Developmentofmoleculardiagnos4ctoolsfortheinvasiveoomycetepathogenPhytophthoratentaculata

N.Luecke,S.Koenig,T.D.Miles*SchoolofNaturalSciences,CaliforniaStateUniversity,MontereyBay.*tmiles@csumb.edu

Abstract

Fig.1.A)Diplacusauran,cus(sCckymonkeyflower)symptomaCcplant(LeI)andhealthyindividual(Right).B)AsecConofthenurseryinwhichhighlysymptomaCcplantsarefoundonthegroundinanareaknowforpoolingofwater

Fig.3.NecrosisonsCckymonkeyflowerrootcrownwithleadinglinesampled.

ThankyoutotheUndergraduateResearchOpportunityCenter forprovidingfundingandsupporttothisproject,USDA-APHISFarmBillforfundingmaterialsandsupplies.Laura Lee Lienk and ChrisCnaMcKnew at theWatershed InsCtute for cooperaCon andsupport,alongwiththefinefolksintheMilesLabforpaCenceandcooperaCon.

References

Acknowledgements

1.  S. Rooney-Latham and C. L. Blomquist. First report of root and stem rot caused byPhytophthoratentaculataonMimulusauran,cusinNorthAmerica.PlantDisease201498:7,996-996

2.  Timothy D. Miles, Frank N. MarCn, and Michael D. Coffey. Development of RapidIsothermal AmplificaCon Assays for detecCon of Phytophthora spp. in Plant Tissue.Phytopathology2015105:2,265-278

3.  Bongsoo Park, Frank MarCn, David M. Geiser, Hye-Seon Kim, Michele A. Mansfield,EkaterinaNikolaeva,Sook-YoungPark,MichaelD.Coffey,JosephRusso,SeongH.Kim,Yilmaz Balci, Gloria Abad, Treena Burgess, Niklaus J. Grünwald, Kyeongchae Cheong,JaeyoungChoi,Yong-HwanLee,andSeogchanKang.PhytophthoraDatabase2.0Updateand new direcCon. Phytopathology 2013 103:12, 1204-1208 hip://www.phytophthoradb.org/species

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Fig.4.A)SchemaCcoftheprimersandprobesrequiredforthedevelopmentofrecombinasepolymeraseamplificaConassays.B)RPAspeciesspecificprimersforseveralPhytophthoraspeciescreaCngspecificitybylocaConwithinpreviouslyidenCfiedgeneorderdifferencesinmitochondrialregions(Milesetal.,2015).

FutureWorkTheuseofaddiConalRPAassayswillidenCfytheepidemiologyofthepathogenandwillinturnbeusedtomapprojectedspreadofdiseaseincorrelaContoelevaCon.ThiswillpredictthemovementpaiernsofthepathogenanddetermineifthereisacorrelaConbetweenpathogenpresence,specifiedelevaConsandareasoftarning.Furthermore,addiConalstudiesaboutsamplingstrategiesonplantrootswillbeconductedusingthesetechniques

ImmunoStrip qPCR qPCR RPA RPA

Assayintendedtarget

AllPhytophthoraspp.(knowntocrossreactwithPythiumspp.)

AllPhytophthora

spp.

Phytophthoratentaculata

only

AllPhytophthora

spp.

Phytophthoratentaculata

only

DetecCon/Numberofsamplestested 22/113 15/113 12/113 15/113 6/113

Timepersample 40minutes 6.5hours 6.5hours 1.1hours 1.1hours

Costpersample($) 12 4 4 4 4

PrimerRegion ELISAbased atp9-nad9 atp9-nad9 trnM-trnP-trnM

atp9-atp9/nad9spacer

Fig.5.AgdiaImmunostripwithposiCvedetecConofPhytophthoraatagenuslevel(2Lines)

Fig.2.DiplacusAuran,cus(SCckyMonkeyFlower)isfeaturedinthispictureinordertodemonstratewhattheplantlookslikeanditsrootsystem.CircledandlabeledA)isthemainrootstalkwherethesoillineisfound.B)representsthetaprootwhichwehavefoundexperimentallyisthetargetedregionfortesCngforapathogen.Itisfoundgenerallywherethemainrootsystemhasanotchandbeginstoturn“hooking”intosoil.C)Thesubrootsystemistheotherpartoftherootsystemtested.ItisadvisednottotestfromthisporConoftheplantasitislesslikelythepathogenwillbepresent.

A

B

C

IntroducCon

ResultsDNAExtrac4onDNA was confirmed in all methods of detecCon, with qPCRfamily specific primers were used and with RPA andImmunosCpsDNAwasvisible(maceratedCssue)qPCRProbeDetec4onThe qPCR method was accurate and was the method mostcommonly used even though this was the most tediousprocess,6.5hours.(seeTable1)GenusandSpeciesSpecificRPAThe RPA assays were accurate in Phytophthora detecCon,aligningwith theqPCRtechnique.ThismethodwasextremelymoreCmeefficientwithresultsachievedin1.1hours.ImmunostripDetec4onAlthough resultswere obtained in aminimal amount of Cme(40 minutes), immunostrips were inaccurate due to crossreacCons with Pythium spp. that provided false posiCves fornon-Phytophthorasamples.

Table1.MethodsusedtodetectPhytophthoratentaculatainfieldsamplesinthisstudy.TableincludesavarietyofaiributessuchasnumberofposiCvesamples,approximateprocessingCme,costpersampleanddetecContarget.

