Sie sind auf Seite 1von 130

United States Department of Agriculture Animal and Plant Health Inspection Service Cooperating State Departments of Agriculture

New Pest Response Guidelines


Ralstonia solanacearum race 3 biovar 2

The U.S. Department of Agriculture (USDA) prohibits discrimination in all its programs and activities on the basis of race, color, national origin, sex, religion, age, disability, political beliefs, sexual orientation, and marital or family status. (Not all prohibited bases apply to all programs.) Persons with disabilities who require alternative means for communication of program information (Braille, large print, audiotape, etc.) should contact USDA's TARGET Center at (202) 720-2600 (voice and TDD). To file a complaint of discrimination, write USDA, Director, Office of Civil Rights, Room 326-W, Whitten Building, 14th and Independence Avenue, SW, Washington, DC 20250-9410 or call (202) 720-5964 (voice or TDD). USDA is an equal opportunity provider and employer. USDA is committed to making its information materials accessible to all USDA customers and employees.

New Pest Response Guidelines


Ralstonia solanacearum race 3 biovar 2
May12,2008 NewPestResponseGuidelines:Ralstoniasolanacearumrace3biovar2was preparedbyJoelFloyd,USDAAPHISPPQNationalIdentificationServices andeditedbyPatriciaS.Michalak,USDAAPHISPPQEmergencyand DomesticPrograms.TheGuidelineswasoriginallylimitedtogeraniumhost plantsandwasissuedonFebruary27,2003. ThisupdatedGuidelinesincludesinformationonpotatohostplants.Citethe updatedreportasfollows:Floyd,J.2008.NewPestResponseGuidelines: Ralstoniasolanacearumrace3biovar2.USDAAPHISPPQEmergencyand DomesticPrograms,Riverdale,Maryland.http://www.aphis.usda.gov/ import_export/plants/manuals/index.shtml

R. L. Dunkle
RichardDunkle,DeputyAdministrator USDA/APHIS/PPQ

October24,2008

EmergencyandDomesticPrograms EmergencyPlanning JoelFloyd,TeamLeader 4700RiverRoad Unit137 Riverdale,Maryland20737 Telephone:3107344396 Joel.P.Floyd@aphis.usda.gov

Ralstonia solanacearum r 3 b 2

Credits

Contributors
PhilipBerger,USDAAPHISPPQCenterforPlantHealthScienceand Technology(CPHST),Raleigh,NorthCarolina RussBulluck,USDAAPHISPPQEmergencyandDomesticPrograms, Raleigh,NorthCarolina WilliamCallison(retired),CaliforniaDepartmentofFoodandAgriculture andtheNationalPlantBoard GeneCross,NorthCarolinaDepartmentofAgricultureandConsumer Services JoannCruse,USDAAPHISPPQ,Madison,Wisconsin ReneeDeVries,USDAAPHISPPQCPHST,NationalPlantGermplasm& BiotechnologyLab,Beltsville,Maryland CharlesDivan,USDAAPHISPPQBiologicalandTechnicalServices, Riverdale,Maryland LynnEvansGoldner,USDAAPHISPPQEmergencyandDomestic Programs(EDP),Riverdale,Maryland MikeFirko,APHISPPQPHPPermits,Registrations,Importersand Manuals(PRIM),Riverdale,Maryland LloydGarcia,USDAAPHISPPQEasternRegionalOffice,Raleigh,North Carolina DavidKaplan,USDAAPHISPPQEDP,Riverdale,Maryland LaureneLevy,USDAAPHISPPQCPHST,NationalPlantGermplasm& BiotechnologyLab,Beltsville,Maryland SteveMarquardt,NorthDakotaStateUniversity,Bismarck,NorthDakota WendyNelson,USDAAPHISPPQEnvironmentalServices(ES),Riverdale, Maryland

May 12, 2008 Emergency and Domestic Programs


Credits.fm

Ralstonia solanacearum r 3 b 2

Consultants

BillyNewton,USDAAPHISPPQEasternRegionalOffice,Raleigh,North Carolina SusanOToole,USDAAPHISPPQES,Riverdale,Maryland MatthewRoyer,USDAAPHISPPQEDP,Riverdale,Maryland LinSchmale,SeniorDirectorofGovernmentRelations,SocietyofAmerican Florists CliffordSmith,USDAAPHISPPQWesternRegionalOffice,Ft.Collins, Colorado JohnStewart,USDAAPHISPPQEasternRegionalOffice,Raleigh,North Carolina GeraldWheeler,USDAAPHISPPQ,GrandRapids,Michigan JimWriter,USDAAPHISPPQEnvironmentalServices,Riverdale, Maryland RichardZink,USDAAPHISPPQCenterforPlantHealthScienceand Technology,Ft.Collins,Colorado

Consultants
CaitilynAllen,DepartmentsofPlantPathologyandWomensStudies, UniversityofWisconsin,Madison,Wisconsin MargeryDaughtrey,CornellUniversity,PlantPathology,LongIsland HorticultureResearchLaboratory,Riverhead,NewYork JohnElphinstone,DepartmentforEnvironment,FoodandRuralAffairs, CentralScienceLaboratory,York,UnitedKingdom

Cover
GeraniumWiltingsymptomsongeraniumscausedbyRalstonia
solanacearumrace3biovar2;courtesyofWisconsinDepartmentof Agriculture,TradeandConsumerServices

PotatoesSymptomsofbrownrot,Ralstoniasolanacearumrace3biovar2,on
potatotubersandplant;courtesyofCentroInternacionaldelaPapa (InternationalPotatoCenterorCIP),Lima,Peru

ii

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Cover

Weed HostBitternightshade,aweedhostofRalstoniasolanacearumrace3
biovar2;courtesyofJoelFloyd,USDAAPHISPPQ

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

iii

Cover

iv

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

Contents
Credits page Ai
Contributorsi Consultantsii Coverii

Contents page iiiii Chapter 1 page 1-1 Introduction page 11


Contents1 Purpose1 RecentUpdates1 Status2 DocumentComprehensiveness2 CommercialSuppliersOrProducts2 PreventionOfInfection2 RegulatoryOfficers2 Growers3 ProgramSafety3 SupportForProgramDecisionmaking3

Chapter 2 page 2-1 PestInformation page 21


Contents1 Introduction1 Classification1 Importance2 Race13 Race33 History3 GeraniumHosts3 CurrentStatus4 SolanaceousHosts4 CurrentStatus5 EconomicImpact6 GeraniumHosts6 SolanaceousHosts6 Hosts6 GeographicDistribution7

Chapter 3 page 3-1 SurveyProcedures

page 31

Contents1 Introduction1 Precautions1 AntimicrobialSoap,Lotion,orDisinfectant1 DisposableGlovesandFootwear2 DetectionSurvey2


