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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.
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
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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
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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).
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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,
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Cohen,Y.1977.Thecombinedeffectsoftemperature,leafwetness,andinoculum
concentrationoninfectionofcucumberswithPseudoperonosporacubensis.
CanadianJournalofBotany,55,pp.1478–1487.
Colucci,S.J.2008.HostRange,FungicideResistanceandManagementof
Pseudoperonosporacubensis,CausalAgentofCucurbitDownyMildew.MS
Thesis,NorthCarolinaStateUniversity.RetrievedfromNCSUInstitutional
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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.
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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–
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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.&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
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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.
Robinson,R.W.andDecker-Walters,D.S.1997.Cucurbits.CABInternational,New
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Runge,F.,Choi,Y.-J.&Thines,M.2011.Phylogeneticinvestigationsinthegenus
PseudoperonosporarevealoverlookedspeciesandcrypticdiversityintheP.
cubensisspeciescluster.EuropeanJournalofPlantPathology,129(2),pp.135–
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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
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phytopathologicalandphylogeneticimplications.PlantPathology,58(5),
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Savory,E.A.etal.2011.Pathogenprofile:Thecucurbitdownymildewpathogen
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Thomas,A.,Carbone,I.&Ojiambo,P.2014.Comparativegenomicanalysisof
Pseudoperonosporacubensistoelucidatethegeneticbasisofhostspecialization.
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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
Isolate Isolatespecies
Host(Scientificname/variety) TissueusedinDNAextraction
IsolateOrigin YearCollected
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
Isolate Isolatespecies
Host(Scientificname/variety) TissueusedinDNAextraction
IsolateOrigin YearCollected
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
hdm257 P.humuli Humuluslupulus Sporangia Oregon,ProvidedbyDavidGent
2008
hdm110-2 P.humuli Humuluslupulus Sporangia Washington,ProvidedbyDavidGent
2006
hdm140 P.humuli Humuluslupulus Sporangia Oregon,ProvidedbyDavidGent
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
Isolate Isolatespecies
Host(Scientificname/variety) TissueusedinDNAextraction
IsolateOrigin YearCollected
hdm224 P.humuli Humuluslupulus Sporangia Oregon,ProvidedbyDavidGent
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
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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