleleoccursat86.5%and87.1%,respectively.Manyalleles

acrossthelocioccuratrelativelylowfrequencies.Nineoftheelevenlocihavean

alleleoccurringat10%orlower.SSR1producedthelargestnumberofalleles.The

eightallelesfoundatSSR16rangedinfrequencyfrom2.0%to46.0%.

Theelectrophoresisresultsalsorevealedamajorityoftheprimersshowed

significantspeciestransferability.Ofthe85primersthatproducedaproductin

morethanjustthepositivecontrolisolate(MSU-1),onlyoneprimerset,SSR94,did

notproduceaproductintheP.humuliisolatesscreened.Whenthe11informative

markerswereappliedtoalargersampleof38P.cubensisisolatesand22P.humuli

isolatesandevaluatedwithfragmentanalysis,twooftheseprimersindicatedthe

abilitytocompletelydifferentiateP.humuliisolatesfromP.cubensisisolates,as

alleleswerenotsharedbetweenP.cubensisandP.humuliisolates.Eightprimers

hadsharedallelesbetweenthetwoPseudoperonosporaspecies,butallelevenhadat

leastoneallelethatwasspecifictoonespecies.

ApairwisepopulationmatrixofNei’sUnbiasedGeneticDistancewas

generatedbyGenAlExtoquantifythegeneticdistancebetweenthetwo

Pseudoperonosporaspeciesdetectedbytheselectedmarkers.Highgenetic

differentiationwasrevealed,withavalueof0.97(Table1.9).

Discussion

Inthisstudy,microsatelliteabundanceandfrequencyintwodownymildew

transcriptomeswerecharacterized.Evolutionarily,microsatellitedistribution

throughoutthegenomeisofinterestbecauseoftheirfrequencythroughoutmost

48

eukaryoticgenomesandtheirhighmutationrates.Thenumberofrepeatingunitsat

agivenlocusandthefrequenciesofeachresultingalleleofferinsightinto

evolutionaryeventsthatshapethecurrentpopulation(Ellegren2004;Schlötterer

2000;Oliveiraetal.2006;Tóthetal.2000).Althoughtheexactrolemicrosatellites

havewithinthegenomeisnotwellunderstood,studieshavesuggestedpossible

contributiontogenomeorganizationandstability(Lietal.2004).Repeating

sequenceswithhighmutationrateslocatedwithingenesmaybeofparticular

interestbecausetheymayhavearoleinfunctionality(Lietal.2004;Oliveiraetal.

2006).ThemostwellstudiedexamplesofeffectsofSSRsincodingregionsarise

fromstudiesonhumandiseases.Inparticular,severalneurologicaldiseasesare

associatedwithunstableSSRrepeats(Orr&Zoghbi2007;Brouweretal.2009).As

ofnowitisuncertainwhetherthatpossibleeffectonfunctionalityhasledto

isolate/populationvariabilityinpathogenicityorvirulence.SeveralSSRswere

locatedwithinpathogenicity-relatedgenes,someofwhichwereexaminedinthis

study.Futurestudiesmaywishtoinvestigatepossibleinvolvementof

microsatellitesinpathogenvirulence.

Interestingcomparisonscanbemadebetweenthepredictedtranscriptomes

ofP.cubensisandH.arabidopsidis,twodownymildewpathogenswithpublically

availablegenomes.ThepercentageofSSRsinexaminedsequenceswassignificantly

differentbetweenP.cubensisandH.arabidopsidis,being12%and15%,respectively.

AlthoughalargerportionofsequencesoriginatingfromtheP.cubensisgenomewas

examinedcomparedtothoseoftheH.arabidopsidisgenome,SSRsweremore

abundantintheH.arabidopsidissequences.Thismaybeattributedtothe

49

differencesinthenumberofsequencesexaminedrelativetothegenomesizeand

qualitybetweenthegenomeassemblies(E.aSavoryetal.2012;Baxteretal.2010).

P.cubensishadmoresequencesexamined(23,522)forasmallerassembledgenome

(64Mb)butlowContigN50scoreof3.96kbp.H.arabidopsidisontheotherhand

hadlesssequencesexamined(14,548)foralargerassembledgenome(79Mb)but

withaContigN50scoreof41.56,considerablyhigherthanthatoftheP.cubensis

genome(Table1.3).Itshouldalsobenotedthatonewouldexpectalowergene

contentinH.arabidopsidis,asthisorganismdoesnotproducezoospores,therefore

hypotheticallywouldnothavetheapproximately500genesassociatedwiththe

flagella(Judelson2012).Withfewdownymildewpathogengenomessequenced

andtheissuesassociatedwithassemblyofgenomesofobligatepathogens,itis

difficulttoknowhowmanygenesareexpectedineachspecies.Theavailabilityofa

diversepanelofdownymildewpathogengenomescouldfurtheranswerthese

questions(Withersetal.2014;Derevninaetal.2015;Sharmaetal.2015).

OtherthandifferencesinSSRabundance,manyfrequencyanddistribution

trendsweresimilarbetweenthetwodownymildewpathogens.Tri-nucleotide

repeatsappearinthegreatestabundanceforbothspecies,acommontrendin

oomycetepathogens(Garnicaetal.2006;Biasietal.2015).Thisistobeexpectedin

codingregions,astri-nucleotiderepeatswouldbelesslikelytocauseframe-shift

mutations,thereforeitisthoughtthatsizeoftherepeatunitissubjecttoselection

pressure(Lietal.2004;Selkoe&Toonen2006;Tóthetal.2000;Metzgaretal.

2000).

50

Manyofthemicrosatellitesidentifiedfellclosetotheminimumsizesetinthe

MISAparameters.Between77%-99%oftheSSRsidentifiedforeachmotif-typehad

thelowestpossiblenumberofrepeatingunitsasspecifiedbytheMISAscript(Fig

1.1).Thisseemstobeatrendobservedinothersurveysofmicrosatellitesin

expressedregions(Caietal.2013;Liuetal.2013;Coulibalyetal.2005).Studies

haveshownthatthenumberofrepeatingbasesinSSRslocatedinexpressedregions

tendtobelow(Lietal.2004;Garnicaetal.2006).Inmicrosatelliteevolution,the

numberofrepeatingunitstendstocorrelatewithmutationrate(Schlötterer2000).

TheprogramPrimer3designedprimersfor76%oftheSSRsidentifiedby

MISA.ThenumberofprimersdevelopedfromtheP.cubensispredicted

transcriptomebyPrimer3foreachrepeat-typegroup(tri-,tetra-,penta-,andhexa-

nucleotiderepeats)followedthesametrendastheidentifiedSSRspredictedby

MISA.Forexample,thefrequencyoftri-nucleotideSSRsidentifiedbyMISAand

developedintoapredictedmarkerwithprimersdesignedbyPrimer3wasroughly

60%.Penta-nucleotiderepeatshadthelowestfrequencyforbothMISAand

Primer3outputs,atapproximately2%(Table1.4).Thissuggeststhatparticular

motiftypesdidnothaveadecreasedorincreasedchanceofhavingprimers

designedinflankingsequencesforSSRamplification.

WhenprimersdesignedbyPrimer3werevalidatedviaPCRreactionsandgel

electrophoresis,itwasfoundthatthepercentageofprimerscorrespondingtothe

Primer3outputwashigh(91%).ThissuggeststhattheP.cubensispredicted

transcriptomeisareliablesourceofinsilicomarkeridentification(E.A.Savoryetal.

2012;Baxteretal.2010).Interestingly,anydeviationfromthePrimer3predicted

51

productsizegreaterthan25basepairswasmainlyduetoalargerproductsize,and

inmostcases,thevalidatedproductsizewastwotimesthesizeofthepredicted

product.Thistrendwasobservedwithtwoofthefiveprimersthatamplifieda

microsatellitecontainedinasecretedRxLReffectorpeptideprotein(TableS1).

Wheresmalldeviationsbetweenpredictedandobservedproductsizemaybedueto

limitedresolutioningelelectrophoresis,thesegreaterdifferencesmayarise

becauseofthedifficultyincapturingtruelengthofrepeatingregionsduringgenome

assembly(Treangen&Salzberg,Steven2012).

IntermsofsuccessfulamplificationandfidelitytoPrimer3predicted

products,therewerenonotabledifferencesbetweenmotiftypes(Table1.6).

However,itwasclearthattri-nucleotiderepeatsweremorelikelytodifferentiate

betweenP.cubensisandP.humuliisolates.Overtheyears,therehavebeenseveral

hypothesesconcerningtherelationshipbetweenthesetwoPseudoperonospora

species(Hadziabdicetal.2013;Mitchelletal.2011;Choietal.2005;Sarrisetal.