Phytophthorabasalrot(PBR)ofplantsisadiseaserecentlyobservedinCentralCaliforniaNaCveplantnurseriesandrestoraConlocaCons.Theprimarycausalagentofthisdiseaseisthe oomycete pathogen Phytophthora tentaculata. Prior to 2012, P. tentaculata wasamong the ~30 Phytophthora species not known to be present in the United States.Previous research had developed an isothermal diagnosCc tool known as recombinasepolymerase amplificaCon (RPA), which had Phytophthora genus specific detecConcapabilitywithresultsobtainablewithinasliileas15minutesdirectlyinthefieldwithoutconvenConalDNAextracCon. AP.tentaculataspecies-specificRPAassaywasdevelopedand specificity validated against pure DNA from 135 Phytophthora taxa. To test thistechniquefordetecConofthepathogeninPBR,113symptomaCcsampleswerecollectedandevaluatedwiththenewRPAassayaswellasthepreviouslyvalidatedTaqManassay,Immunostrip and convenConal culturing and baiCng techniques. Results were similaracrossthevariousamplificaConplaqorms,withqPCRbeingthemostsensiCveingeneral.Finally,spaCalmodelswerecreatedtotestifelevaConcorrelatedwiththepresenceofthepathogen. These results indicate that P. tentaculata is oIen present in low-lying areaswhen infectedplantmaterial isused for restoraCon.This informaConwillassist inmoreefficient,rapid,sensiCve,specificpathogendetecConformanagementdecisionsinafieldandnursery serng aswell as understanding pathogendistribuCon and spreadwithin afield.

MainObjec4ves:UClizefourdifferentmethodsofdetecContoidenCfyP.tentaculataatarestoraConsite:1.  1) Determine the amount of Cme required for each method: qPCR, genus/species

specificRPAassaysandAgdiaImmunostrips.2.  2)invesCgatetheeaseandfeasibilityofeachmethod3.  3)correlatedetecConresultsofeachmethodacrossdifferentplaqorms.

In 2012 the fungal plant pathogen, Phytophthora tentaculata, known to causebasal rot was detected in California naCve plant nurseries. NaCve plant Nurseries aregenerallynonprofitorganizaConswithmainfocusoncommunitybasedrestoraCon.

P. tentaculata currently has a known host range of five plants species, but thenumberisexpandingandextensivehostrangestudiesarebeingconducted(Lathametal.,personalcommunicaCon).

CalStateMontereyBay’sWatershedInsCtuterecentlydetectedthispathogenonnaCveplantspeciesatthenursery,ReturnoftheNaCves(RON),andwithinarestoraConsite. The spread of this pathogen in a natural serng could negaCvely affect many ofCalifornia’splantcommuniCes.

BestManagementPracCces(BMPs)havebeenrecommendedbutareoIenmoreexpensiveandlaboriouswhichcanproseissuesfornonprofitorganizaCon

ManynaCveplantnurseriesdonothaveaccesstotesCngmethodsandobtainingresults from outside laboratories can be tedious. Currentmethods of detecCon can becumbersome and inaccurate. However, the recent development of a rapid moleculardetecCon technique, Recombinase PolymeraseAmplificaCon (RPA), has been developedforthePhytophthoragenus(Milesetal.,2015)

A B

DiscussionAIerundergoingfourseparatetestsresultswouldindicatetheRPAisthebestopConforrapid detecCon of a pathogen in a wild serng or in the case of a nursery. TheImmunostripswereusefulforgeneralpathogendetecContheywereunspecific(seeTable1).qPCRprovidesagreatamountofinformaConandshouldbeusedforvalidaConaswellasquanCficaConbutisCmeconsumingandrequiresa considerableupfrontinvestment.ValidaConusingpearbaiCngandclassicculturingtechniquehoweverprovedtobedifficultduringdryseason.ThesetypesofassaysarenecessaryformakingdecisionstosaveCmeandmoneyaswellasassistwiththoroughunderstandingofthepathogenandpreventitfromspreadingoutsideofnurseriesandrestoraConsites.

Methods-SampleswerecollectedatarestoraConsiteadjacenttoCalStateMontereyBayproperty.-SymptomaCcDiplacusauran,acusrootcrownswerecollectedundersterilecondiCons.-DNAwasextractedfromtherootcrownusingPlantDNAextracConkit(Qiagen,3hours)-AssembledqPCRmastermixcontained(30min)-ReacConswereranintheBioRadCFX96qPCRmachine(3hours)-GroundasecConoftherootcrownin5mLofGeneralExtracConBuffer2(AgdiaInc.)(10min)-Assembleamastermix(withRPAprimers/probe)andrunreacConreacCon(25min)-5mLofSimpleExtracConBufferwasaddedtorootcrownsecConinExtracConpouch(Agdia)-SamplewasemulsifiedunClahomogenousmixturewasachieved(10min)-Immunostripaddedtoemulsifiedliquid(30min)-PosiCveIdenCficaCon:presenceoftwolines

Fig.6.MapoftheLighAightersitethathastheposiCvesamples(Red)testedwithqPCRandallothersamplescollected(Black).Developmentofthesemodelsassistsinunderstandingthepathogensmovementincertainterrain.ThissitemainlyconsistsofabasinaboutthreesquareacreswithdifferentdepressionsatdifferentelevaCons.