May 12, 2008 Emergency and Domestic Programs
RalstoniaBook2TOC.fm

Ralstonia solanacearum r 3 b 2

iii

Procedure2 GeraniumHosts3 SolanaceousHosts4 GeneralSurvey4 TargetedSurvey4 DelimitingSurvey6 GeraniumHosts6 SolanaceousHosts6 ListofFacilities6 WorkingwithGrowersorFarmManagers7 TracebackAndTraceForwardSurvey7 TracebackSurvey7 AnalyzingInformation7 PositiveDetectioninPotatoes8 TraceForwardSurvey8 WaterSourcesandIrrigationMethods8 InvestigationsatProcessors9 MonitoringSurvey9 GeraniumHosts9 SolanaceousHosts9 VisualInspectionForDetectionSurvey10 GreenhouseandNurseryCrops10 Geraniums10 OtherHostsinGreenhousesorNurseries11 DiscardedPlants11 PotatoCroppingSystems11 PotatoPlants11 PotatoTubers12 TomatoesandOtherSolanaceousCrops13 VisualInspectionForDelimitingSurvey14 GreenhouseandNurseryCrops14 PotatoCroppingSystems14 TuberSampling14 DelimitingSurveysinaGeneralGrowingArea15 SamplingofWaterSources15 TomatoandOtherSolanaceousHosts15 DataCollection15

Chapter 4 page 4-1 Identification page 41


Contents1 Introduction1 Authorities1 PlantProtectionAct2 AgriculturalBioterrorismProtectionAct2 ProcedureForSamplingAndIdentification3 ScreeningNurseryandFieldHosts3 Labeling,Numbering,andRecordKeeping3 StorageandShipping4 PPQForm3914 SaturdayDelivery5 NotificationofStateOfficialsofSubmissionsandResults5 SampleProcessingTime5

iv

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

DiagnosticScreeningLaboratories6 AlternativeLaboratory6 RequiredPermits6 IntrastateTransfer7 InterstateTransfer8 DiagnosticTestsToDetermineGenusAndSpecies8 ValidatedScreeningTestKits8 ELISATestKits9 WaterTesting9 Culturing9 ConfirmatoryTestingToRaceAndBiovar9 PositiveConfirmations10

Chapter 5 page 5-1 RegulatoryProcedures

page 51

Contents1 Introduction1 InstructionsToOfficers1 QuarantineActionsRequired2 Geraniums2 PotatoCroppingSystems2 EmergencyQuarantineAction2 AccesstoPrivateProperty3 RegulatoryRecords3 OverviewOfRegulatoryProgramAfterAUnitedStatesDetection3 DeterminingOriginofInfections3 Geraniums3 PotatoCroppingSystems4 DistributionofListsofPositiveandSuspectProperties4 Geraniums4 PotatoCroppingSystems5 FurtherInvestigationsAtNurseriesWithSuspectGeraniums6 PlacingHolds7 Nurseries7 PotatoStorageFacilitiesandGrowerFields7 RecordKeeping8 Geraniums8 PotatoCroppingSystems8 PlantstoHold9 SuspectGeraniums9 Variety9 Shipment9 PotentiallyInfectedPlants9 SuspectPotatoCroppingSystems11 GrowerRequirementsUnderQuarantine11 IssuingPPQForm523EmergencyActionNotification11 GeraniumAndPotatoVolunteersAndCullPiles12 HarvestingEquipment,StorageAreas,AndConveyances12 RegulatoryRequirementsOfSeedProducers12 RegulatoryRequirementsForProcessors12 RegulatedArticles13 CultivatedHostPlants13 WeedHostPlants13

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

PotsandMedia14 ToolsandEquipment14 RemovingAreasFromQuarantine14 RequirementsForPotatoCroppingSystemsUnderQuarantine15 PropertieswithPositiveTestingorAssociatedFields15 FieldsAdjacenttoPositiveTestingorAssociatedFields,orSharingWater15 StorageFacilities15 VolunteerPotatoesandCullPiles16 HarvestingEquipment,StorageAreas,andConveyances17 RegulatoryRequirementsofSeedProducers17 RegulatoryRequirementsforProcessors17 UseOfChemicals18

Chapter 6 Control

page 6-1 page 61

Contents1 Introduction1 LawsPertainingtoPestControl1 ControlRecords2 ControlDecisionsandOversight2 HandlingOfSamples2 PlantDisposalAndDestructionMethods3 BurningorIncineration3 SteamTreatmentUnderPressure3 SoilFumigation3 ApprovedLandfill3 DisinfectionProcedures4 ToolsandEquipment5 ExposedIrrigationSystems5 HoldingPonds5 OutdoorSoilorHoldingAreas5 ApprovedDisinfectants6

Chapter 7 page 7-1 EnvironmentalCompliance

page 71

Contents1 Introduction1 Disclaimer1 NationalEnvironmentalPolicyAct2 EndangeredSpeciesAct2 FederalInsecticide,fungicide,AndrodenticideAct3 OtherLaws3 EnvironmentalMonitoring3

Glossary page 81 References page 11 Appendix A page A-1 Resources page A1


DiagnosticToolsandEquipment1 EnvironmentalCompliance1 EnvironmentalServices1

Appendix B page B-1 FormsandQuestionnaires


vi

page B1
May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

Contents1 PPQForm391SpecimensForDetermination2 AddendumtoPPQForm3914 QuestionnaireForNurseryAndPotatoCroppingSystemOwnersOrManagers5 Location5 Questions5 PPQForm523EmergencyActionNotification7 Instructions8 Procedure8 ReleasingPlants8 DocumentingActionsTaken9 DisposalMemorandum9 PPQForm526PermittoMoveLivePlantPlantsorNoxiousWeeds10 11

Appendix C page C-1 DiagnosticValidationsandIsolationMethods

page C1

Contents1 Introduction1 StatusofCommercialDiagnosticKits1 Nonplate,Rapid,ImmunoassayDetection2 AdgenSpotCheckLFTM2 AgdiaImmunoStrip2 CSLPocketDiagnosticTM3 ImmunoplateAssays(ELISA)3 AgdiaPathoScreenELISA3 ComparisonOfNonplateImmunoassayAndPlateImmunoassay4 ValidationOfCentralScienceLaboratoryTaqmanAssay5 ValidationOfSampleAndDnaPreparationMethods5 PreparationOfMediaForIsolation6 SelectiveMediumforRalstoniasolanacearum6 SemiselectiveMediumforRalstoniasolanacearum6 IsolatingBacteriaFromTissueSamples8 MethodA8 MethodB8 MethodC8

Appendix D page D-1 DiagnosticLaboratories

page D1

Contents1 Introduction1 DeterminationOfRaceAndBiovar1 DeterminationOfGenusAndSpecies2 StateandCooperatingUniversityDiagnosticLaboratories3 PlantDiagnosticNetworksandPrivateLaboratories10

Appendix E page E-1 PelargoniumintheNurseryTrade page E1 Appendix F page F-1 ComparisonofSymptoms page F1 Appendix G page G-1 Disinfectants page G1

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

vii

viii

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

1
Ralstonia solanacearum r 3 b 2

Chapter 1
Introduction

Contents
Purpose page 11 Status page 12 DocumentComprehensiveness page 12 CommercialSuppliersOrProducts page 12 PreventionOfInfection page 12 ProgramSafety page 13 SupportForProgramDecisionmaking page 13

Purpose
UseNewPestResponseGuidelines:Ralstoniasolanacearumrace3biovar2asa guidewhendesigningaprogramtodetect,diagnose,contain,control,or eradicatethispathogen.Personnelwithinstatedepartmentsofagriculture andothersconcernedwithdevelopinglocalsurveyorcontrolprograms shouldfindtheGuidelinesuseful. UnitedStatesDepartmentofAgricultureAnimalandPlantHealthInspection ServicePlantProtectionandQuarantine(USDAAPHISPPQ)developedthe Guidelinesthroughdiscussion,consultation,oragreementwithstaffat USDAAgriculturalResearchService(ARS),universities,industries,andstate departmentsofagriculture. TheGuidelineswillbeupdatedasnewinformationbecomesavailable.Specific emergencyprogramsshouldbebasedoninformationavailableatthetimeof theincident.