2009;Rungeetal.2011).Highmorphologicalandsequencesimilarityhavemade

thesetwoeconomicallyimportantpathogensdifficulttodiscriminate.Thisbecomes

especiallyproblematicinregionswherebothcucurbitcropsandhopyardsare

prevalentandsporetrapsareusedtoscoutfordisease(Gentetal.2009).New

moleculartoolsthatcandifferentiatebetweenthesePseudoperonosporamayserve

asamuchneededdiagnostictool(Withersetal.2014).However,manymoreofthe

examinedmicrosatellitesrevealedthatallelesweresharedbetweentheP.cubensis

andP.humuliisolates.Thisinformationmayalsobeusefulinfutureinvestigations

52

ongeneflowbetweenthesespecies,speciationinPseudoperonospora,andother

evolutionaryrelationships.

Twoofthepolymorphicmarkersevaluatedinthisstudywerelocatedin

putativegenesassociatedwithpathogenicity.SSR97andSSR102arelocatedin

Crinkler(CRN)familyproteins.CRNproteinsaretypesofeffectors,molecules

deployedbypathogenstoovercomehostresistancesotheycaninfectthehostplant

andcausedisease.P.cubensisinfectscucumberleaves,CRNfamilyproteinsarefirst

expressedthreedayspostinoculation,andareconsistentlyexpressedthrougheight

dayspost-inoculation(E.A.Savoryetal.2012).Interestingly,theyarefoundin

oomyceteplantpathogensofbiotrophic,hemibiotrophic,andnecrotrophiclifestyles

(Derevninaetal.2015;Stametal.2013;Lévesqueetal.2010).Previousstudies

haveshowntheyhaveinvolvementininducingcelldeath,butvariationin

functionalityhasbeenobservedandspecificpathwayshavenotbeendetermined

(Stametal.2013).Asfurtherresearchisconductedoneffectors,itmaybeofuseto

knowthepresenceofmicrosatelliteslocatedinrelevantgenes.Forafield

pathogenomicsapproach,microsatellitesoccurringinvirulence-associatedgenes

couldbeusedtotrackvirulenceacrosspopulations(Hubbardetal.2015).

Whentheidentifiedpolymorphicmarkerswereappliedtoalarger,diverse

panelofP.cubensisisolates,itwasfoundthattheaveragePICwas0.53andaverage

heterozygocitywas0.44.Inarecentstudy,researchersfromMichiganused

microsatellitesminedfromtheP.cubensispredictedtranscriptometoevaluateP.

cubensispopulationsacrossrelativelysmallgeographicregionsandoverthecourse

ofonegrowingseason.Thepopulationanalyzedintheirstudy,hadanaverage

53

geneticdiversityof0.69andaveragePICof0.64,andwereabletousetheirmarkers

todetectstructureanddeterminevariationinpopulationsfromdifferentcounties

inMichiganandOntario,aswellasvariationbetweenisolatescollectedfrom

differenttimepoints(Naegeleetal.2016).MarkersusedintheMichiganstudywere

selectedfortheirpolymorphismamongisolatesintheGreatLakesregion,whereas

themarkersinthecurrentstudywereselectedforpolymorphismamongisolates

primarilyfromNorthCarolina.DifferencesingeneticdiversityandPICvalues

betweenthisstudyandoursarelikelyduetotheprimersselected.However,the

resultsoftheMichiganstudysuggestifthemarkersidentifiedinourstudywere

appliedtoamuchlargernumberofisolates,structurecouldalsobedetectedin

otherdistinctgeographicregioninthesoutheasternUSandalsooverthecourseofa

growingseason.

Theinformativenatureofthesemicrosatellitesisparticularlypromisingin

thatmorepopulationstudiesareneededtounderstandthefinerdetailsofP.

cubensispopulations.Inparticular,a2012studyconductedbyQuesadaetal.used

sequencedatatosurveyP.cubensisonaglobalscaleanddetectedsomegeographic

andhostdifferentiation,withcertaingeneticclustersoccurringmorefrequentlyin

certaincontinentsorhosts.Italsofoundhighgeneticdiversityincertainregionsof

theUnitedStates,particularlyinGeorgia,NorthCarolina,andIndiana(Quesada-

Ocampoetal.2012).However,thispreviousstudyhadlimitedsamplingincertain

regions.Also,usingmoregeneticregionsmightcapturemorediversitythanwas

seenintheresultsofthisstudy.Ifmarkerswereusedthatcoulddetectdifferences

inalargesamplingofisolatesfromadistinctgeographiclocation,particularlya

54

locationwithhighgeneticdiversity,detailsonpopulationstructureonafinerscale

couldbedetermined.

AsEuroperecentlyexperiencedashiftinP.cubensisvirulence(Lebeda&

Cohen2011),similarlytowhattheUSexperiencedin2004-2005(Holmesetal.

2015),researchersusedAFLPstostudyP.cubensispopulationsacrossEuropeand

foundtwomaincladesseparatingCentralandWesternEuropefromthe

Mediterranean(Sarrisetal.2009).AlaterstudyusingISSRsandSRAPswasableto

determinethatisolatesfromIsraelaredistinctfromgroupsintheCzechRepublic

andTurkey.Althoughinformativeonageographiclevel,thestudieshadhopedto

finddifferentiationbasedonhostoforigin,matingtypes,andpathotypes,andcalled

fortheimportanceofadifferentmolecularmarker(Polatetal.2014).Theuseof

microsatellitesdevelopedinourstudymaybeanothercost-effectivetoolthatcan

aidourunderstandingofP.cubensisdiversity.

Overall,thepublicallyavailablepredictedtranscriptomesofdownymildews

offerawealthofinformationtocontributeinsighttodownymildewgenomicsas

wellasavenuesforapplicationinaddressingquestionsaboutthepopulation

biologyofdownymildewpathogens.MajorsimilaritiesinSSRfrequencyand

distributioninpredictedgeneswereobservedbetweenH.arabidopsidisandP.

cubensisdespitedifferencesinlifecycleandhostrange.Primer3wasabletoprovide

asubstantialsourceofmarkersfromP.cubensistranscriptsthathadhighfidelityto

predictedproducts.Amajorityofthemarkersweretransferabletoanother

economicallyimportantPseudoperonosporaspecies,andcouldpotentiallybe

transferabletoothergeneraofdownymildewpathogens.Thesemarkers

55

demonstratedalargegeneticdifferentiationbetweenthetwoPseudoperonospora

species.Finally,manyofthemarkersidentifiedwereabletodetectdiversitywithin

asmallpanelofP.cubensisisolates.Analysisoflocalpopulationsusingmolecular

markersisessentialwhenresolvingspecificaspectsofpathogendispersalthrougha

region.Ultimately,thesemarkerscanprovidetheinsightnecessarytooptimize

diseasemanagementstrategiesforthisdevastatingpathogen.

Acknowledgements

WethankallthemembersoftheQuesadalabfortheirvaluablehelp;weespecially

thankcolleagueslistedinTable1.1andTable1.2forprovidingsomeoftheisolates

usedinthisstudy.

56

TablesandFigures

Table1.1.IsolatesusedininitialSSRscreen

Isolate IsolateSpecies

Host(Scientificname/variety)

TissueusedinDNAextraction

IsolateOrigin

KIN2-1-4 P.cubensis Cucumissativus,cvStraight8

Infectedleaftissue

LenoirCounty,NC

WAY2-2A-1S P.cubensis Cucumissativus,cvSVR14763462

Sporangia HaywoodCounty,NC

WAY2-3-1* P.cubensis Cucumismelo,cvHalesBestJumbo

Infectedleaftissue

HaywoodCounty,NC

WAY2-4-3* P.cubensis Cucurbitapepo,cvTableAce

Infectedleaftissue

HaywoodCounty,NC

CLAY5_2 P.cubensis Cucurbitamaxima,cvBigMax

Infectedleaftissue

JohnstonCounty,NC

WAY2-6-2* P.cubensis Cucurbitamoschata,cvWalthambutternut

Infectedleaftissue

HaywoodCounty,NC

CLE2-7-3 P.cubensis Citrulluslanatus,MickyLee

Infectedleaftissue

RowanCounty,NC

CLE-11-12* P.cubensis Momordicacharantia

Infectedleaftissue

RowanCounty,NC

WAY-12-9* P.cubensis Momordicabalsamina

Infectedleaftissue

HaywoodCounty,NC

MSU-1* P.cubensis Cucumissativus,cvVlaspik

Sporangia OH,ProvidedbyBradDay(Tianetal.2011)

