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Development of molecular diagnos4c tools for the invasive oomycete pathogen Phytophthora tentaculata N. Luecke, S. Koenig, T.D. Miles* School of Natural Sciences, California State University, Monterey Bay. *[email protected] Abstract Fig. 1. A) Diplacus auran,cus (sCcky monkey flower) symptomaCc plant (LeI) and healthy individual (Right). B) A secCon of the nursery in which highly symptomaCc plants are found on the ground in an area know for pooling of water Fig. 3. Necrosis on sCcky monkey flower root crown with leading line sampled. Thank you to the Undergraduate Research Opportunity Center for providing funding and support to this project, USDA-APHIS Farm Bill for funding materials and supplies. Laura Lee Lienk and ChrisCna McKnew at the Watershed InsCtute for cooperaCon and support, along with the fine folks in the Miles Lab for paCence and cooperaCon. References Acknowledgements 1. S. Rooney-Latham and C. L. Blomquist. First report of root and stem rot caused by Phytophthora tentaculata on Mimulus auran,cus in North America. Plant Disease 2014 98:7, 996-996 2. Timothy D. Miles, Frank N. MarCn, and Michael D. Coffey. Development of Rapid Isothermal AmplificaCon Assays for detecCon of Phytophthora spp. in Plant Tissue. Phytopathology 2015 105:2, 265-278 3. Bongsoo Park, Frank MarCn, David M. Geiser, Hye-Seon Kim, Michele A. Mansfield, Ekaterina Nikolaeva, Sook-Young Park, Michael D. Coffey, Joseph Russo, Seong H. Kim, Yilmaz Balci, Gloria Abad, Treena Burgess, Niklaus J. Grünwald, Kyeongchae Cheong, Jaeyoung Choi, Yong-Hwan Lee, and Seogchan Kang. Phytophthora Database 2.0 Update and new direcCon. Phytopathology 2013 103:12, 1204-1208 hip:// www.phytophthoradb.org/species A B S Fig. 4. A) SchemaCc of the primers and probes required for the development of recombinase polymerase amplificaCon assays. B) RPA species specific primers for several Phytophthora species creaCng specificity by locaCon within previously idenCfied gene order differences in mitochondrial regions (Miles et al., 2015). Future Work The use of addiConal RPA assays will idenCfy the epidemiology of the pathogen and will in turn be used to map projected spread of disease in correlaCon to elevaCon. This will predict the movement paierns of the pathogen and determine if there is a correlaCon between pathogen presence, specified elevaCons and areas of tarning. Furthermore, addiConal studies about sampling strategies on plant roots will be conducted using these techniques ImmunoStrip qPCR qPCR RPA RPA Assay intended target All Phytophthora spp. (known to cross react with Pythium spp.) All Phytophthora spp. Phytophthora tentaculata only All Phytophthora spp. Phytophthora tentaculata only DetecCon/Number of samples tested 22/113 15/113 12/113 15/113 6/113 Time per sample 40 minutes 6.5 hours 6.5 hours 1.1 hours 1.1 hours Cost per sample ($) 12 4 4 4 4 Primer Region ELISA based atp9-nad9 atp9-nad9 trnM-trnP- trnM atp9-atp9/ nad9 spacer Fig. 5. Agdia Immunostrip with posiCve detecCon of Phytophthora at a genus level (2 Lines) Fig. 2. Diplacus Auran,cus (SCcky Monkey Flower) is featured in this picture in order to demonstrate what the plant looks like and its root system. Circled and labeled A) is the main root stalk where the soil line is found. B) represents the taproot which we have found experimentally is the targeted region for tesCng for a pathogen. It is found generally where the main root system has a notch and begins to turn “hooking” into soil. C) The sub root system is the other part of the root system tested. It is advised not to test from this porCon of the plant as it is less likely the pathogen will be present. A B C IntroducCon Results DNA Extrac4on DNA was confirmed in all methods of detecCon, with qPCR family specific primers were used and with RPA and ImmunosCps DNA was visible (macerated Cssue) qPCR Probe Detec4on The qPCR method was accurate and was the method most commonly used even though this was the most tedious process, 6.5 hours. (see Table 1) Genus and Species Specific RPA The RPA assays were accurate in Phytophthora detecCon, aligning with the qPCR technique. This method was extremely more Cme efficient with results achieved in 1.1 hours. Immunostrip Detec4on Although results were obtained in a minimal amount of Cme (40 minutes), immunostrips were inaccurate due to cross reacCons with Pythium spp. that provided false posiCves for non-Phytophthora samples. Table 1. Methods used to detect Phytophthora tentaculata in field samples in this study. Table includes a variety of aiributes such as number of posiCve samples, approximate processing Cme, cost per sample and detecCon target. Phytophthora basal rot (PBR) of plants is a disease recently observed in Central California NaCve plant nurseries and restoraCon locaCons. The primary causal agent of this disease is the oomycete pathogen Phytophthora tentaculata. Prior to 2012, P. tentaculata was among the ~30 Phytophthora species not known to be present in the United States. Previous research had developed an isothermal diagnosCc tool known as recombinase polymerase amplificaCon (RPA), which had Phytophthora genus specific