Recent Updates
Significantchangestothisdocumentasaresultofnewinformationwillbe providedinthissection.Updatestothecurrentversionincludethefollowing: Thediagnosticlaboratorywithproperauthorizationstodofinal confirmatorytestingforRalstoniasolanacearumrace3biovar2isthe USDAAPHISPPQMolecularDiagnosticsLaboratory(MDL)in Beltsville,MD.MDLhasthenecessarypermitsandregistrationsto possessthisselectagent.SeeIdentificationonpage 41. GeographicDistributiononpage 27wasupdated.

1
May 12, 2008 Emergency and Domestic Programs Introduction.fm Ralstonia solanacearum r 3 b 2 1-1

Chapter 1 Status

Status
Ralstoniasolanacearumisaplantpathogenicbacteriumthatcauseswilt diseases.StrainsofRalstoniasolanacearumcauseeconomicdamagetodiverse agriculturalcropsandarewidespread(Hayward,1991;Sequeira,1994). Ralstoniasolanacearumrace3biovar2isofparticularconcernintheUnited Statesisbecauseofitsaffectoneggplant,geraniums,potatoes,andtomatoes. Whilerace1isendemictothesoutheasternUnitedStateswhereitcanaffect tomatocrops,Ralstoniasolanacearumrace3biovar2isnotknowntooccurin theUnitedStatesandisconsideredofquarantineimportance.

Document Comprehensiveness
Thisdocumentisnotintendedtobecompleteandexhaustive,butprovidesa foundationbasedupontheliteratureavailabletoassistfurtherwork.Some keyarticleswerenotavailableatthetimeofwriting,andnotallspecialists andmembersoftheresearchcommunitywereconsultedfortheiradvice.

Commercial Suppliers Or Products


Referencestocommercialsuppliersorproductsshouldnotbeconstruedasan endorsementofthecompanyorproductbyUSDA.

Prevention Of Infection
Tominimizetheeffectsofthisdiseaseandcontrolactionsthatmaybe necessary,regulatoryofficersandgrowersshoulddevelopandimplement effectivesanitationprocedurestoensurethatthepathogendoesnotspread withintheirgreenhouseornurseryfacilities,associatedsupportbuildings, equipmentorvehicles.

Regulatory Officers
Federalandstateregulatoryofficersmustconductinspectionsandapply controlmeasurestoensurethatthediseaseorpathogendoesnotspread withinorbetweengreenhousesornurseries,associatedsupportbuildings, equipment,vehicles,orfields,anddoesnotescapeintootherproduction systems.
Important Since inspectors could inadvertently spread Ralstonia solanacearum race 3 biovar 2 or other pathogens during the inspection process, federal and state regulatory officers conducting inspectionsbefore entering and upon leaving each greenhouse and nursery locationmust follow sanitation guidelines to prevent spreading contaminated plant material, soil, or water to other facilities. See Precautions on page 3-1 for more information on this topic.

1-2

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Chapter 1 Program Safety

Growers
Geraniumgrowerscanminimizetheimpactofthedisease,includingthe regulatoryimpact,byfollowingthesepractices: Submitfortestingplantsthatdisplaywiltsymptomsanddonotrecover afterwatering. Avoidsubirrigationandsharedwateringsystems. Usestrictsanitarypracticeswhenhandlingorpropagatingplants. Donotplaceplantsbeneaththedriplineofhanginggeraniums. Maintainvarietiesofgeraniumsfromforeignsourcesseparate. Disinfectwatersystemsandregularlytreatirrigationwaterfromany source. Keepgreenhouses,areasaroundgreenhouses,andirrigationwater holdingoroverflowpondsfreeofweeds. Domesticgrowerscanimplementthepracticestominimizecontaminationof rootedcuttingsforsaleintheUnitedStates. GrowerscanconsultthedocumentMinimumSanitationProtocolsfor OffshoreGeraniumCuttingProduction,developedbyUSDAAnimaland PlantHealthInspectionService(APHIS)andusedbyoffshoregeranium suppliers,formoreinformation.

Program Safety
Safetyofthepublic,andprogrampersonnel,isapriorityconsiderationin preprogramplanningandtraining,andthroughoutprogramoperations. Safetyofficersandsupervisorsmustenforceonthejobsafetyprocedures.

Support For Program Decisionmaking


USDAAPHISPPQCenterforPlantHealthScienceandTechnology (CPHST)providestechnicalsupporttoemergencypestresponseprogram directorsconcerningriskassessments,surveymethods,controlstrategies,and otheraspectsofpestresponseprograms.

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

1-3

Chapter 1 Support For Program Decisionmaking

1-4

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

2
Ralstonia solanacearum r 3 b 2

Chapter 2
Pest Information

Contents
Introduction page 21 Classification page 21 Importance page 22 History page 23 EconomicImpact page 26 Hosts page 26 GeographicDistribution page 27

Introduction
UseChapter2PestInformationtolearnmoreabouttheclassification, importance,history,hostrange,anddistribution,ofRalstoniasolanacearum race3biovar2.

Classification
UseTable 21asanaidtotheclassificationofRalstoniasolanacearumrace3 biovar2.
TABLE 2-1 Classification of Ralstonia solanacearum race 3 biovar 2 (continued)
Domain Phylum Class Order Family Strain Synonyms Approved common names (APS, 2003) Bacteria Proteobacteria Betaproteobacteria Burkholderiales Ralstoniaceae Ralstonia solanacearum (Smith 1896) Yabuuchi, et al. 1995 [race 3, biovar 2] Bacillus solanacearum (Smith 1896), Pseudomonas solanacearum (Smith) Smith 1914, Burkholderia solanacearum (Yabuuchi, et al. 1992) bacterial wilt (pepper, potato, tomato), southern wilt (geranium), brown rot (potato)

1
May 12, 2008 Emergency and Domestic Programs Pest Information.fm Ralstonia solanacearum r 3 b 2 2-1

Chapter 2 Importance

TABLE 2-1 Classification of Ralstonia solanacearum race 3 biovar 2 (continued)


Other common names bacterial wilt of potato, bacterial wilt of solanaceous crops, brown rot of potato, brown rot of solanaceous crops, southern bacterial blight of tomato, southern bacterial wilt (CABI, 2003)

Importance
Ralstoniasolanacearumisaplantpathogenicbacteriumthatcauseswilt diseases.StrainsofR.solanacearumcauseeconomicdamagetodiverse agriculturalcropsandarewidespread(Hayward,1991;Sequeira,1994) (Table 22).OfparticularconcernintheUnitedStatesisR.solanacearumrace3 biovar2becauseofitsaffectoneggplant,geraniums,potatoes,andtomatoes. Whilerace1isendemictothesoutheasternUnitedStateswhereitcanaffect tomatocrops,R.solanacearumrace3biovar2isnotknowntooccurinthe UnitedStatesandisconsideredofquarantineimportance.
TABLE 2-2 Host Range and Distribution of Strains of Ralstonia solanacearum [Adapted from Daughtrey (2003) and Denny and Hayward (2001)]
Race 1 2 3 4 5 Biovar 1, 3 or 4 1 2 3 or 4 5 Hosts Wide Musa spp. Potato, other Solanaceae, geranium, others Ginger Mulberry Geographic Distribution Asia, Australia, Americas Caribbean Islands, Brazil, Philippines Worldwide except United States and Canada Asia China