MSU2-B* P.cubensis Cucumissativus,cvVlaspik

Sporangia MI,ProvidedbyMaryHausbeck

sant2-5* P.humuli Humuluslupulus,cvSantiam

Infectedleaftissue

HendersonCounty,NC

HDM-490* P.humuli Humuluslupulus,cvPacificGem

Sporangia Japan,ProvidedbyDavidGent

*IsolatescommonbetweenTable1.1andTable1.2

57

Table1.2.PseudoperonosporaisolatesusedforSSRscreeningwithfragmentanalysis

Isolate Isolatespecies

Host(Scientificname/variety) TissueusedinDNAextraction

IsolateOrigin YearCollected

Kin2-2a-4 P.cubensis Cucumissativus,cvSVR14763462 Infectedleaftissue LenoirCounty,NC 201314cle2-1-6a P.cubensis Cucumissativus,cvStraight8 Infectedleaftissue RowanCounty,NC 201414cle-1-7 P.cubensis Cucumissativus,cvStraight8 Infectedleaftissue RowanCounty,NC 201414kin2-1-3B P.cubensis Cucumissativus,cvStraight8 Infectedleaftissue LenoirCounty,NC 2014way2-3-1* P.cubensis Cucumismelo,cvHalesBestJumbo Infectedleaftissue HaywoodCounty,NC 201314kin2-3-2a P.cubensis Cucumismelo,cvHalesBestJumbo Infectedleaftissue LenoirCounty,NC 201414cle2-3-6a P.cubensis Cucumismelo,cvHalesBestJumbo Infectedleaftissue RowanCounty,NC 2014cle2-3-10 P.cubensis Cucumismelo,cvHalesBestJumbo Infectedleaftissue RowanCounty,NC 2013way2-4-3* P.cubensis Cucurbitapepo,cvTableAce Infectedleaftissue HaywoodCounty,NC 201314cle2-4-3a P.cubensis Cucurbitapepo,cvTableAce Infectedleaftissue RowanCounty,NC 2014cle2-4-8 P.cubensis Cucurbitapepo,cvTableAce Infectedleaftissue RowanCounty,NC 2013way2-4-7 P.cubensis Cucurbitapepo,cvTableAce Infectedleaftissue HaywoodCounty,NC 2013cle-5-7 P.cubensis Cucurbitamaxima,cvBigMax Infectedleaftissue RowanCounty,NC 201314kin2-5-5a P.cubensis Cucurbitamaxima,cvBigMax Infectedleaftissue LenoirCounty,NC 2014kin-5-4 P.cubensis Cucurbitamaxima,cvBigMax Infectedleaftissue LenoirCounty,NC 2013kin-5-9 P.cubensis Cucurbitamaxima,cvBigMax Infectedleaftissue LenoirCounty,NC 2013way2-6-2* P.cubensis Cucurbitamoschata,cvWaltham

butternutInfectedleaftissue HaywoodCounty,NC 2013

14cle2-6-8a P.cubensis Cucurbitamoschata,cvWalthambutternut

Infectedleaftissue RowanCounty,NC 2014

14kin-6-6 P.cubensis Cucurbitamoschata,cvWalthambutternut

Infectedleaftissue LenoirCounty,NC 2014

cle2-6-6 P.cubensis Cucurbitamoschata,cvWalthambutternut

Infectedleaftissue RowanCounty,NC 2013

cle-7-3 P.cubensis Citrulluslanatus,MickyLee Infectedleaftissue RowanCounty,NC 2013

58




cle2-7-12 P.cubensis Citrulluslanatus,MickyLee Infectedleaftissue RowanCounty,NC 2013way-7-10 P.cubensis Citrulluslanatus,MickyLee Infectedleaftissue HaywoodCounty,NC 2013cle-11-12* P.cubensis Momordicacharantia Infectedleaftissue RowanCounty,NC 2013way-11-7 P.cubensis Momordicacharantia Infectedleaftissue HaywoodCounty,NC 2013way-12-9* P.cubensis Momordicabalsamina Infectedleaftissue HaywoodCounty,NC 2013way-12-6 P.cubensis Momordicabalsamina Infectedleaftissue HaywoodCounty,NC 201314way-13-3a P.cubensis Cucurbitafoetidissima Infectedleaftissue HaywoodCounty,NC 2014MSU-1* P.cubensis Cucumissativus,cvVlaspik Sporangia Homerville,Ohio,Provided

byBradDay(36)2007

MSU2-B* P.cubensis Cucumissativus,cvVlaspik Sporangia MI,ProvidedbyMaryHausbeck

2013

sw003 P.cubensis Cucumismelo Sporangia SouthCarolina,ProvidedbyPeterOjiambo

1982

NY10 P.cubensis Cucumissativus Sporangia Suffolk,NewYork,ProvidedbyChristineSmart

2013

NY8 P.cubensis Cucumismelo Sporangia Ontario,NewYork,ProvidedbyChristineSmart

2013

NY60 P.cubensis Cucumissativus Sporangia Seneca,NewYork,ProvidedbyChristineSmart

2013

SCD3 P.cubensis Cucurbitamoschata Sporangia SouthCarolina,ProvidedbyPeterOjiambo

2012

FL2013E1 P.cubensis Citrulluslanatus Sporangia Florida,ProvidedbyPeterOjiambo

2013

CA081 P.cubensis Cucumissativus Sporangia California,ProvidedbyPeterOjiambo

2008

SL1010 P.cubensis Cucurbitapepo Sporangia Israel,ProvidedbyYigalCohen

2013

SANT2-5* P.humuli Humuluslupulus,cvSantiam Sporangia HendersonCounty,NC 2014Cas5 P.humuli Humuluslupulus,cvCascade Infectedleaftissue HendersonCounty,NC 2014

59




HDM-501ba P.humuli Humuluslupulus,cvPacificGem Sporangia Oregon,ProvidedbyDavidGent

2012

HDM-499 P.humuli Humuluslupulus,cvPacificGem Sporangia Oregon,ProvidedbyDavidGent

2013

hdm503ac P.humuli Humuluslupulus Sporangia Vermont,ProvidedbyDavidGent

2013

hdm481j-1 P.humuli Humuluslupulus,feral Sporangia NewYork,ProvidedbyDavidGent

2011

hdm457e3 P.humuli Humuluslupulus Sporangia Oregon,ProvidedbyDavidGent

2011

hdm254 P.humuli Humuluslupulus Sporangia Oregon,ProvidedbyDavidGent

2008


2008

hdm110-2 P.humuli Humuluslupulus Sporangia Washington,ProvidedbyDavidGent

2006


2006

hdm482 P.humuli Humuluslupulus Sporangia NewYork,ProvidedbyDavidGent

2011

hdm506cb P.humuli Humuluslupulus Sporangia NewYork,ProvidedbyDavidGent

2013

hdm484A P.humuli Humuluslupulus Sporangia CzechRepublic,ProvidedbyDavidGent

2012

502aa P.humuli Humuluslupulus Sporangia Oregon,ProvidedbyDavidGent

2013

hdm490* P.humuli Humuluslupulus Sporangia Japan,ProvidedbyDavidGent

2012

hdm247 P.humuli Humuluslupulus Sporangia Washington,ProvidedbyDavidGent

2008

60





2008

gal P.humuli Humuluslupulus,cvGalena Sporangia HendersonCounty,NC 2014zeus P.humuli Humuluslupulus,cvZeus Sporangia HendersonCounty,North

Carolina2014

nug P.humuli Humuluslupulus,cvNugget Sporangia HendersonCounty,NorthCarolina

2014

14wayhop14 P.humuli Humuluslupulus,cvPacificGem Infectedleaftissue HaywoodCounty,NC 2014*IsolatescommonbetweenTable1.1andTable1.2

61

Table1.3.NumberanddistributionofSSRsintranscriptsequencesaccordingto

MISA

P.cubensis H.arabidopsidisSizeofGenomeAssembly(Mb) 64.33 78.90ContigN50(kbp) 3.96 41.56Totalnumberofsequencesexamined 23,522 14,548Totalsizecoveredbyexaminedsequences(Mb)

26.90 13.94

TotalnumberofSSRsidentified 2,738 2,119Perfect 2,638

(96.4%)a1,964(92.7%)a

Compound 100(3.7%)a 155(7.3%)aNumberofSSR-containingsequences 2,398

(10.2%)b1,691(11.6%)b

NumberofsequencescontainingmorethanoneSSR

280(1.2%)b 316(2.2%)b

Totalrelativeabundance(SSRs/Mb)c 101.79 152.01Totalrelativedensity(bp/Mb)d 1421.66 2322.89

aPercentageoftotalSSRsidentifiedbPercentageoftotalnumberofsequencesexaminedcRelativeabundanceisdefinedasthetotalnumberofSSRsperMbofsequenceanalyzeddRelativedensityisdefinedasthetotalsequencelength(bp)contributedbySSRsperMbofDNAoftotalsequenceanalyzed

