New Disease-Causing Organisms in Alfalfa: Aphanomyces Euteiches Race 2 and Phytoplasma

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1 Introduction New Disease-Causing Organisms in Alfalfa: Aphanomyces Euteiches Race 2 and Phytoplasma by R.D. Peters and C.R. Grau Department of Plant Pathology University of Wisconsin Madison AlfalfaistheprimaryforagecropinWisconsinandisakeyelementinthestatesdairyindustry.The yieldofnewvarietiesisgreaterthanthatofvernalandotheroldervarietiesduetogeneticgainsmade bybreedersovertheyears.nevertheless,overallyieldshavedeclinedsteadilyinwisconsinduringthe past30years(wiersmaetal.1997),eventhougheffortshavebeenmadetobreedforresistancetoa widevarietyofmajorpathogensofalfalfasuchasverticillium,phytophthora,andaphanomyces.there areseveralpossibleexplanationsforthislackofprogressincludingchangingclimaticandsoilconditions andthedifficultiesinherentindealingwithsuchageneticallydiversecrop.fromapathologist s perspective,newdiseasejcausingorganismsornewstrainsofpreviouslyjdescribedpathogensmayalso playaroleinlimitingyieldgainsforwisconsinalfalfagrowers.attheuniversityofwisconsinjmadison, weareconductingresearchonalfalfadiseases,particularlythosecausedbyorganismsthathavenot beenstudiedpreviouslywithrespecttotheirpresenceandinfluenceonthealfalfacrop.whatfollowsis ashortsummaryofthebiologyandpotentialimpactoftwonewpathogens:aphanomyces3euteiches Race2andphytoplasma. Aphanomyces,Root,Rot AphanomycesrootrotiscausedbythepathogenAphanomyces3euteiches.Itismostsevereinflooded soilconditionsandoftenassociatedwithotherrootjrottingpathogenssuchasphytophthora3 medicaginis(thepathogencausingphytophthorarootrot).infectionwithaphanomycescanresultin thedeathofseedlings,butmoreoftenresultsinstunted,chlorotic(yellow)plants.rootsand hypocotylsdeveloplighttodarkbrownlesions,butunlikeotherrootjrotpathogenswhichcause seedlingcollapse,hypocotylsinfectedwithaphanomycestendtoremainrigid,resultinginstuntedbut uprightseedlings(grau1990).regrowthafterharvestorwinterdormancyisreducedininfectedplants (Grau1990). Aphanomycessurvivesasoospores(sexualspores)inthesoilorininfectedplanttissues.Flooded conditionsencourageoosporestogerminateproducingfreejswimmingzoospores(zoosporesarealso producedfromgerminatingasexualprimaryspores).itisthezoosporesthatencystonhostroots causinginfectiontooccur.soiltemperaturesof75j82 Fareoptimumforinfectionandthe developmentofdisease(grau1990).

2 SoilsinfestedwithAphanomycesinoculumcanbedetectedbyplantingbaitplantsintopotscontaining thefieldsoilofinterestandmaintainingfloodedconditionsfor5days.plantswithsymptomsarethen removedfromthepotsandroottissuefromtheseplantsisplatedoutontoaselectivegrowthmedium toobtainpureculturesofthepathogen(pfenderetal.1984).theorganismgrowswelloncornmeal agaratroomtemperature,butmustbetransferredeverymonthorsotomaintainitsviability(grau 1990).Usingaspecializedtechnique(MitchellandYang1966),pureculturescanbeusedtoproduce zoosporeswhichcanthenbeutilizedtoinoculateplantseedlingsinthegreenhouseorgrowthroomto determinetheresistanceresponsesofgermplasmtospecificisolatesofthepathogen. StudieshavenotedthatisolatesofAphanomycestendtobemostvirulent(i.e.causethemostdisease) onthecropsfromwhichtheyhavebeenisolated(malvicketal.1998).inotherwords,isolatesfrom alfalfatendtocausemorediseaseonalfalfathanonpeasandviceversa.exceptionsdooccur,and someisolatescancauseseverediseaseonmorethanonecrop(malvicketal.1998).theuseof moleculartoolshasdocumentedgenotypicdiversityamongisolatesofaphanomycesobtainedfrom peas(malvickandpercich1998)andsignificantgenotypicdiversityamongisolatesobtainedfrom differenthostspecies(malvicketal.1998).thislevelofdiversity(whichindicatesthattherearegenetic differencesamongpopulationsofthepathogen;theyarenotallthesame)inpopulationsof Aphanomycesprovideschallengesforbreedingprograms,becausespecificresistancesmaynotbeactive