ThebacteriaofallracesofRalstoniasolanacearumcanbetransmittedthrough contaminatedsoil,irrigationwater,equipment,orpersonnel.Thebacteria maybespreadbythefollowingmeans: Sharedwaterirrigationsystems Transplantingandpropagatinginfectedplants Takingcuttingswithoutdisinfectingimplementsbetweenplants Pinchingbudsofplantswithoutsanitizing Transferofcontaminatedsoil(onequipmentorshoes)todiseasefree areas

2-2

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Chapter 2 History

Thepathogenisnotreadilyspreadbetweenplantsbythefollowingmeans: Splashingofwater Leaftoleafcontact Aerialtransfer Infectionoccurstypicallythroughtherootsandwoundinginrootareas,a normalphysiologicalprocessasrootletsgrow.Bacteriaarenormally concentratedinthelowerstemportionsoftheplant.Spreadcanbecontrolled ingreenhousesbythestrictapplicationofsanitationpractices. Thespeciesisdifferentiatedintofiveracesaccordingtohostrange (Buddenhagen,Sequeira,andKellman,1962),andintobiovarclassifications accordingtophysiologicaltests(Hayward,1964;Hawyard,1991).

Race 1
StrainsofRalstoniasolanacearumrace1biovars1and3havebeenfoundto occurongreenhouseornamentalsinthenorthernhemisphere(CABI,2003). Race1alsohasawiderhostrangeandisendemictothesoutheasternUnited States.

Race 3
Ralstoniasolanacearumrace3isfoundworldwideexceptintheUnitedStates andCanada.HostsofR.solanacearumrace3biovar2areusuallyrestrictedto cultivatedsolanaceousspeciessuchaspotatoandtomato.Race3hasbeen reportedoccasionallyoneggplant(Solanummelongena),pepper(Capsicum annum)andsolanaceousweeds(Martin&French,1995).Someinfectedweed hostsremainsymptomless.Theseweedhostsmayenablerace3biovar2to surviveinalatentformwithinthehostorintheirrootareasinthesoil(Janse, etal.2003).

History
Geranium Hosts
IntheUnitedStates,a1979studyofhosttestswithastrainofRalstonia solanacearumisolatedfromPelargoniumxhortorum(=P.zonalehybridsor geranium)indicatedthepresenceofrace3becausetheisolatewasnot pathogenicontobacco(Strider,etal.1981).However,somereportsoffindings ofR.solanacearumrace3biovar2onPelargoniumspp.fromWesternAustralia andTanganyikaarequestionable(Pittman,1933;Wallace,1934). In1995,KimandOlson(2003)receivedastrainofRalstoniasolanacearumrace3 biovar2fromaConnecticutsource.Thestrainwasisolatedfromageranium plantfromGuatemala.Theyconfirmedthepresenceofasimilarstrainon wiltedgeraniumplantsfromgreenhousesinPennsylvaniain1999and2000 andfromDelawarein1999;theplantswereimportedfromGuatemala,too.

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

2-3

Chapter 2 History

In1999,Ralstoniasolanacearumrace3biovar2wasreportedinNewJersey, NewYork,Ohio,Pennsylvania,SouthDakotaandWisconsinoncommercially growngeranium(Pelargoniumzonale)importedfromGuatemala(Hudelson,et al.1999and2002;Nameth,1999;SPRO,2002).Thesefindingsresultedina reviewofforeignsiteproductionfacilitypracticesthatexportgeranium cuttingstotheUnitedStates. Alsoin1999,thebacteriumwasdetectedintheUnitedKingdomonimported geraniumcuttingsproducedinKenyafortheEuropeanmarket(Janse,etal. 2003).DuringSeptemberDecember2000,symptomsofbacterialwiltwere observedinseveralgeraniumnurseriesinBelgiumandGermany(Janse,etal. 2003). InFebruary2003,geraniumplantsimportedfromKenyatotheUnitedStates wereimplicatedasthesourceofRalstoniasolanacearumrace3biovar2that resultedindetectionsin127individualnurseriesin27states.Importsfrom Kenyawerehaltedandphytosanitaryrequirementsimplementedforall geraniumimportsfromcountriesthathaveR.solanacearumrace3biovar2.In early2004,anotherdetectionwasmadeintheUnitedStates,thistimetraced backedtoproductiongreenhousesinGuatemala.Thisprogram,again conductedbyfederalandstateregulatoryofficials,resultedincontrolactions in453nurseriesin41states.Bothactionsresultedinthedestructionof approximatelytwomilliongeraniumplantseach. In2003and2004,detectionsintheUnitedStateshavebeenlimitedtowithin greenhouses.Theinfectionswerecontainedandplantsweredestroyed.The diseasewaseradicatedthroughindustrypracticesorcontrolprograms.
Current Status

Inlate2003,acertificationprogramforgeraniumsimportedtotheUnited Stateswasimplemented.Theprogramrequiresspecificcleanculturepractices andregulartesting.GeraniumplantsexportedtotheUnitedStatesrequire phytosanitarycertificationandastatementthattestingshowedgeraniums shippedtotheUnitedStatesarefreeofRalstoniasolanacearumrace3biovar2.

Solanaceous Hosts
In1880,astrainofbacterianowrecognizedasRalstoniasolanacearumrace 1infectedtobaccoinGranvilleCounty,NorthCarolina,causingsymptoms ofbacterialwilt1.Thediseasebecamesoseverethatgrowersexperienced lossesof25100%,andtheeconomiesofentirecommunitiesweredestroyed (MeltonandShew,2000).Atthattime,thediseasewasnamedGranvillewilt forthecountyinwhichitwasfirstobserved.Aroundthesametime,an unidentifieddiseasebegantoaffectsouthernpotatogrowersandtomato growersinMississippi(Kelman,1953).

Tobacco is not affected by race 3 (Janse, 1991).

2-4

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Chapter 2 History

Whenscientistsfirstrecognizedbacterialwiltsofsolanaceouscrops,they madenoattempttodifferentiatebetweenracesorstrains.Thehostdifferential testtoseparateRalstoniasolanacearumraces1,2,and3,wasdevelopedinthe 1960s;limitedinformationregardingracedesignationwasavailablebefore thattime. Sincethe1950s,scientistshaverecognizedthatinfectionsofRalstonia solanacearumraces1and3havehinderedmanysolanaceouscropsinboth tropicalandtemperateregions.Race1hascausedthegreatesteconomic damagetopotato,tobacco,andtomatocropsinthesoutheasternUnited States.InIndonesia,Brazil,Colombia,andSouthAfrica,races1and3caused thegreatesteconomicdamage(CABI,2006).InthePhilippinesduringthe period19661968,races1and3causedlossesof15%,10%,and25%,in tomato,pepper,andtobaccocrops,respectively(Zehr,1969). InIndia,infectionoftomatoeswithRalstoniasolanacearumraces1and3can resultinthecompletelossofacrop.InIsrael,Volcani&Palti(1960)reported thatpotatolossesweregreaterinthespringcropthantheautumncropdueto hightemperaturesduringtuberformationandmaturity.Extensivelossesof potatowerereportedinGreeceduringtheperiod19511953(Zachos,1957).
Current Status