62

Table1.4.Percentage,relativeabundance,andrelativedensityofSSRsindowny

mildewtranscripts

Motiflength

Count Percentagea Relativeabundanceb

Relativedensityc

P.cubensis di 563 20.56% 20.93 205.44 tri 1675 61.18% 62.27 732.99 tetra 305 11.14% 11.34 133.10 penta 47 1.72% 1.75 26.02 hexa 148 5.41% 5.50 89.45 H.arabidopsidis di 252 11.89% 18.08 172.74 tri 1511 71.31% 108.40 1207.53 tetra 221 10.43% 15.85 178.19 penta 29 1.37% 2.08 26.54 hexa 105 4.96% 7.53 114.06

aPercentagewascalculatedforeachorganismonthebasisofthecorrespondingtotalSSRscountbRelativeabundanceisdefinedasthetotalnumberofSSRsperMbofsequenceanalyzedcRelativedensityisdefinedasthetotalsequencelength(bp)contributedbySSRsperMbofDNAoftotalsequenceanalyzed

63

Table1.5.Mostcommonrepeatmotifsidentifiedfromperfectandcompound

microsatellitesintwodownymildewtranscriptomes

P.cubensis H.arabidopsidisMotif Count Motif Count

AGC/CTG 451 AGC/CTG 495CG/CG 400 AAG/CTT 330AAG/CTT 359 ACG/CGT 227CCG/CGG 238 AGG/CCT 149ACG/CGT 172 AG/CT 119ACC/GGT 164 ATC/ATG 107AGG/CCT 104 ACC/GGT 82ATC/ATG 104 AC/GT 76AG/CT 87 AAC/GTT 55AC/GT 74 CG/CG 48AAC/GTT 69 CCG/CGG 40CCGG/CCGG 33 ACAG/CTGT 29CCCG/CGGG 32 ACT/AGT 24AGCC/CTGG 28 AAGG/CCTT 19AAGC/CTTG 27 AGCG/CGCT 17

64

Fig1.1.Frequencyofnumberofrepeatsbymotif-typeandsourcetranscriptome

65

Table1.6.ResultsofmarkervalidationviagelelectrophoresisMotiftype Number

evaluatedA B C D E F

tri 23 5 15 1 0 2 1

tetra 15 0 9 1 4 1 2

penta 15 0 2 3 2 8 0

hexa 23 4 10 2 0 7 2

function-associated

24 2 15 1 1 5 4

ANumberofSSRprimersthatamplifiedacross8ormoreP.cubensisisolates.

Productsshowedpolymorphismandwasconfirmedwithfragmentanalysis

BNumberofSSRprimersthatamplifiedacrossatleast8P.cubensisisolates.All

productsizeswerethesame.

CNumberofSSRprimersthatproducednoproduct.

DNumberofSSRprimersthatonlyproducedaproductforthepositivecontrol,

MSU-1.

ENumberofSSRprimersthatdidnotamplify4ormoreP.cubensisisolates.

Amplifiedproductshadoneormoreofthefollowingcharacteristics:complex

bandingpattern,non-specificamplification,multi-locusamplification.

FNumberofSSRprimersthatproducedaproductatleast100basepairslargerthan

thePrimer3predictedproduct

66

Table1.7.PolymorphicSSRsinP.cubensis

SSRName

ForwardPrimer Motif GeneAnnotation ProductSize

SSR1 F:TAGCTGCTGTGGATGTGACG (AAG)12 Conservedgene

ofunknown

function

292

R:TACTTTCTCTGGGCAGCTGC

SSR29 F:GGAAGAAGAGGGCGACACAA (CAA)8 AP-1complex

subunitbeta

128

R:GATCTATGCTGGGTGCTGCT

SSR34 F:AGGTGCAAGGTCTGATGACG (AGA)7 TIMELESS

interacting

protein

149

R:TCCTTCACTCTCCCTGTCGT

SSR57 F:GACAAAAACGTGGACACCCG (GGCGGT)4 ATP-binding

Cassette(ABC)

Superfamily

241

R:TGGACCTTTTCCCCCATTGG

SSR85 F:GGAGGAGGAGGAGGAGGAAG (AGA)7 Digestiveorgan

expansionfactor

287

R:TCAACGTCGGGATCTTGACG

SSR97 F:TGTTTCCGGTGAAGATCGCA (GA)5 Crinkler(CRN)

familyprotein

237

R:GCTTCCACGATGAACGCATC

SSR16 F:TCAGCCTTCTAATGCCCAGC (CAACAG)6 Multiplebanded

antigen

254

R:GTTGCTGTTGTTGCTGCTGT

SSR79 F:TGGCATGGCTTCGTACATGT (TCT)7 Tankyrase2

272

R:TAGTGGTGAGGAGGGGTCTG

SSR88 F:CAAATGCCCATGGGAATGCC (AATGCA)3 Conservedgene

ofunknown

function

114

R:ACTCATCTGCGCGATCTGAG

SSR66 F:AGCGTCGTTCACCAAGATGT (TGGAGG)3 TypeII(General)

Secretory

Pathway(IISP)

Family

244

R:CAGTGTCGTTGGCTGTTTCG

SSR102 F:CAAAAAGCGCGATATCGGCA (AGA)4 Crinkler(CRN)

familyprotein

277

R:CCCAACCACGTCTTCTTCGA

67

Table1.8.StatisticsofpolymorphicprimersacrossP.cubensisisolates.CalculationswereperformedinPowermarker(Liu&Muse2005).SSRName

No.ofobs.

Alleles HeterozygosityY PICZ

SSR79 38430,436

0.45 0.40

SSR85 38

285,300,318,321,

384,390 0.71 0.57

SSR97 38241,253

0.79 0.54

SSR102 38

288,291,294,300,

306,309 0.61 0.70

SSR57 38

230,248,254,266,

284 0.18 0.33

SSR66 38261,267,297,303

0.39 0.73

SSR34 38162,165,189

0.16 0.27

SSR88 38118,124,132

0.58 0.48

SSR29 38

131,137,140,155,

161,164,173 0.32 0.40

SSR16 38236,266,272,278

0.50 0.69

SSR1 38

275,278,305,311,

314,317,332,341 0.18 0.72

YHeterozygosityistheproportionofheterozygousindividualsinthepopulationatagivenlocus

ZPICisthePolymorphismInformationContentisameasureofpolymorphismata

givenlocus

68

Fig1.2.Allelefrequenciesperlocus.Frequenciesareproportionsofeachalleleatagivenlocus.FiguregeneratedbyGenAlEx

(Peakall&Smouse2006).

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0.800

0.900

1.000

430 285 318 384 241 288 294 306 230 254 284 267 303 165 118 130 137 155 164 236 272 275 305 314 332

SSR79 SSR85 SSR97 SSR102 SSR57 SSR66 SSR34 SSR88 SSR29 SSR16 SSR1

Frequency

Locus

69

Table1.9.PairwisePopulationMatrixofNei’sUnbiasedGeneticDistance.Figure

generatedbyGenAlEx(Peakall&Smouse2006).

P.cubensis P.humuliP.cubensis 0.000 P.humuli 0.971 0.000

Nei’sUnbiasedGeneticDistance=-1*Ln(Nei’sUnbiasedIdentity)

70

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CHAPTERIV

PopulationStructureandDiversityofPseudoperonosporacubensison

CommercialandNon-CommercialCucurbitsinNorthCarolina

Abstract

Pseudoperonosporacubensis,thecausalagentofcucurbitdownymildew,isan

airborne,obligate,oomycetepathogenthatre-emergedin2004andsincethen

causesfoliardiseaseandyieldlossesinallmajorcucurbitcropsintheUnitedStates

(US).Approximately60speciesintheCucurbitaceaefamilyhavebeenreportedas

hostsofP.cubensis.Commercialhostsincludingcucumber,cantaloupe,pumpkin,

squash,andwatermelonaregrowninNorthCarolina(NC)andmanynon-

commercialhostspeciesaregrownonasmallscaleinNCoroccurinthewildas

weedyplants.Littleisknownaboutthecontributionofwildandnon-commercial

cucurbitstotheyearlyepidemic.SinceP.cubensishasrelativelyhighgenetic

diversityinNC,andhostspecializationexistswithinthepathogenpopulation,this

studyaimedtodeterminethegeneticstructureofP.cubensisoncommercialand

non-commercialcucurbitsinNC.Tensimplesequencerepeat(SSR)ormicrosatellite

markerswereappliedto344isolatesfromsixcommercialandthreenon-

commercialcucurbitsfromthreelocationsrepresentingdifferentgrowingregions

acrossNC.Populationanalysesrevealedthegreatestgeneticdifferentiationbyhost

species,whichsupportstheconceptofhost-drivendifferentiation.Thelowest

geneticdifferentiationoccurredamongisolatesfromMomordicabalsamina,

Cucurbitapepo,andCucurbitamoschata.Furthermore,thehighestgenetic

78

differentiationoccurredbetweenisolatesfromCucumisversusCitrullusand

commercialCucurbitahosts.Surprisingly,theisolatesfromCucurbitafoetidissima,a

wildperennialcucurbit,aremoresimilartoisolatesfromCucumisthantheyareto

isolatesfromcommercialCucurbitaspecies.Thesefindingssuggestthatwildand

non-commercialcucurbitsareplayingaroleinP.cubensisdiversification,and

possiblycontributinginoculumtoepidemicsofdownymildewsondifferentgenera

ofcommercialcucurbithosts.