againstallstrainsofthepathogen. Fungicidescontainingmetalaxyl(i.e.Apron)arenotactiveagainstAphanomycesandtherefore,avoiding poorlydrainedsoilsandusingresistantvarietiesarethemainmethodsusefulforcontrol.waphj1 alfalfagermplasmwasreleasedbythewisconsinagriculturalexperimentstationin1989(grau1992). ThisgermplasmisresistanttoisolatesofAphanomycescommonlyfoundinWisconsin(Race1). ResistancetoRace1ofAphanomyceshasbeenwidelyincorporatedintocommercialalfalfavarieties (Malvick1998).Around1990,isolatesthatwerehighlyvirulent(i.e.causedsignificantdisease)to breedinglineswithresistancetorace1wererecoveredfromsoilscollectedineasternandsouthern states.similarisolateswerealsofoundinscatteredlocationsinwisconsin.suchisolateshavesince beencoined Race2"isolatesandrepresentanewformofthepathogen.Race2isolatesof AphanomyceshavenowbeenfoundinIdaho,Maryland,Minnesota,Mississippi,NorthCarolina, Tennessee,Virginia,Iowa,Kentucky,andWisconsin(Malvick1998).Race2isthereforewidely distributedintheunitedstates.thedistributionofrace2populationswithinastateisalsoimportant. InWisconsin,southwesterncountiesseemtohaveahigherincidenceofRace2isolatesthanother areas.thelancasterresearchstationishighlyinfestedwithrace2populationsandmostisolatesfrom easterniowaarealsorace2.bycontrast,fieldsatthemarshfieldresearchstationhavealower incidenceofrace2,althoughthisincidenceappearstobeincreasing. AlfalfabreedingprogramsaredevelopinglinesthatareresistanttoRace2ofAphanomyces(Malvick 1998).Fortunately,theselinesalsoappeartobehighlyresistanttoRace1isolatesaswell.Race2 resistantmaterialwouldshowthemostpronouncedeffectswhererace2populationsofthepathogen predominate.however,eveninregionswhererace1populationspredominatenow,race2may becomemoreprevalentinthefutureifthereisselectionpressurebythecrop(i.e.growingonlyrace1 resistantvarieties).thetimeframeforthedevelopmentofresistantpopulationsofthepathogenis unclear.however,thevariabilitythatispresentinpopulationsofaphanomycesimpliesthatbreeders andgrowersmustcontinuetobevigilanttomeetthedemandsofcontrollingachangingpathogen population.,,

3 Phytoplasma Phytoplasmasmaybestbedescribedasbacteriathathavelosttheircellwalls.Theyareonlyenclosed byathinmembranewhichmakesthemverypliableandabletoassumeavarietyofshapes(thinkofa balloonfilledwithwaterthatcanbesqueezedintodifferentshapes).theyexistexclusivelyinthe phloem(thecarbohydratejconductingtissueoftheplant)andaretransferredfromplanttoplantby insectvectors,primarilyleafhoppers.sincetheirdiscoveryinthelate1960s,phytoplasmashavebeen foundinhundredsofdifferentplantspeciesandcauseavarietyofdiseases.symptomsofinfectionmay includeyellowing,stuntedgrowthandslowdecline(particularlyinassociationwithmanytreespecies), andabnormalgrowthsuchasproliferation(anabnormallylargenumber)ofstemsandbudsand vegetativegrowth(suchasleaves)fromfloralparts(phyllody).althoughsomestrainsofphytoplasma arefoundonlyononecrop,others,suchastheorganismcausingasteryellows,caninfectawiderange ofhostspecies. Oneofthemajordifficultiesofworkingwithphytoplasmaistheinabilitytogrowtheorganismin culture.thismakesdetectionmoredifficult.traditionaltechniquesthatrelyonplantsymptomsto determineinfectionarenotalwaysreliable.anexcellentcollaborationhasbeenestablishedbetween ourlabatu.ofwisconsinjmadisonanddr.ingjminglee,usda,beltsville,marylandrelativeto phytoplasmasinsoybean.thiscollaborationhasbeenextendedtocoveralfalfadiseaseproblems.dr. LeeisaleadingresearcherinphytoplasmadetectionandtaxonomyandhaspioneeredgroundJbreaking researchinthisarea(leeetal.1998).notonlyhasdr.leeassistedintheestablishmentofphytoplasma detectionandidentificationprotocolsatu.ofwisconsinjmadison,hehasperformedreplicate experimentstoconfirmandvalidateresultsobtainedinourlaboratory. AsurveywasconductedduringSeptembertoNovember,1998todeterminetheincidenceof