Today,Ralstoniasolanacearumrace1isendemicinsomeareasofthesouthern UnitedStates,wheremanagementofthediseasehasreducedlossesto12% ofproductionor$1015millionannuallyintobacco,withsimilarlossesin tomatoandpepper. Ralstoniasolanacearum,thecausalagentofpotatobrownrot,hasbeen describedasoneoftheworldsmostdestructiveplantbacterium.Ithas affectedatleast3.7millionacresofpotatoesinmorethan80countries,with estimatedannuallossesofmorethan$950million(DEFRA,2003a).Losses duespecificallytoR.solanacearumrace3biovar2cannotbequantifiedsince reliabledataareunavailable.Nevertheless,intheearly1990sthispathogen causedseriouslossesintheEuropeanpotatoindustry. Duetorecentoutbreaks,insuranceclaimsintheNetherlandsin1998and 1999asaresultofpotatobrownrotcausedbyRalstoniasolanacearumrace3 biovar2wereestimatedatU.S.$2.7million(Meiners,2000).Inaddition,the U.S.floricultureindustryincurredsubstantiallosseswhenthebacteriumwas recentlydetectedandconfirmedingeraniumcuttingsimportedfromKenya andGuatemala.Foralimitednumberofnurseries,thecostofdisposaland replacementofrootedgeraniumplugsduetorace3biovar2wasestimatedto bearound$221,632,withtotallossesfor70nurseriesof$1.4million(L.Garret, USDAAPHISCPHSTeconomist,personalcommunication).However,the totalnumberofestablishmentsthatsufferedlossesduetoR.solanacearumrace 3biovar2isunknownatthistime,thusaccuratelossesduetothisbacterium cannotbecalculated.

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

2-5

Chapter 2 Economic Impact

Estimatedlossestothepotato,tomato,pepper,eggplant,andgeranium industriesaregreat.Insolanaceouscrops,economiclossesduetoRalstonia solanacearumcanbeaslowas2%,ormayreach100%incertainregionsofthe world,suchasSoutheastAsia(CABI,2006).Combinedlossestopotato, tomato,pepperandeggplantcropsthatarelikelytobeaffectedbyR. solanacearumrace3biovar2canreach$70,191,390annually2.Theseestimates arebasedupontotalvalueoftheacresplantedtothesecommoditiesin2001 (ERS,2003).

Economic Impact
Geranium Hosts
LikeXanthomonaspelargonii,infectionbyRalstoniasolanacearumrace3biovar2 cancausemortalityingeraniums.However,growerscanmanageRalstonia solanacearumbycullingandfollowingpropergreenhousesanitationpractices. TheeconomicimpactofRalstoniasolanacearumrace3biovar2ongrowers, importers,anddistributorsintheUnitedStatesisduetotheregulatory actionsnecessarytoeradicatethediseaseoncedetected.

Solanaceous Hosts
Ralstoniasolanacearumrace3biovar2isaseriouspathogenthatcausesbrown rotorbacterialwiltofpotato(Hayward,1991;Janse,1996).Bacterialwiltof potatohasbeenestimatedtoaffect3.75millionacresin80countries.Annual globaldamageestimatesexceed$950million(DEFRA,2003a).Itisadaptedto coolertemperaturesandwouldbeparticularlydamagingtopotato productionregionsoftheUnitedStates. Whilerace1causeslossestotomatocropsinFlorida(Momol,etal.2003),the affectofrace3ontomatoesandothersolanaceouscropsintheUnitedStatesis unknown.Ralstoniasolanacearumrace3biovar2causesmortalityingeranium plants,butXanthomonaspelargoniiisagreaterconcernofgrowers.However, theregulatoryaffectcanbemoresignificantforgrowerswiththediseasein theirfacility.

Hosts
Excludinggeranium(Pelargoniumspp.),mosthostsofRalstoniasolanacearum race3biovar2areinthefamilySolanaceae.Themajorcrophostplantspecies includepotato,tomato,peppersandeggplant.Othercrophostshavebeen implicatedbylaboratorytesting.WeedhostsincludemanySolanumspp.See Janse,etal.(2003)foracompletereviewofhosts.SeePelargoniuminthe NurseryTradeonpage E1formoreinformationonnamingofgeraniums.

Excluding geranium, and based upon a minimal damage of 1.5%.

2-6

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Chapter 2 Geographic Distribution

Geographic Distribution
Geographicdistributionrecordslistedbelowareofconfirmedrace3orof presumedrace3basedonpathogenicitytopotatoandtomato(CABI,2003). North AmericaMexico(CABI,2003). EPPO RegionBelgium,Estonia,France,Germany,Hungary,Ireland, Netherlands,Latvia,Lebanon,Lithuania,Moldova,RussianFederation, Slovakia,Slovenia,UnitedKingdom,FormerYugoslavia,FormerUSSR (CABI,2003). AsiaBangladesh,China(Fujian,GuangdongGuangxi,Hebei,Jiangsu, Taiwan,Zhejiang),India(HimachalPradesh,MadhyaPradesh,Maharashtra, Manipur,Meghalaya,TamilNadu,Tripura,UttarPradesh,WestBengal), Indonesia(Java),Iran,Japan(Kyushu),Nepal,Pakistan,Philippines,SriLanka (CABI,2003),RepublicofKorea(NIAST,2002),Malaysia(Possier,etal.,1999), Taiwan,Thailand(HoritaandTsuchiya,2001). AfricaBurundi,Egypt,Ethiopia,Kenya,Libya,Nigeria(Cook,etal.1994), Runion,Rwanda,SouthAfrica,Uganda(Hayward,1964;Tusiime,etal.1998), Zaire,Zambia,Zimbabwe(CABI,2003). South AmericaArgentina,Bolivia,Brazil(Goias,Parana,Pernambuco,Rio GrandedoSul,SantaCatarina,SaoPaulo),Chile,Colombia,Peru,Uruguay, Venezuela(CABI,2003). Central America and CaribbeanCuba(French,1998),CostaRica, DominicanRepublic,ElSalvador(French,1998),Guadeloupe,Guatemala, Haiti,Honduras,Nicaragua,Panama,WestIndies(CABI,2003). OceaniaAustralia(NewSouthWales,SouthAustralia,Victoria)New Caledonia,PapuaNewGuinea(CABI,2003).

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

2-7

Chapter 2 Geographic Distribution

2-8

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

3
Ralstonia solanacearum r 3 b 2

Chapter 3
Survey Procedures

Contents
Introduction page 31 Precautions page 31 DetectionSurvey page 32 DataCollection page 315 DelimitingSurvey page 36 TracebackAndTraceForwardSurvey page 37 MonitoringSurvey page 39 VisualInspectionForDetectionSurvey page 310 VisualInspectionForDelimitingSurvey page 314

Introduction
UseChapter3SurveyProceduresasaguidetoconductingasurveyforRalstonia solanacearumrace3biovaringeraniums,potatoes,andothersolanaceous hosts. ConductadetectionsurveytodetermineifRalstoniasolanacearumrace3biovar ispresentorabsentinadefinedgreenhouse,nursery,orfieldarea.Aftera newdetectionintheUnitedStates,orwhenadetectioninanewareais confirmed,conductadelimitingsurveytodefinethegeographiclocationof thedisease.Conductamonitoringsurveyifyouhaveappliedacontrol procedureandneedtomeasureitseffectiveness.