Introduction

Pseudoperonosporacubensisisanobligateoomyceteplantpathogenthat

resultsinfoliardestructionofcucurbitcropswhenitcausesdownymildew.

EconomiclosseshavebeenreportedinmanypartsoftheworldincludingEurope,

theMiddleEast,andAsia(Lebeda&Cohen2011).Thecucumber-producingregions

intheUnitedStates(US)havesufferedparticularlydramaticlosses,startingin2004

whenthepathogenre-emergedafter35years.Cucurbitdownymildewwasonce

controlledprimarilyviahostresistanceincucumberandwithmodestfungicide

applicationsinothercucurbits,butapopulationshiftrenderedresistantvarieties

andcommonlyusedfungicidesineffective.Approximately$20milliondollarsin

lossesoccurredin2004andsincethen,diseasemanagementrequiressubstantial

andfrequentfungicideapplications(Coluccietal.2006;Holmesetal.2015;Cohen

etal.2015).Thepathogenremainsahighriskfordevelopingresistanceto

fungicidesandovercominghostresistance,inpart,becauseofitshighrateof

asexualreproductionandhighlevelofgeneticvariation(Quesada-Ocampoetal.

79

2012).P.cubensishasashortincubationperiodof4-12days(Cohen1977;Savoryet

al.2011)andwhenitsporulatesontheabaxialsurfaceoftheleaf,itcanproduceup

to7x104sporangiapercubiccentimeter(Lebeda&Cohen2011).Sporangiacan

travelapproximately1,000milesviaaircurrentstravellingfromfieldtofieldas

cucurbithostsareplantedandtemperaturesbecomeconduciveforinfection.

(Ojiambo&Holmes2011;Ojiamboetal.2015).

DownymildewpathogenssuchasPseudoperonosporahumuli,Bremia

lactucae,Plasmoparaobducens,Plasmoparahalstedii,Peronosporabelbahrii,and

Peronosporatabacinatypicallycausediseaseononlyafewplantspeciesusually

withinthesamegenus.Interestingly,P.cubensishasaverybroadhostrangeandis

capableofinfectingover60differentspeciesofplantsintheCucurbitaceaefamily

fromverydiversegenera(Runge&Thines2010;Lebeda&Cohen2011).

DifferencesinvirulenceofP.cubensisondiversehostshasbeendescribed,with

isolatesofthepathogenbeingclassifiedintodifferentracesandpathotypesbased

ontheabilitytoinfectasetofhostdifferentials(Runge&Thines2010;Rungeetal.

2012).However,theobligatenatureofthispathogenmakesthisclassification

systempronetofalsenegativesanddifficulttoexecuteandreproduce.Population

geneticsstudieshaveshowngeneticdifferentiationofP.cubensisisolatesis

associatedwithhostandgeographicallocation.Forexample,Quesada-Ocampoetal.

(2012)usedfivenuclearandtwomitochondrialgenesandfoundthatisolatesfrom

EuropegenerallybelongedtodifferentgeneticclustersthanisolatesfromNorth

America.Theyalsofoundthatisolatesinonegeneticclusteroccurredmore

frequentlyinisolatesfromhostsotherthancucumber,andthatanothercluster

80

predominantlycontainedisolatesfromcucumber.Finally,itshowedthatcertain

geographicregionsintheUS,suchasGeorgiaandNorthCarolina,haverelatively

highgeneticdiversity(Quesada-Ocampoetal.2012).Nonetheless,thatstudywas

notabletodetermineiftheobservedgeographicstructurewasanartifactof

populationstratificationdrivenbyhostduetodifferencesinhostavailabilityonthe

diverseregionssampled.

Thecontributionofnon-commercialandwildcucurbithostsasasourceof

inoculumorasadiversifyingfactorofP.cubensisislargelyunknown.Rungeand

Thines(2009)foundthattheperennialwildhop,Bryoniadioica,isahostforP.

cubensisinEuropebutwereunabletodeterminearoleinoverwinteringsincethe

studywasperformedinalaboratorysetting.Inlaboratoryinoculations,Lebeda

(1992)determinedthatamajorityofthe56accessionsofwildcucurbitsscreened

weresusceptibletothepathogen.RecentreportsintheUSrevealedthathostssuch

asMomordicabalsamina,M.charantia,andCucurbitafoetidissimawereinfected

withP.cubensisinfieldsettingswithnaturalinoculum(Wallaceetal.2014;Wallace

etal.2015).SincedozensofreportedhostsforP.cubensisarefoundinthewildor

aregrownonasmallscaleinareaswithhighcucumberproductionintheUS,

studiesthatexaminethepotentialofwildandnon-commercialcucurbitsto

contributetotheyearlydownymildewepidemicareneeded.Non-commercial

cucurbitscouldbeinvolvedinoverwinteringsurvivalofthepathogen,serveas

greenbridgesforpathogendispersal,providesitesofsexualreproduction,actas

pathogenreservoirs,andcontributetothegeneticdiversityofthepathogen(Cohen

etal.2015;Ojiamboetal.2015;Holmesetal.2015).

81

Inthisstudy,weevaluatedhowdifferentcucurbithostsaffectedthe

populationstructureofP.cubensisbyusingstandardizedfieldplotsplantedwith

differentcommercialandnon-commercialcucurbitsandpreviouslydeveloped

microsatellitemarkers(Wallace,Chapter3).SpecificallyweaimedtodetermineifP.

cubensisisolatesaregeneticallystructuredbyhostgenus(Cucumis,Cucurbita,

Citrullus,Momordica),hostspecies(Cucumissativus,Cucumismelo,Cucurbitapepo,

Cucurbitamaxima,Cucurbitamoschata,Citrulluslanatus,Cucurbitafoetidissima,

Momordicacharantia,andMomordicabalsamina),geographicallocation(Kinston,

Piedmont,andWaynesville),andthetimeofproduction(summerandfall)andhost

speciesateachlocation(Kinston,Piedmont,andWaynesville).NorthCarolinaisa

majorproducerofallmajorcucurbitcropsincludingcantaloupe,squash,pumpkin,

gourds,andwatermelon(NCDA&CS2013),whichhaslikelyresultedinthe

previouslyreportedhighgeneticdiversityofP.cubensisinthesate(Quesada-

Ocampoetal.2012).NorthCarolinaisinthegeographicalrangeofseveralwildand

non-commercialcucurbitsthatarenaturallyinfectedbyP.cubensis(Wallaceetal.

2014;Wallaceetal.2015).

MaterialsandMethods

Fieldplots,isolatecollection,andDNAextraction

Pseudoperonosporacubensisisolateswerecollectedfromthreeuniformplots

inthreedistinctgeographicregionsinNC:TheLowerCoastalPlainTobacco/

CunninghamResearchStationinKinston,TheNCDA&CSPiedmontResearch

StationinSalisbury,andtheMountainResearchStationinWaynesville.Eachof

82

theseresearchstationsarelocatedingeographicallydistinctlocationsinthestate.

Kinstonislocatedintheeast,innercoastalplainwithslowsurfacewatermovement

duetoflatgeographyandloosesoils.Salisburyisinthecentral,Piedmontregionof

NorthCarolinacontainingahillygeography,manystreams,andclaysoilwithhard

rockbeneath.Waynesvilleislocatedinthemountainsinthewesternpartofthe

state,characterizedbymorerain,acoolerclimate,andvariablesoilsdueto

dramaticvariationinclimate,geography,andelevation(Carbone,G.&Hidore,J.

2008).TheseplotswerepartoftheCDMIPM-PIPEsentinelplotnetwork(Ojiambo

etal.2011)andcontainedtenplantsofeachcommercialcucurbitcropincluding

CucumissativuscvStraight8(cucumber),CucumismelocvHale’sBestJumbo

(cantaloupe),CucurbitapepocvTableAce(acornsquash),CucurbitamaximacvBig

Max(pumpkin),CucurbitamoschatacvWalthamButternut(butternutsquash),and

CitrullusLanatuscvMickyLee(watermelon).Theplotsalsocontainedfiveplantsof

eachnon-commercialorwildcucurbitcropincludingMomordicacharantia(bitter

melon),Momordicabalsamina(balsamapple),andCucurbitafoetidissima(buffalo

gourd).Anadditional13isolatesofM.charantiaandM.balsaminawerecollected

fromtheHorticulturalCropsResearchStationinClinton,andtheMountain

HorticulturalCropsResearchandExtensionCenterinMillsRiverin2013.Asplots

weremonitoredfordisease,symptomaticleaveswerecollectedatfourtimeranges,

Summer2013,Fall2013,Summer2014,andFall2014.Betweensummerandfall

seasons,allcommercialcucurbitswereremovedfromthesentinelplotsandre-

planted.Diseasewasconfirmedbyobservationofsporulatinglesionsundera

83

dissectingmicroscope.Individualsporulatinglesionswereexcisedandstoredin

microfugetubesat-80˚C.