phytoplasmainalfalfaplantings.sampleswereobtainedfromfieldsneararlington,evansville, Marshfield,WestMadison,Lancaster,Whitewater,HancockandagrowerfieldwestofMadison. Samplesconsistedofgrowingtipsfromupperregionsofplants.Sampleswereplacedintolabeled plasticbagsandthenplacedinstyrofoamcoolersforreturntouwjmadisonuponwhichsampleswere frozenatj20 C.SampleswereprocessedbyextractionofDNAusingtheprotocolof(Zhangetal.1998). ThisprocessseparatestheDNA(whichcontainsthegenesweareinterestedin)fromtherestofthe plantmaterial.afterthepurifieddnawasobtained,nestedpcr(polymerasechainreaction)was carriedoutusingtwouniversalprimerpairsaccordingtotheprotocolofgundersonandlee(1996).the PCRprocessallowsustodetecttheDNAfromonlythephytoplasmaspresentinasampleand distinguishesphytoplasmadnafromalltheothertypesofdnapresentinthesample(suchasplant, bacteria,andfungaldna).thisnewtechnique(pcr)iscostlyandtimejconsuming,butallowsusto detectorganismssuchasphytoplasmasinalfalfa(andothercrops)whichhaveprobablybeenpresentin plantingsformanyyears,butwereverydifficulttodetectwithtraditionalmethods.forfurther classificationofphytoplasmas,restrictionenzymedigestswereperformed(thephytoplasmadnawas cutwithenzymes)andcomparisonofrflp(restrictionfragmentlengthpolymorphisms)weremadewith knownpatternsdescribedbyleeetal.(1998).inotherwords,afterwecutthephytoplasmadnawith enzymes,thepatternsthatwereproducedwerecomparedwithpatternsofotherphytoplasmasfrom aroundtheworldtofurtheridentifywhatphytoplasmaswearedealingwithinwisconsin. Resultsindicatedthatphytoplasmasororganismscloselyrelatedtophytoplasmasarewidespreadin alfalfaplantingsinwisconsin(seetable1).

4 Table,1.,IncidenceofsamplesPCRJpositiveforphytoplasmainWisconsin. Location Number of samples Number of samples PCR-positive for phytoplasma Percent of samples PCR-positive for phytoplasma Arlington Evansville Marshfield West Madison Lancaster Whitewater Hancock Growers field west of Madison TOTAL Restrictiondigestwork(cuttingwithenzymes)onthePCRproducts(amplifiedDNA)ofthe12positive samplesiscontinuing,butinitialresultshaverevealedthepresenceofphytoplasmasbelongingtothe asteryellowsgroupingaswellasotherorganismsthatarenotcurrentlyrecognizedasphytoplasmasin thetaxonomicsystemdevelopedbyleeetal.(1998).thisisthefirstreportofthepresenceoftheaster yellowsorganisminalfalfa.asteryellowshasbeenreportedinotherleguminouscropssuchasclover, broadbean,burjclover(medicago3hispida),andsoybean(mccoyetal.1989,m.e.lee,personal communication).thepresenceofcloverproliferationstrainsisalsolikelybasedonitspresenceinsweet clover(mccoyetal.1989)andinsoybeansinwisconsin(m.e.lee,personalcommunication).more thanonephytoplasmastrainmayalsooccurinasingleplant(almaetal.1996,biancoetal.1993,leeet al.1995).inthecurrentstudy,thepresenceofphytoplasmaorrelatedorganismswasassociatedwith plantsamplesdisplayingsymptomsofpurplingofthefoliage,interveinalchlorosis(yellowingbetween plantveins),andleafpuckering.purplingandchloroticdiscoloration(yellowing)aresymptoms commonlyassociatedwithinfectionbyasteryellowsphytoplasmasinothercrops(khuranaetal.1988, Zitter1991).However,thepresenceofthesesymptomsdidnotalwaysimplythepresenceofthe pathogen,aspcrnegativeresultswerealsoobtainedfromplantsamplesshowingthesesymptoms. Morerigoroussurveys,arerequiredtoassessthefullextentofphytoplasmaincidenceinalfalfa plantingsinwisconsin. Alfalfaisdistinguishedfromotheragriculturalcropsinhavingaperennialhabit.Inthisway,itcanbe comparedtomanytreespeciesthathavebeenshowntoharborthephytoplasmaorganismformany seasons,resultinginchronicdisease.mostourpcrpositivealfalfasamplesoriginatedfromplantingsin theirthirdorfourthyear.thismayindicatethattheperennialnatureofthecroppredisposesitto phytoplasmainfectionbyallowingalongertimejframefortransmissionbyleafhoppervectorsandfor thebuildjupofinoculuminhosttissues.forestresearchershavenotedthatphytoplasmasareoften concentratedinroottissues(seemüller,1988).ourresearchhasalsoshownthatpcrpositiveresults canbeobtainedinbasalstem,crown,androottissuesaslateasdecember(samplingthroughthe wintermonthswillcontinuetodetermineifthepathogencanbedetectedinthesetissuesthroughout thewinter).severalplantsobtainedfromthearlingtonresearchstationinearlydecember,yielded root,crown,andbasalstemtissuethatwaspcrpositivewithuniversalphytoplasmaprimers.itis probablethatphytoplasmascanoverwinterinthesetissuesasitdoesinrootsoftreespecies (Seemüller,1988)andinperennialweeds.