Precautions
Whenvisitingsitestoconductsurveysorcollectsamples,regulatoryofficials musttakestrictmeasurestopreventcontaminationbyplantpathogens betweengreenhouses,nurseries,andpotatofacilitiesduringinspections. Acceptablemethodsofprotectionincludetheuseofaneffectiveantimicrobial soap,lotion,ordisinfectantusedaccordingtothelabelinstructions,or disposableglovesandbootieschangedbetweenvisits.SeeDisinfection Proceduresonpage 64formoreinformationondisinfectionoftools, irrigationsystems,ponds,andsoil.

Antimicrobial Soap, Lotion, or Disinfectant


Washhandswithanapprovedmicrobialsoap.Ifnotusingamicrobialsoap, washhandswithregularsoapandwarmwatertoremovesoilanddebris. Afterward,useanalcoholbasedantimicrobiallotionsuchasPurelloran

May 12, 2008 Emergency and Domestic Programs Survey Procedures.fm

Ralstonia solanacearum r 3 b 2

3-1

Chapter 3 Detection Survey

equivalentproductthatcontains63%ethylalcohol.Purellhasdemonstrated efficacyagainstvariousresilientPseudomonasspp.andsoiseffectiveagainst Ralstoniasolanacearum. Ifhandsarefreeofsoilordirt,analcoholbasedantimicrobiallotioncanbe appliedwithoutwashing.Unlikesomeantimicrobialsoaps,anantimicrobial lotionislesslikelytoirritatethehandsandtherebyimprovecompliancewith handhygienerecommendations.

Disposable Gloves and Footwear


Ifsoapsarenotused,thendisposablelatexornitrileglovescanbeusedasan alternative.Glovesmustbechangedbetweenvisitstogreenhouses,nurseries, andpotatofacilities. Footwearshouldbedisinfectedusingafootbathorotherappropriatemethod whenmovingbetweengreenhousesornurseries.Iffootbathsarenotavailable usedisposablebootiesavailablefromhospitalsuppliers.Bootiesmustbe changedbetweenvisitstogreenhouses,nurseries,andpotatofacilities.

Detection Survey
Thepurposeofadetectionsurveyistodetermineifapestexistsinanarea. Thiscanbeextremelybroadinscopeaswhenassessingthepresenceofthe diseaseinawideareaoritcanberestrictedtodiscoveringifaspecificpestis presentinacertainarea. Basedstrictlyonanegativeresultinadetectionsurvey,itisnotvalidtoclaim thatapestdoesnotexistinanareaifresultsarenegative.However,negative resultsarevaluableforprovidingcluesastomodeofdispersal,temporal occurrence,orindustrypractices,particularlywhenconsideredwithresults fromsimilarareasorproximities.
Important Detection surveys for geraniums infected by Ralstonia solanacearum race 3 biovar or other cultivated hosts in nurseries should be conducted by state inspectors in conjunction with federal PPQ inspectors. PPQ inspectors may inspect nurseries without a state inspector if they have permission of the nursery owner or manager and have advised the state of their visit.

Procedure
FollowthisprocedurewhenconductingadetectionsurveyforRalstonia solanacearumrace3biovar2: 1. Usevisualinspectiontoexaminecultivatedhostplants(geraniums, potatoes,andothersolanaceoushosts)andsolanaceousweedsfor wiltingsymptoms.Besuretoincludeplantsinthefieldthatwere startedingreenhousesornurseriesinassociationwithimported geraniums.SeeVisualInspectionForDetectionSurveyonpage 310 formoreinformationoninspectionprocedures.

3-2

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Chapter 3 Detection Survey

2. Collectsamplesofsymptomaticplants.SeeProcedureForSampling AndIdentificationonpage 43forinstructions. 3. Submitsamplesfortesting.SeeStorageandShippingonpage 44for instructions. 4. UseTable 31todetermineiffurtheractionisrequired.


TABLE 3-1 Decision Table for Testing Plant Samples in a Detection Survey
And conduct further inspection of the following: Suspect geranium and potato shipments from infested greenhouses or fields, respectively, on hold. See Delimiting Survey on page 3-6 for more information. Any other hosts in nurseries suspected of having material at risk for R. solanacearum race 3 biovar 2. See Delimiting Survey on page 3-6 for more information.

If: Samples test positive for the species Ralstonia solanacearum by ELISA or culture

And: Their origin is in the southern areas of the United States where race 1 occurs1

Then: Conduct monitoring and further tests at a qualified, authorized diagnostic laboratory to determine race and biovar. See Monitoring Survey on page 3-9 and Identification on page 4-1 for more information.

Samples test negative for the species Ralstonia solanacearum by ELISA or culture
1

Release plants from regulatory control.

While race 1 of Ralstonia solanacearum occurs in the southeastern United States on many of the same hosts as race 3, it is indistinguishable from race 3 without further diagnostic testing.

Suspectshipmentsofgeranium,orpotatoandothersolanaceouscrops (includingvegetabletransplants),arethoseheldbecauseoftheirassociation withapositivedetectionfromanothersourcecountry,rootingstation, nursery,potatoorothersolanaceouscropfieldorpotatostoragefacility.Be suretofollowsanitationproceduresduringnurseryvisits.SeePrecautionson page 31formoreinformationconcerningsanitation.

Geranium Hosts
Usevisualinspectiontoconductadetectionsurveyingreenhouseand nurseryfacilities.SeeVisualInspectionForDetectionSurveyonpage 310 formoreinformationonthistopic. Randomsamplingofgeranium(andpotato)plantslackingsymptomsisnot recommendedatthistimebecauseofalackofsamplingmethodologyand processingcapability.Duetolatency,infectedplantsmaynotshow symptoms.Thereiscurrentlynoreliabletestforasymptomaticplants, thereforesymptomologyisthemostreliableindicatorofpossibleinfection.1
May 12, 2008 Emergency and Domestic Programs Ralstonia solanacearum r 3 b 2 3-3

Chapter 3 Detection Survey

Solanaceous Hosts
Usevisualinspectiontoconductadetectionsurveyinsolanaceoushosts. DetectionsurveysforRalstoniasolanacearumrace3biovar2inpotatogrowing areascanbegeneral,ortargetedtoareasconsideredtobeatgreaterrisk.See VisualInspectionForDetectionSurveyonpage 310formoreinformation.
General Survey

Conductageneralsurveyatlowrisksites.Lowrisksitesincludefieldswith littleornohistoryofsolanaceoushostproduction. Detectionsurveysinpotatoes,tomatoes,andothersolanaceoushosts,should betargetedatparticularcropproductionpracticeswherethestageof productionorgeographicareamaypresentsomeriskofinfectionandspread ofthedisease.


Proximity of Potatoes to Historically Positive NurseriesConductsurveys

Targeted Survey

inpotatofieldsif: Localgreenhouseseitherimportedgeraniumsthattestedpositivefor thepathogenorreceivedsuspectvarietiesofgeraniumsassociatedwith importedgeraniumswhichtestedpositiveforthepathogen. Potatofieldsarelocateddownstreamoradjacenttopositivefacilities thathadholdingorrecirculatingpondsforirrigationwater. Potatofieldsmayhavebeenirrigatedwithsurfacewaterorotherwater sourcesdownstreamfrompotatoprocessingplantswheretheeffluent fromprocessingisnottreated.