Infectedtissuewasdisruptedwith425-600µMacid-washedglassbeads

(SigmaLifeSciences)and2.3mmZircona/Silicabeads(BioSpecProducts,Inc.

Bartlesville,OK)inanOmniBeadRuptor24(OmniInternational,Inc.,Kennesaw,

GA).SDS-pageextractionbufferwasusedtoextractDNAandwaspurifiedvia

phenol-chloroformextractionsandethanolwashesadaptedfrompreviouswork

(Ahmedetal.2009).DNAquantity(ng/µL)andquality(260/280)weremeasured

withNanodropND1000spectrophotometerandNanoDrop2.4.7csoftware

(NanoDropTechnologiesInc.,WilmingtonDE).

ProductAmplification,FragmentAnalysis,andGenotyping

DNAfromeachP.cubensisisolatewasamplifiedwith10SSRmarkers

identifiedandcharacterizedinChapterIII(Table2.1).Productswereamplifiedwith

eitherthePolymeraseChainReactions(PCR)describedinCulley(Culleyetal.2013)

orwithaTouchdownPCRreactionasdescribedpreviously.

PCRproductslabeledwithdifferentfluorescentdyeswerepool-plexedand

diluted20-foldin96-wellplates.Sampleswerepreparedforthe3730xlDNA

Analyzer(AppliedBiosystems,FosterCity,CA)byaddingHiDiFormamideand

Liz600sizestandardtoeachwell.SamplesweresubmittedtotheNorthCarolina

StateUniversityGenomicScienceLaboratoryforgenotyping.

84

PopulationAnalysis

Rawdatageneratedbythe3730xlDNAAnalyzerwereindividuallyanalyzed

bycallingpeaksandbinningthemtoallelesestablishedinChapterIII.TheGeneious

MicrosatellitePlug-In(Kearseetal.2012)wasusedtovisualize,process,andstore

data.AlleleswereindividuallybinnedinGeneious.PowerMarkerversion3.25(Liu&

Muse2005)wasusedtogeneratesummarystatisticsanddiversitymeasuressuch

asheterozygosityandthePolymorphicInformationContent(PIC).GenAlEx(Peakall

&Smouse2006)wasusedtogeneratepairwisepopulationmatricesofNei’s

unbiasedgeneticdistance.(Peakall&Smouse2006).

Results

Threehundredforty-fourPseudoperonosporacubensisisolateswere

evaluatedwithtenSimpleSequenceRepeats(SSRs),ormicrosatellites.Two

hundredninety-eightoftheisolateswerefromcommercialhosts,with63from

Cucumissativus,58fromCucumismelo,46fromCucurbitapepo,46fromCucurbita

maxima,47fromCucurbitamoschata,and38fromCitrullusmelo.Theremaining46

werecomprisedof30isolatesfromMomordicacharantia,10isolatesfrom

Momordicabalsamina,and6isolatesfromCucurbitafoetidissima.Theseisolates

werecollectedacrossthesummerandfallseasonsof2013and2014fromlocations

inEastern,Central,andWesternNorthCarolina(Table2.2).

Nei’sunbiasedgeneticdistance(D)detectedrelativelylowlevelsofgenetic

differentiationwhenisolatesweregroupedbylocation.Threehundredandthirty

oneisolateswereevaluatedwith94isolatesfromKinston,143isolatesfrom

85

Piedmont,and94isolatesfromWaynesville.ThethirteenisolatesfromClintonand

MillsRiverwerenotincludedinthisanalysis.ADvalueof0.008wasdeterminedfor

bothKinston-WaynesvillecomparisonsandPiedmont-Waynesvillecomparisons.A

slightlyhigherdifferentiationwasdeterminedforKinstonvsPiedmontisolateswith

D=0.019(Table2.3).

Highergeneticdifferentiationwasdetectedwhenisolatesweregroupedby

timeandlocation.Onceagain,thethirteenisolatesfromClintonandMillsRiver

wereexcludedfromthisanalysis.Isolatesweregroupedintothefollowing

categories:KinstonSummer2013(n=13),KinstonFall2013(n=30),Kinston

Summer2014(n=18),KinstonFall2014(n=33),PiedmontSummer2013(n=49),

PiedmontFall2013(n=45),PiedmontSummer2014(n=25),PiedmontFall2014

(n=25),WaynesvilleSummer2013(n=45),WaynesvilleFall2013(n=26),

WaynesvilleSummer2014(n=17),WaynesvilleFall2014(n=6)(Table2.4).The

overallNei’sGeneticDistancevaluescomparingthesecategorieswererelatively

low,withthehighestbeing0.183,comparingisolatesfromKinstonSummer2013

andPiedmontFall2014.Sixlocation-timepointcombinationshadaDvalue>0.1.

Thesegroupswerefromdifferentlocationsandyears.Thelowestgenetic

differentiation(D=0)betweengroupsoccurredwithisolatesfromthesameyearor

withinthesamelocation.

Whengenediversity,orexpectedheterozygositywasexaminedateachtime-

locationcombination,anincreaseingenediversitywasseeninKinstonin2013and

inWaynesvillein2014.InKinston2013,genediversityincreasedfrom0.53inthe

86

summerto0.63inthefall.InWaynesville2014,genediversityincreasedfrom0.46

inthesummerto0.51inthefall(Table2.5).

Whenisolatesweregroupedbyhostgenera,Nei’sunbiasedgeneticdistance

revealedthegreatestdifferentiationbetweenisolatesfromCucumisandisolates

fromMomordicaat0.173.ThelowestvalueofdifferentiationwasbetweenCitrullus

isolatesandMomordicaisolates,with0.019(Table2.6).

Whentheisolatesweregroupedbyhostspecies,thevalueswererelatively

larger,revealingahigherdegreeofdifferentiationatahostspecieslevel.Someof

thegreatestdifferentiationexistedbetweenisolatesfromCucurbitapepoand

Cucumissativus(D=0.330)CucurbitamoschataandCucumissativus(D=0.324)and

CitrulluslanatusandCucumissativus(D=0.309).Thethreelowestdifferentiation

values(D=0)werebetweenisolatesfromCucurbitamoschataandMomordica

balsamina,CucurbitapepoandMomordicabalsamina,andCucurbitamoschataand

Cucurbitapepo(Table2.7).

Isolateswerealsogroupedbyhostspeciesandlocationtoverifyif

differentiationbasedonhostspecieswasmaintained.Nei’spairwisecomparisonof

geneticdifferentiationrevealedthatisolatesfromthesamehostspecieshadlowD

valuesbetweengeographiclocations,manyhavingavalueofD=0andthehighest

beingD=0.026(CitrullusisolatesfromPiedmontvs.Waynesville)(Table2.8).

Discussion

Pseudoperonosporacubensis,beinganaeriallydispersedpathogencapableof

long-distancetransportshouldhavetheopportunityforextensivemigrationand

87

geneflow.Thereisnoevidenceofthepathogenpersistingyear-roundintheUS

abovethe30°latitudeasthecommercialhostsdonotsurviveinthewinterandthe

pathogenneedsalivinghosttosurvive(Lebeda&Cohen2011;Cohenetal.2015).

ManylinesofevidencesupportthatP.cubensisisactiveinthewintermonthsin

southernFlorida,andtheninthespringandsummerthepathogenisdispersed

northwardfromfieldtofieldashostsbecomeavailable(Ojiambo&Holmes2011).

However,theroleofthepathogen’ssexualstageandinfectionofwildcucurbithosts

addpotentialsourcesofinoculumandgeneticdiversityforthepathogen(Ojiambo

etal.2015).

WithpreviousstudiesshowingdistinctpopulationsinNorthCarolina

(Quesada-Ocampoetal.2012)andgeneticstructurewithinasmallgeographic

region(Naegeleetal.2016),thisstudyevaluatedthecontributionofhost,time,and

locationongeneticdifferentiationofP.cubensisinNorthCarolina.

Geneticdifferentiationbygeographiclocation

Wedidnotobservestrongsupportofdifferentiationbasedonlocation.This

suggeststhatotherfactorssuchasgrowingseasonandhosthaveagreaterrolein

geneticdifferentiationofP.cubensis.InMichiganandOntario,significantgenetic

differentiationcouldbeseenbetweencertaincounties,however,inthatstudy,P.

cubensisisolatescamefromcucumberandspaghettisquashhostsonly,possibly

contributingtothegeneticstructureofthepopulations.