5 OurdiscoveryofPCRpositivelateseasonbasaltissueshasseveralpotentialepidemiological consequences.currentdogmastatesthatleafhoppersblownintowisconsinfromthesouthernu.s.are largelyresponsiblefortransmissionofphytoplasmaanddiseasespread.however,thepresenceofthe organisminoverjwinteredtissuewouldleadtotheavailabilityofinoculuminearlyspringwhichcould thenbetransmittedbylocalleafhopperpopulations.themajorvectorinvolvedintransmissionis unclear,howevertheasterleafhopper(macrosteles3fascifrons)wouldbeaprimecandidate.although thepotatoleafhopperisnotstrictlyaphloemfeeder,itssheernumbersandshotgunapproachto feedinginalfalfaalsomakesitacandidatefortransmission.researchisneededtodeterminethevector speciesinvolvedintransmissionandthetimingoftransmissionevents.wehaveacollectionofinsects obtainedduringthe1998fieldseasonwhichisawaitinganalysisforthepresenceofphytoplasma.we arealsogrowingplantsobtainedfromwisconsinfieldsinthegreenhousetoactasasourcefor transmissionstudies.wearehopingtobeginestablishmentofleafhopperpopulationsforthesestudies injanuary.transmissionfrominfectedtononjinfectedhostsmayalsobepossibleusingdodder (Cuscuta3spp.)toactasabridgebetweenplants(dodderisaparasiticplant)orviadirectgraftingofhost tissues(chiykowski1988).sincekochspostulates(rulesthatmustbefulfilledtoprovethatanorganism causesadisease)aredifficulttocompleteinanonjculturableorganismsuchasphytoplasma, transmissionelectronmicroscopywillbeutilizedtoconfirmpcrdatarelativetothesuccessand mechanismsoftransmission(chenetal.1989).inaddition,fieldtrialsestablishedattheagricultural ResearchStationinLancasterincludemanyalfalfavarieties(includingglandularhairvarieties)and chemicalsprayregimenswhicharebeingassessedforimpactrelativetoleafhopperinjury.samples fromtheseplotshavealsobeengatheredtodeterminephytoplasmaincidence.todate,wehavefound samplesthatarepcrpositiveforphytoplasmainbothleafhopperjsusceptibleandresistantvarieties.it ispossiblethatglandularjhairedvarieties,althougheffectiveforcontrollingpotatoleafhopperfeeding, donotcontrolotherleafhopperspeciesorinsectsthatareinvolvedinthespreadofphytoplasmas.the effectsofchemicalspraysonvarioustypesofinsectspeciesandontheincidenceofphytoplasmasalso needstobedetermined. Alfalfaplantsstorecarbohydrates(starchesandsugars)intheirrootsandcrowns.Thesecarbohydrate reservesareusedforregrowthinthespringandaftereachcutting.carbohydratereservesare replenishedwhentheplantreaches6to8inchestall(undersanderetal.1994).managementpractices suchascuttingtoofrequentlycandepletecarbohydratereservesandlowerstandpersistence.similarly, thepresenceofphytoplasmainthephloemofinfectedplantscouldaffectthemovementandavailable amountofcarbohydratereservesfortheplantleadingtoreducedproductivityandstandpersistence. Thepresenceofphytoplasmaoverseveralyearsinafieldcouldbepartiallyresponsiblefortheobserved declinethatoftenoccursinalfalfaplantingsovertime.itisoftennotedthatsuchdeclineisnot associatedwithanyofthetraditionalalfalfapathogensand,inthepast,hasbeenblamedonvariety geneticsorenvironmentalconditions.itisourhypothesisthatphloemjlimitedphytoplasmaarehaving adelayedyetsignificantimpactonforageyieldduetoutilizationandinterruptionwithtransportof carbohydrates.suchanimpactwouldonlybenoticedovertime.tothisdate,directexperimental evidencesupportingthishypothesisislackinghowever,itisinterestingtonotethatthegrowersfield fromwhichsampleswereobtainedwestofmadisonwasshowingseriousdeclineinitssecondyear. Symptomsofcommonpathogenswerenotapparentinthesesamples,yettheywerePCRpositivewith theuniversalphytoplasmaprimers.althoughcauseandeffecthasnotbeenproven,thisobservation providesimpetustoperformfurtherresearchtotestthishypothesis.asteryellowsphytoplasmashave significantagronomiceffectsonawidevarietyofcropsincludingcarrotandlettuce(errampallietal. 