Tomato and Other Solanaceous HostsSimilartopotatofieldsurveys,

visualobservationsofwiltintomato,pepper,andeggplantcropscanbeused indetectionsurveys.Conductsurveysoftomatoandothersolanaceoushosts if: Hostsweregrowninthesameproductiongreenhousesasimported geraniums,sincetheymayrepresentahighriskpathwayfor introducinginfectionintoafieldorhomegarden. Fieldsarelocateddownstreamornearnurseryfacilitiesthathad importedgeraniumswhichtestedpositiveforRalstoniasolanacearum race3biovar2,orhadvarietiesofsuspectgeraniumsthatwere associatedwithimportedgeraniumsthattestedpositiveforthe pathogen.
Tomato TransplantsTomato,eggplant,andpepperplantsfor

commercialproductionareoftengrownastransplantsinonearea andshippedtoanotherareaforfieldplanting.Theseavenuesof

If available, testing of asymptomatic potato tubers might prove to be more reliable. Latent infections may be present for several years before field symptoms become visible. In the absence of symptoms, if a positive detection occurs in potato seed certification screening, normal traceback or trace forward investigations will indicate potential high-risk sites to survey and test. A potato sampling scheme over a large area may be the most efficacious method of sampling.

3-4

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Chapter 3 Detection Survey

potentialpathogendisseminationcanbemonitoredforthepresence ofwilt.Sendsamplesofsymptomatichostplantstoadiagnostic laboratorytodetectRalstoniasolanacearumrace3biovar2.


Potato Seed TubersThemostlikelywayforRalstoniasolanacearumrace3

biovar2tospreadinpotatogrowingareasisthroughinfectedtubersusedfor seed.Seedtubersareregularlysampledandscreenedasapartofpotatoseed certificationprograms.Theseprograms,conductedatvariousinstitutions undertheauspicesofstateuniversityorstatedepartmentsofagriculture, assurethatcommercialpotatoseedtubersareinspectedforavarietyofpotato diseasesbeforereceivingcertification. Seedpotatoinspectorsregularlylookforbacterialringrot(Clavibacter michiganensesubsp.sepedonicus).LikeRalstoniasolanacearumrace3biovar2, diagnosticlaboratorytestsareanintegralpartofconfirmingthepresenceof bacterialringrotinsymptomatictubers.Ifdiagnostictestingisperformedto confirmthepresenceorabsenceofbacterialringrot,testsshouldalsobe conductedtoruleoutthepossibilitythatR.solanacearumrace3biovar2isthe causalagent.
Solanaceous Weed HostsRalstoniasolanacearumrace3biovar2cansurvive

inwaterwaysandonweedhosts,includingblacknightshade(Solanumnigrum L.)andbitternightshade(S.dulcamaraL.)(Figure 31).Forexample,bitter nightshadegrowinginriverwaterinEuropeactedasasymptomless, secondaryhostinwhichthepathogenoverwinteredandmultipliedtofurther contaminateirrigationwatersupplies(Janse,1996).Inconjunctionwith detectionandmonitoringsurveys,collectpotentialweedhostsandsendto theappropriatediagnosticlaboratory,aswithcultivatedhostsofR. solanacearumrace3biovar2.Inparticular,weedhostsshouldbecollected aroundholdingpondsandrunoffareasinornearfieldswithhoststhathave testedpositive.

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

3-5

Chapter 3 Delimiting Survey

FIGURE 3-1 Bitter Nightshade, a Weed Host of Ralstonia solanacearum race 3 biovar 2 [Image courtesy of Joel Floyd, USDAAPHISPPQ]

Delimiting Survey
AfteranewdetectionintheUnitedStates,orwhenadetectioninanewareais confirmed,conductadelimitingsurveytodeterminetheextentofthe infestationorsuspectareasinwhichtoconductfurtherinvestigations.

Geranium Hosts
Commercialproductionofgeraniumsislimitedtogreenhouses,nurseries, andretailgardencenters.Lookforplantsconnectedtocirculatingirrigation systems,waterrunoff,andcollectionponds,todetermineiftheinfectionhas spreadtonewareasonoroffthegrowerspremises. Tracebackandtraceforwardinvestigationswillhelpdeterminenewfacilities needinginspection.Developlistsofpotentiallyinfectedfacilitieswithinand outsideastate.Theselistsarenottobesharedoutsideregulatorycooperators. Iftraceforwardstoresidencesfrompositivenurseriescanbeidentified,then thesegeraniumsshouldalsobeinspectedwhenpossible.

Solanaceous Hosts
List of Facilities

Alistoffacilitiesassociatedwithfieldgrownpotatoesfromlotstesting positiveforRalstoniasolanacearumrace3biovar2willbecompiled.Stateswill distributetheliststofieldoffices.


Important Do not share lists of facilities with individuals outside our regulatory cooperators. Grower names and field locations on these lists are strictly confidential, and any distribution of lists beyond appropriate regulatory agency contacts is prohibited.

3-6

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Chapter 3 Traceback And Trace Forward Survey

Working with Growers or Farm Managers

Whennotifyinggrowersorfarmmanagersonthelist,besuretoidentify yourselfasaUSDAorstateregulatoryofficialconductinganinvestigationof facilitiesthatmayhavereceivedRalstoniasolanacearuminfectedmaterial. Itisimportanttounderstandthattherearemanydifferenttypesofmarkets andfacilities(i.e.,seedlots,processing,orfreshmarket)thatmayhave handledinfestedandpotentiallyinfestedplantmaterialandsoil.Some growersoperateonhundredsofacreswithmanydifferentfieldsizesandseed lots,andstoretubersinhugewarehousesforlongperiodsoftime.


ProcedureFollowthesestepswhenconductinganinvestigation:

1. Requestthatthegrowerormanagerproduceanydocumentation includinginvoices,shippinglistsassociatedwiththepositivetested suspecttubersonthelistoffacilities. 2. Makecopiesoftherecords. 3. Locateanysuspectmaterialstillonthepremises. 4. Takephotographsoftheareawheresuspectplantmaterialislocated. Includephotographsdepictingconditions,includingtheproximityof neighboringplants,thatmayindicatebroadercontamination. 5. Makearecordofthenumberofplants,type,andlocationincludingan accountingofsuspectplantsfromthatshipmentnotonthepremises. 6. Inspecttheareaforthepresenceofcullpilesanddetermineifany harvesting/processingequipmenthasbeensharedwithotherfarms.

Traceback And Trace Forward Survey


Usetracebackandtraceforwardsurveystodetermineprioritiesfordelimiting surveyactivitiesafteraninitialU.S.detection.Tracebacksurveysattemptto determinethesourceofinfection.Traceforwardsurveysattempttodefine furtherpotentialdisseminationthroughmeansofnaturalandartificialspread (commercialorprivatedistributionofinfectedplantmaterial).Onceapositive detectionisconfirmed,surveysareconductedtodeterminetheextentofthe infestationorsuspectareasinwhichtoconductfurtherinvestigations.

Traceback Survey
Analyzing Information

Tracebackinformationgatheredfromseedlottagsandinvoicescanbeusedto determinetheoriginoftheseedtubers.Withtimelysubmittedrecordsfrom growersandtuberstoragefacilities,planningstaffsonsitecanconstruct prioritizedlistsforfurthersurveys.Informationavailablefromlocalwater companiesorfarmorganizationscanbeobtainedtoconstructmapsofwater sources,irrigationchannels,andconnectionsinareaswithsuspecttubersand potentiallyinfectedtubers.Usethisinformationtoassesstheneedforfurther surveysoffieldsassociatedwithirrigationwater.