Geneticdifferentiationandgenediversitybytimeofproduction

Therealsowasnotstrongdifferentiationamongisolatesacrosstimeand

locationwhencomparingsummerandfallisolatesfromagivenlocation.Inmost

88

cases,thehighestdifferentiationobservedwasbetweenyearsanddifferent

locations.Theexceptiontothistrendwasarelativelyhighdifferentiationbetween

isolatesfromWaynesvilleinthefallof2013andisolatesfromWaynesvillecollected

inthesummerof2014.

Whengenediversity,orexpectedheterozygositywasobservedovertimeand

space,wegenerallysawsimilarvaluesfromthesummertothefallgrowingseason.

InKinston2013andWaynesville2014,however,P.cubensisisolatesincreasedfrom

thesummertothefallgrowingseasonwithinasingleyear,andineachgeographic

location.Anincreaseingenediversityovertimeistobeexpectedsincethe

pathogenhashadlongertodisperseamongdifferentregions,asobservedin

MichiganandOntario(Naegeleetal.2016).InMichiganandOntario,itwasfound

thatasthegrowingseasonprogressed,thegeneticdiversityofP.cubensisincreased,

likelyduetoaccumulationofinoculuminanarea.BecauseofWaynesville’slocation

inthemountains,cucurbitcropsarepositionedtobeexposedtoinoculumcoming

fromthesouthwest.Theircucurbitproductionisalsolessthanthatofthecentral

andcoastalregionsofNorthCarolinaanddiseaseseverityisgenerallylower.It

shouldalsobenotedthattherewasadifferenceinpathogensamplinginthe

Waynesvillesentinelplotbetweenthefallof2013andsummerof2014.Inthefallof

2013,P.cubensisisolateswerecollectedfromallcommercialhosts,whereasinthe

summerof2014,P.cubensisisolateswereobtainedfromCucumisspecies,andtwo

non-commercialcucurbits,C.foetidissima,andM.balsamina.Theroleofhost

speciesmayplayalargerroleindifferentiationthangeographiclocationand

samplingdate.

89

Geneticdifferentiationbycucurbithost

Previouspopulationstudieshaveshownevidenceofgeneticdifferentiation

inP.cubensisbasedontheoriginalcucurbithost(Quesada-Ocampoetal.2012;

Naegeleetal.2016).ThehostoriginisthoughttoplayaroleinP.cubensis

morphologyandpathogenicity(Runge&Thines2010;Rungeetal.2012).Quesada

etal.(2012)determinedrelativelyhighgeneticdiversityofP.cubensisinNorth

Carolina.Thestudyalsoshoweddifferentiationbetweenisolatesfromcucumber

andisolatesfromtheothercommercialcucurbithostssuchascantaloupe,squash,

pumpkin,andwatermelon.Thisdifferentiationcouldhavebeenbetterresolvedwith

agreaternumberofisolatesfromeachhostspeciesfromeachdistinctregion.

Naegeleetal.(2016)alsoobserveddifferentgeneticclusterpatternsinisolates

fromspaghettisquashcomparedtoisolatesfromcucumber.However,thisstudy

onlyexaminedtwohostspecies.InNorthCarolina,allcommercialcucurbitsare

growninthestate,andmanywildandnon-commercialcucurbithostsarealso

foundintheregion.Withrobustsamplingfromeachhost,wecouldeffectively

evaluatetheroleofcucurbithostonP.cubensisdiversity.

Whenisolatesweregroupedbygenus,theNei’sgeneticdistance(D)values

showedrelativelysmallvaluesthatwerecomparabletothatofthetime-location

grouping.Thesecomparisonsdid,however,showthegreatestdifferentiation

betweenisolatesfromCucumisspeciesandisolatesfromMomordicahosts.The

lowestdifferentiationwasbetweenisolatesfromCitrullusandisolatesfrom

Momordica.Thislowgeneticdistancevalue(D=0.019)suggestsahigherexchange

ofgeneticmaterialbetweenisolatesfromthesehosts.

90

Isolateswerealsogroupedbyhostspecies,andthehighestvaluesofgenetic

distancewereseenwhenapairwisepopulationmatrixwascreated.InsomecasesD

valueswerethreetimeshigher.Thegreatestgeneticdistancewasbetweenisolates

fromCucumissativusandisolatesfromCucurbitapepo,Cucurbitamoschata,Citrullus

lanatus,andMomordicabalsamina.

SincewehaveseengreaterNei’sgeneticdistancevalueswhenisolatesare

groupedbyhostspecies,thisfactorseemstobecontributingthemostto

diversificationofP.cubensisinNorthCarolina.Cucurbithostplaysaroleinselection

ofthispathogen.EvidenceshowsthatP.cubensispopulationsarechangingover

spaceandtimeinNorthCarolina.Someevidencefordifferentiationbasedontime-

locationcombinationswasseen,butbecausethesegroupscontainedisolatesfrom

multiplehostspecies,theremightbebetterresolutionofdifferentiationbytimeand

location.Thesamplesizeofeachhostateachtimepointateachlocationwillhaveto

beincreasedtobeadequatelyevaluated.

TheeffectofhostspeciesonP.cubensisdifferentiationwassupportedbylow

differentiationvaluesbetweenisolatesfromthesamehostspeciesfromdifferent

locations.P.cubensisisolatesfromthesamehostwerenotgreatlydifferentiated

betweendifferentgeographicregions.Theseresultsshowthatthedifferentiation

seenwhengroupedbyhostspeciesaloneisnotconfoundedbyhavingisolatesfrom

multiplegeographiclocations.

Thisstudywasalsoabletodeterminethatwildandnon-commercial

cucurbitsareplayingaroleingeneticdifferentiation.IsolatesfromMomordicaare

highlydifferentiatedfromCucumisisolatesandaremostlikelytosharegenetic

91

materialwithisolatesfromCucurbitaandCitrullus.Cucurbitafoetidissima,onthe

otherhandshowshigherdifferentiationfromcommercialCucurbitahosts,butlower

differentiationfromisolatesfromCucumishosts.Thisfindingmayhave

implicationsforearlyarrivalofdiseaseintemperateregionsbecauseC.foetidissima

isperennialanditisnotclearwhetherthepathogencanoverwinterinthishost.The

closelyrelatedorganism,P.humuli,issystemicandisabletooverwinterinits

perennialhost,Humuluslupulus(Royle,D.J.&Kremheller,H.TH.1981).Because

Cucumisspeciesusuallybecomeinfectedearlierintheseasonthancommercial

Cucurbitaspecies,C.foetidissimamayposeathreatforthediseasetooccurearlier

thananticipated.Thiscanbeasignificantmanagementhurdlewhenmanygrowers

relyonforecastingdatafromtheCDM-IPMPipe,whichdoesnotfactorin

overwinteringsources.Overall,differentwildandnon-commercialcucurbitswill

contributetotheepidemiconageneticleveldifferently,andhavethepotentialto

playaroleintheepidemic.

92

Table2.1.Polymorphicsimplesequencerepeatmarkers

SSRName Primer Motif Heterozygosity PIC NumberofAlleles

ExpectedProductSize

SSR1 F:TAGCTGCTGTGGATGTGACG (AAG)12 0.172 0.641 17 292 R:TACTTTCTCTGGGCAGCTGC SSR29 F:GGAAGAAGAGGGCGACACAA (CAA)8 0.416 0.571 22 128 R:GATCTATGCTGGGTGCTGCT SSR34 F:AGGTGCAAGGTCTGATGACG (AGA)7 0.281 0.469 12 149 R:TCCTTCACTCTCCCTGTCGT SSR57 F:GACAAAAACGTGGACACCCG (GGCGGT)4 0.317 0.586 18 241 R:TGGACCTTTTCCCCCATTGG SSR85 F:GGAGGAGGAGGAGGAGGAAG (AGA)7 0.395 0.626 16 287 R:TCAACGTCGGGATCTTGACG SSR97 F:TGTTTCCGGTGAAGATCGCA (GA)5 0.733 0.519 17 237 R:GCTTCCACGATGAACGCATC SSR16 F:TCAGCCTTCTAATGCCCAGC (CAACAG)6 0.535 0.726 10 254 R:GTTGCTGTTGTTGCTGCTGT SSR79 F:TGGCATGGCTTCGTACATGT (TCT)7 0.445 0.519 12 272 R:TAGTGGTGAGGAGGGGTCTG SSR88 F:CAAATGCCCATGGGAATGCC (AATGCA)3 0.674 0.523 9 114 R:ACTCATCTGCGCGATCTGAG SSR102 F:CAAAAAGCGCGATATCGGCA (AGA)4 0.701 0.693 15 277 R:CCCAACCACGTCTTCTTCGA Mean 0.467 0.587 14.8

93

Table2.2.PseudoperonosporacubensisisolatesevaluatedHostSpecies 2013SummerHarvest 2013FallHarvest 2014SummerHarvest 2014FallHarvest KINX CLEY WAYZ KINX CLEY WAYZ KINX CLEY WAYZ KINX CLEY WAYZ

Cucumissativus 3 6 7 9 9 3 6 5 6 7 1 N/A

Cucumismelo N/A 4 5 5 7 6 5 8 7 8 2 1

Cucurbitapepo N/A 5 5 7 7 5 1 3 N/A 6 6 2

Cucurbitamaxima 8 6 2 1 6 5 N/A N/A N/A 8 10 N/A

Cucurbitamoschata 2 4 2 8 5 1 5 8 N/A 4 5 3

Citrulluslanatus N/A 13 8 N/A 11 6 N/A N/A N/A N/A N/A N/A

Cucurbitafoetidissima 1 1 4

Momordicacharantia 11 10

Momordicabalsamina 6

XLowerCoastalPlainTobacco/CunninghamResearchStation,Kinston,NC,LenoirCountyYNCDA&CSPiedmontResearchStation,Salisbury,NC,RowanCountyZMountainResearchStation,Waynesville,NC,HaywoodCountyNote:Tabledoesnotinclude13isolates:6isolatesofP.cubensisonM.charantiafromtheHorticulturalCropsResearchStationinClintonNC,3isolatesofP.cubensisonM.charantiaand4isolatesofP.cubensisonM.balsaminafromtheMountainHorticulturalCropsResearchandExtensionCenterinMillsRiverNC.