1991)andbarley(Chiykowski1991)andcancausesignificantlossesincropssuchascucurbits,celery, onion,andlegumes(provvidenti1996).inaddition,phytoplasmashavebeenassociatedwithslow

6 declineofvariousshrubsandtreespecies(leeetal.1995,sinclair1995,sinclairetal.1990,sinclairet al.1994). Thechronicpresenceofphytoplasmainalfalfawouldalsohaveimplicationsforothercrops.Field observationsimplythatsoybeansplantednearalfalfatendtodisplaygreatersymptomsofphytoplasma infectionthansoybeansinotherlocations.inthismanner,alfalfacouldbeactingasareservoirhostfor infectionofothercrops.phytoplasmapresenceinsuchabridgehostmaynotonlyhaveanagronomic effectonthereservoirhostitself,butalsoonsurroundingcrops. Researchinourlabhasalsoshownthatalfalfaseedharvestedfromplantswithlowseedyieldcan produceplants(afterplantinginagrowthchamber)thatarepcrpositivewithuniversalphytoplasma primers(m.e.lee,personalcommunication).althoughmoreresearchisrequired,thepotentialforseed transmissionofphytoplasmawouldnotonlyhavesignificantepidemiologicalandagronomic consequences,butwouldalsochallengecurrentscientificthinkingwhichstatesthatsuchtransmission cannotoccur.thisresearchwouldthereforehavesignificantbasicandappliedaspects.thisresult warrantsfurtherresearchintotheroleofseedtransmissionintheepidemiologyofthe phytoplasma/alfalfapathosystem. AmajorprojecthasbeenproposedattheUniversityofWisconsinJMadisontodeterminethe distribution,characterization,modesoftransmissionandimpactofphytoplasmainalfalfainwisconsin. TheprincipleinvestigatorsareCraigR.Grau(PlantPathology),DavidB.Hogg(Entomology),JohnL. Wedberg(Entomology),DanielJ.Undersander(Agronomy),JerryD.Doll(Agronomy),RobAlleman (GraduateStudent),andRickD.Peters(PlantPathology).Studiesareproposedtodeterminethe distributionofphytoplasmaonaregional,individualfield,andindividualplantbasis.weedswillalsobe screenedaspotentialsourcesofthepathogen,sincetheycanactasreservoirsforsubsequentcrop infection.theinitialcornerstoneofanyipmprogramiscorrectdiagnosis.moleculartoolswillbe utilizedtoaccuratelydetectandcharacterizetheorganisminalfalfa.thesetoolsshouldassistgrowers directlyinthediagnosisofdiseasescausedbyphytoplasmasandwillaidalfalfabreedersindeveloping programstobreedforresistancetothesepathogens.insectcollectionswillbemadeineachyearofthe studytodeterminetheprincipleinsectvectors(primarilyassumedtobeleafhoppers)responsiblefor spread.thesestudieswillbelinkedwithfieldstudiesaddressingcontrolpracticessuchaschemical insectcontrolandhostresistance(glandularjhairedvarieties)toexaminetheeffectofcurrentipm practicesonphytoplasmainfectionandtodevelopaneffectiveipmprogramthattakesphytoplasma infectionintoaccount.othermodesoftransmissionwillbeexaminedtodetermine,forexample,the importanceofseedtransmissionintheepidemiologyofdisease.finally,infectedplantsproducedvia infectedseed,viadirecttransmissioninthegreenhouseusinggrafting,dodderorinsecttransmission systems,orvianaturallyjoccurringinoculumwillbeusedingreenhouseandfieldstudiestodetermine theimpactofphytoplasmainfectionontheyieldandqualityofalfalfa. Summary Aphanomyces,Race,2 o! PopulationsofRace2ofAphanomycesarewidespreadintheU.S.A.andinWisconsin. o! TheprevalenceofRace2populationsinsouthwesternWisconsinhaslimitedthe performanceofrace1resistantalfalfavarietiesinthatregion. o! Race2resistance,whichalsoconfersresistancetoRace1,isbeingincorporatedinto futurealfalfavarieties.