detecCon capability with results obtainable within as liile as 15 minutes directly in the field without convenConal DNA extracCon. A P. tentaculata species-specific RPA assay was developed and specificity validated against pure DNA from 135 Phytophthora taxa. To test this technique for detecCon of the pathogen in PBR, 113 symptomaCc samples were collected and evaluated with the new RPA assay as well as the previously validated TaqMan assay, Immunostrip and convenConal culturing and baiCng techniques. Results were similar across the various amplificaCon plaqorms, with qPCR being the most sensiCve in general. Finally, spaCal models were created to test if elevaCon correlated with the presence of the pathogen. These results indicate that P. tentaculata is oIen present in low-lying areas when infected plant material is used for restoraCon. This informaCon will assist in more efficient, rapid, sensiCve, specific pathogen detecCon for management decisions in a field and nursery serng as well as understanding pathogen distribuCon and spread within a field. Main Objec4ves: UClize four different methods of detecCon to idenCfy P. tentaculata at a restoraCon site: 1. 1) Determine the amount of Cme required for each method: qPCR, genus/species specific RPA assays and Agdia Immunostrips. 2. 2) invesCgate the ease and feasibility of each method 3. 3) correlate detecCon results of each method across different plaqorms. In 2012 the fungal plant pathogen, Phytophthora tentaculata, known to cause basal rot was detected in California naCve plant nurseries. NaCve plant Nurseries are generally nonprofit organizaCons with main focus on community based restoraCon. P. tentaculata currently has a known host range of five plants species, but the number is expanding and extensive host range studies are being conducted (Latham et al., personal communicaCon). Cal State Monterey Bay’s Watershed InsCtute recently detected this pathogen on naCve plant species at the nursery, Return of the NaCves (RON), and within a restoraCon site. The spread of this pathogen in a natural serng could negaCvely affect many of California’s plant communiCes. Best Management PracCces (BMPs) have been recommended but are oIen more expensive and laborious which can prose issues for nonprofit organizaCon Many naCve plant nurseries do not have access to tesCng methods and obtaining results from outside laboratories can be tedious. Current methods of detecCon can be cumbersome and inaccurate. However, the recent development of a rapid molecular detecCon technique, Recombinase Polymerase AmplificaCon (RPA), has been developed for the Phytophthora genus (Miles et al., 2015) A B Discussion AIer undergoing four separate tests results would indicate the RPA is the best opCon for rapid detecCon of a pathogen in a wild serng or in the case of a nursery. The Immunostrips were useful for general pathogen detecCon they were unspecific (see Table 1). qPCR provides a great amount of informaCon and should be used for validaCon as well as quanCficaCon but is Cme consuming and requires a considerable upfront investment. ValidaCon using pear baiCng and classic culturing technique however proved to be difficult during dry season. These types of assays are necessary for making decisions to save Cme and money as well as assist with thorough understanding of the pathogen and prevent it from spreading outside of nurseries and restoraCon sites. Methods - Samples were collected at a restoraCon site adjacent to Cal State Monterey Bay property. - SymptomaCc Diplacus auran,acus root crowns were collected under sterile condiCons. -DNA was extracted from the root crown using Plant DNA extracCon kit (Qiagen, 3 hours) -Assembled qPCR master mix contained (30 min) -ReacCons were ran in the BioRad CFX96 qPCR machine (3 hours) -Ground a secCon of the root crown in 5mL of General ExtracCon Buffer 2 (Agdia Inc.) (10 min) -Assemble a master mix (with RPA primers/probe) and run reacCon reacCon (25 min) -5mL of Simple ExtracCon Buffer was added to root crown secCon in ExtracCon pouch (Agdia) -Sample was emulsified unCl a homogenous mixture was achieved (10 min) -Immunostrip added to emulsified liquid (30 min) -PosiCve IdenCficaCon: presence of two lines Fig. 6. Map of the LighAighter site that has the posiCve samples (Red) tested with qPCR and all other samples collected (Black). Development of these models assists in understanding the pathogens movement in certain terrain. This site mainly consists of a basin about three square acres with different depressions at different elevaCons.

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Page 1: Development of molecular diagnosc tools for the invasive ... Presentations/Miles... · Development of molecular diagnosc tools for the invasive oomycete pathogen Phytophthora tentaculata

Developmentofmoleculardiagnos4ctoolsfortheinvasiveoomycetepathogenPhytophthoratentaculata

N.Luecke,S.Koenig,T.D.Miles*SchoolofNaturalSciences,CaliforniaStateUniversity,MontereyBay.*[email protected]

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

A B

S

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.

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