94

Table2.3.PairwisepopulationmatrixofNei’sunbiasedgeneticdistanceofP.

cubensisisolatesgroupedbygeographicregion

KinstonX PiedmontY WaynesvilleZ

Kinston 0.000 Piedmont 0.019 0.000 Waynesville 0.008 0.008 0.000XLowerCoastalPlainTobacco/CunninghamResearchStation,Kinston,NC,LenoirCountyYNCDA&CSPiedmontResearchStation,Salisbury,NC,RowanCountyZMountainResearchStation,Waynesville,NC,HaywoodCounty

95

Table2.4.PairwisepopulationmatrixofNei’sunbiasedgeneticdistanceofP.cubensisisolatesgroupedbytimeandlocation

KINX

Summer2013

KINXFall2014

KINXSummer2014

KINXFall2014

PIEDYSummer2013

PIEDYFall2013

PIEDYSummer2014

PIEDYFall2014

WAYZSummer2013

WAYZFall2013

WAYZSummer2014

KINXSummer2013 0.000 KINXFall2013 0.036 0.000 KINXSummer2014 0.003 0.023 0.000 KINXFall2014 0.054 0.028 0.004 0.000 PIEDYSummer2013 0.109 0.060 0.049 0.028 0.000 PIEDYFall2013 0.066 0.036 0.040 0.025 0.017 0.000 PIEDYSummer2014 0.031 0.016 0.000 0.000 0.050 0.035 0.000 PIEDYFall2014 0.183 0.094 0.090 0.030 0.051 0.051 0.060 0.000 WAYZSummer2013 0.092 0.050 0.038 0.010 0.019 0.027 0.026 0.024 0.000 WAYZFall2013 0.076 0.075 0.037 0.019 0.021 0.030 0.035 0.045 0.026 0.000 WAYZSummer2014 0.006 0.036 0.008 0.055 0.114 0.089 0.026 0.192 0.093 0.099 0.000

WAYZFall2014 0.107 0.051 0.030 0.000 0.030 0.025 0.008 0.000 0.000 0.015 0.120

*ShadinggroupscomparisonsmadewithinageographicregionXLowerCoastalPlainTobacco/CunninghamResearchStation,Kinston,NC,LenoirCountyYNCDA&CSPiedmontResearchStation,Salisbury,NC,RowanCountyZMountainResearchStation,Waynesville,NC,HaywoodCounty

96

Table2.5.ExpectedheterozygosityofP.cubensisisolatesovertimeandlocation

Time-Location SampleSizeExpectedHeterozygosity(GeneDiversity)

KinstonSummer2013 13 0.53

KinstonFall2013 30 0.63

KinstonSummer2014 18 0.55

KinstonFall2014 33 0.49

PiedmontSummer2013 49 0.65

PiedmontFall2013 45 0.64

PiedmontSummer2014 25 0.56

PiedmontFall2014 24 0.54

WaynesvilleSummer2013 45 0.63

WaynesvilleFall2013 26 0.63

WaynesvilleSummer2014 17 0.46

WaynesvilleFall2014 6 0.51

97

Table2.6.PairwisepopulationmatrixofNei’sunbiasedgeneticdistanceofP.

cubensisisolatesgroupedbyhostgenera

Cucumis Cucurbita Citrullus MomordicaCucumis 0.000

Cucurbita 0.220 0.000 Citrullus 0.247 0.019 0.000

Momordica 0.117 0.053 0.044 0.000*LowestdifferentiationD=0.00–0.138indicatedinyellow*ModeratedifferentiationD=0.139–0.276indicatedinorange*HighestdifferentiationD=0.277–0.414indicatedinred

98

Table2.7.PairwisepopulationmatrixofNei’sunbiasedgeneticdistanceofP.cubensisisolatesgroupedbyhostspecies

*LowestdifferentiationD=0.00–0.138indicatedinyellow*ModeratedifferentiationD=0.139–0.276indicatedinorange*HighestdifferentiationD=0.277–0.414indicatedinred

Cucumissativus

Cucumismelo

Cucurbitapepo

Cucurbitamaxima

Cucurbitamoschata

Citrulluslanatus

Cucurbitafoetidissima

Momordicacharantia

Momordicabalsamina

Cucumissativus 0.000

Cucumismelo 0.012 0.000

Cucurbitapepo 0.344 0.239 0.000

Cucurbitamaxima 0.196 0.115 0.019 0.000

Cucurbitamoschata 0.337 0.228 0.000 0.014 0.000

Citrulluslanatus 0.303 0.189 0.026 0.021 0.021 0.000

Cucurbitafoetidissima 0.010 0.000 0.153 0.065 0.146 0.139 0.000

Momordicacharantia 0.127 0.069 0.136 0.071 0.131 0.083 0.071 0.000

Momordicabalsamina 0.275 0.180 0.000 0.005 0.000 0.009 0.108 0.085 0.000

99

Table2.8.PairwisepopulationmatrixofNei’sunbiasedgeneticdistanceofP.cubensisisolatesgroupedbycommercialhost

speciesandlocation

Cucumissativus Cucumismelo Cucurbitapepo Cucurbitamaxima Cucurbitamoschata Citrulluslanatus

KINX PIEDY WAYZ KINX PIEDY WAYZ KINX PIEDY WAYZ KINX PIEDY WAYZ KINX PIEDY WAYZ PIEDY

Cucumissativus

KINX 0 PIEDY 0.001 0 WAYZ 0 0.021 0

Cucumismelo

KINX 0 0.018 0 0 PIEDY 0.028 0.056 0.006 0.012 0 WAYZ 0.026 0.05 0.015 0.01 0.017 0

Cucurbitapepo

KINX 0.329 0.342 0.303 0.279 0.199 0.181 0 PIEDY 0.378 0.414 0.339 0.312 0.196 0.216 0.004 0 WAYZ 0.315 0.349 0.272 0.256 0.173 0.165 0 0.003 0

Cucurbitamaxima

KINX 0.111 0.135 0.092 0.08 0.038 0.033 0.04 0.061 0.027 0 PIEDY 0.223 0.262 0.191 0.176 0.087 0.11 0.016 0.008 0.008 0.003 0 WAYZ 0.309 0.348 0.246 0.247 0.127 0.153 0.006 0 0 0.023 0 0

Cucurbitamoschata

KINX 0.328 0.358 0.288 0.274 0.171 0.179 0 0 0 0.032 0 0 0 PIEDY 0.345 0.392 0.29 0.283 0.173 0.18 0 0.001 0 0.047 0.012 0 0 0 WAYZ 0.277 0.333 0.209 0.22 0.104 0.134 0 0 0 0 0 0 0 0 0

Citrulluslanatus

PIEDY 0.326 0.377 0.261 0.259 0.148 0.172 0.065 0.025 0.038 0.066 0.024 0 0.031 0.024 0 0WAYZ 0.3 0.343 0.242 0.23 0.157 0.135 0.017 0.03 0.013 0.037 0.013 0.007 0.023 0.011 0 0.026

XLowerCoastalPlainTobacco/CunninghamResearchStation,Kinston,NC,LenoirCountyYNCDA&CSPiedmontResearchStation,Salisbury,NC,RowanCountyZMountainResearchStation,Waynesville,NC,HaywoodCounty

100

*Shadinggroupscomparisonsmadebetweenisolatesfromasinglehostspeciesacrossgeographicregions*LowestdifferentiationD=0.00–0.138indicatedinyellow*ModeratedifferentiationD=0.139–0.276indicatedinorange*HighestdifferentiationD=0.277–0.414indicatedinred

101

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ABSTRACT WALLACE, EMMA CATHERINE. Population Structure of ...

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