7 Phytoplasma o! PhytoplasmasarebacteriaJlikepathogensthathavenotpreviouslybeenassociatedwith alfalfainwisconsin. o! Phytoplasmasbelongingtotheasteryellowsgroupandotherunrecognizedgroupshave beendetectedinalfalfafieldsinwisconsinusingnewmoleculartools(pcr). o! PCRiscostlyandtimeJconsuming,butverysensitiveandallowsorganisms(suchas phytoplasmas)tobedetectedthatweredifficulttodetectinthepast. o! Symptomsofinfectionwithphytoplasmaincludeyellowingandpurplingoffoliageand stuntingwhicharelikesymptomscausedbyinsectjfeeding,nutritionaldisordersand climaticeffects. o! Phytoplasmasaretransmittedbyinsects(mostlikelyleafhoppers)andpossiblyseed. o! Webelievethatthepresenceofphytoplasmasinthefoodtransporttissuesoftheplant isinterferingwiththetransportanduseofcarbohydratereservesleadingtoloweryields andlossofpersistence. o! AmajorprojecthasbeenproposedattheUniversityofWisconsinJMadisonto determinethedistribution,characterization,modesoftransmissionandimpactof phytoplasmainalfalfainwisconsin., References Alma,A.,R.E.Davis,M.Vibio,A.Danielli,D.Bosco,A.Arzone,andA.Bertaccini.1996.Mixedinfectionof grapevinesinnorthernitalybyphytoplasmasincluding16srrnarflpsubgroup16srijbstrains previouslyunreportedinthishost.plantdis.80:418j421. Bianco,P.A.,R.E.Davis,J.P.Prince,I.JM.Lee,D.E.Gunderson,A.Fortusini,andG.Belli.1993.Doubleand singleinfectionsbyasteryellowsandelmyellowsmlosingrapevineswithsymptomscharacteristicof flavescencedoree.riv.pat.veg.,s.v.3:69j82. Chen,T.A.,J.D.Lei,andC.P.Lin.1989.Detectionandidentificationofplantandinsectmollicutes.Pages 393J424inThe3Mycoplasmas3Volume3V:3Spiroplasmas,3Acholeplasmas,3and3Mycoplasmas3of3Plants3and3 Arthropods,R.F.WhitcombandJ.G.Tully,eds.AcademicPressInc.,SanDiego,CA.653p. Chiykowski,L.N.1988.MaintenanceofyellowsJtypemycoplasmalikeorganisms.Pages123J134inTree3 Mycoplasmas3and3Mycoplasma3Diseases,C.Hiruki,ed.,TheUniversityofAlbertaPress,Edmonton,AB. 245pp. Chiykowski,L.N.1991.Reactionofadditionalbarleycultivarstotwoasteryellowsstrains.Can.PlantDis. Surv.71:143J145. Errampalli,D.,J.Fletcher,andP.L.Claypool.1991.Incidenceofyellowsincarrotandlettuceand characterizationofmycoplasmalikeorganismisolatesinoklahoma.plantdis.75:579j584. Grau,C.R.1990.Aphanomycesrootrot.Pages10J11inCompendium3of3Alfalfa3Diseases:3Second3Edition, D.L.StutevilleandD.C.Erwin,eds.APSPress,St.Paul,MN. Grau,C.R.1992.RegistrationofWAPHJ1alfalfagermplasmwithresistancetoAphanomycesrootrot. CropSci.32:287J288.

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