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

3-7

Chapter 3 Traceback And Trace Forward Survey

Positive Detection in Potatoes

Tracebacksurveysforpositivepotatodetectionsshoulddeterminethesource ofinfectionbyexaminationoranalysisofthefollowing: Certifiedseeddocumentation Potatostoragefacilities Irrigationpracticesandwatersources Potatoprocessingplantsandwaterhandlingpractices Proximitytogreenhouseoperationshousingimportedgeraniums Anyotherpotentialmovementofplantmaterial,water,ormachinery thatcouldcontributetotracingthesourceofcontamination

Trace Forward Survey


Targetseedortubersforconsumptionassociatedwithpositivetestinglots. Consideranydistributionchannelsorirrigationwaterthatmightbepathways forfurtherdisseminatingthepathogen,includingbutnotlimitedtothe following: Associatedseedlotsonafarm,instorageareas,orbins,thatmayhave comeincontactwithpositivetestinglots Harvestingequipmentorothervehiclemovementhistoryinfieldsand nearbyfieldsplantedwithpositivetestedlots Irrigationorassociatedwaterwaysrunningtootherareasfromfields withpositivetestinglots Weedhostsaroundcontaminatedwaterwaysassociatedwithpositive testingfields
Water Sources and Irrigation Methods

Forfieldsthattestpositiveoraredirectlyassociated,gatherinformationon irrigationwaterdistributionsystemsdocumentingthewatersourceforeach area.Thisinformationcanbeobtainedfromlocalcommercialwater companiesorirrigationdistrictorganizations.Mapsofirrigationdistribution systemsshouldbeavailabletooverlayonagriculturefieldmaps.


Water SourcesWatersourcescanincludethefollowing:

Drylandfarmingwithoutirrigationsystems Groundwater,whichispumpedfromwells Surfacewater,distributedthroughcanalsandirrigationditches


Irrigation MethodsIrrigationmethodscanincludethefollowing:

Flood Centerpivot Solidsetwithpipeandfixedrisersforsprinklers Siderollwithpipesandsprinklersthatcanberolledonlargewheels

3-8

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Chapter 3 Monitoring Survey

Investigations at Processors

Thefollowinginformationcanbeusedtodeterminetheextentofdistribution ofsuspecttubersorcontaminationbycontaminatedwatertovariouslocations intheUnitedStates.CoordinatewithyourStatecontactsandregionalofficeto gatherthisinformation.Additionalresourcesmaybeneededtoquickly gatherthisinformationandanalyzeitinordertoholdsuspecttuber shipmentsandpotentiallyinfectedmaterial.


Destination of PotatoesDeterminethedestinationofpotatoesfromknown

positivefieldsbydocumentingdistributionchannelsthatincludewashing facilitiesfortableconsumptionpotatoesandprocessors.Washingoperations canoftenincludeflumes,orwaterwithinaprocessingplantusedtocarry potatoesfromoneareatothenext.Thewaterfromflumesisoftenrecirculated andheldinpondsortanksontheproperty.


Movement of WaterDocumentthemovementofwaterwithinafacilityand

taketuberandwatersamplesatfacilitiesthathandledsuspectandpotentially infestedtubers.
Processing EnvironmentDeterminetheprocessingmethods,temperatures

tubersaresubjectedto,andthedispositionofwastematerialfromthe processingplant.Ifwastematerialisariskforfurtherdisseminationofthe pathogen,itwillbesubjecttoregulatoryactions.

Monitoring Survey
Conductamonitoringsurveyifyouhaveappliedacontrolprocedureand needtomeasureitseffectiveness.SeeControlonpage 61formore informationoncontroloptions.

Geranium Hosts
Onceanurseryhasdestroyedallsuspectgeraniumsanddisinfectedgrowing areas,followupmonitoringisnecessary.Besuretofollowsanitation proceduresduringnurseryvisitsinamonitoringsurvey. Visualinspection,duringthesameorsubsequentgrowingseason,is appropriateinordertoexaminehostsforsymptoms.Ifsamplingprotocolsare available,samplingofwaterfromirrigationsystemsorpondsmayalsobe includedwithadditionalinspectionsofhostcropareasinrunoffareas adjacenttopositivetestingnurseries.SeeVisualInspectionForDetection Surveyonpage 310andVisualInspectionForDelimitingSurveyonpage 314formoreinformationoninspection.

Solanaceous Hosts
OnceRalstoniasolanacearumrace3biovar2hasbeenconfirmedfroma particularfield,andinfectedandpotentiallyinfectedplantshavebeen destroyed,followupmonitoringwillbenecessary.Usethefollowingtools: Visualinspectioninthefield

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

3-9

Chapter 3 Visual Inspection For Detection Survey

Collectionofsamplesfromcullpiles,soil,potentialweedhosts,and water,forseveralyearsandmultipletimesperseason SeeVisualInspectionForDetectionSurveyonpage 310andVisual InspectionForDelimitingSurveyonpage 314formoreinformationon inspectionofhostplants.

Visual Inspection For Detection Survey


Greenhouse and Nursery Crops
UsevisualinspectionasatoolwhensurveyingforRalstoniasolanacearumrace 3biovaringreenhouseandnurserycrops. Conductavisualinspectioninnurseryfacilitiesbylookingforplantswith typicalwiltingsymptoms.Theabsenceofwiltsymptoms,however,doesnot necessarilymeanRalstoniasolanacearumrace3biovar3isnotpresentinthe facility.Recentinformationsuggeststhatsomeinfectedplantsmaynot expresssymptoms,evenwhenheldforprolongedtemperatures.This conditionisknownaslatency.
Geraniums

WiltingsymptomsingeraniumscausedbyRalstoniaspp.aresimilartowilting symptomscausedbyotherbacterialpathogenssuchasXanthomonas campestrispv.pelargonii,theagentofbacterialblight. Therefore,whenwiltingsymptomsarepresent,donotmakeafielddiagnosis suchasforXanthomonascampestrispv.pelargonii.Alwayscollectasamplefor testingattheappropriatediagnosticlaboratory.SeeComparisonof Symptomsonpage F1forphotosoftypicalwiltingsymptomsingeraniums causedbybothorganisms.


Ralstonia solanacearum race 3 biovar 2Theprimarysymptomin geraniumsofinfectionbyRalstoniasolanacearumrace3biovar2iswiltingof leavesand/orabnormalyellowingoflowerleaves.Xanthomonascampestrispv. pelargoniicausesthesamesymptoms,aswellasleafspots.

PlantsinfectedbyRalstoniaspp.alsoexhibitthefollowingsymptoms: Bacterialstreamingwhensymptomaticplantsareplacedinwater Vasculardiscolorationofthestem Rootschangingtoabrowncolor


Xanthomonas campestris pv. pelargoniiIngeraniums,Xanthomonas campestrispv.pelargoniicauseswiltingofleavesand/orabnormalyellowingof lowerleaves,aswellasleafspots.However,vasculardiscolorationisless pronouncedorabsent,androotsremainwhite.

3-10

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Chapter 3 Visual Inspection For Detection Survey

Other Hosts in Greenhouses or Nurseries

Visualsurveysofotherhostsingreenhousesornurseriesshouldbeconducted duringgeraniuminspections.Symptomologyinotherinfectedhostssuchas tomato,eggplant,andpeppermayalsoincludeseverewilting.Weedhosts, however,generallydonotdisplaywiltsymptoms. Inaddition,inspectnurserywasteareasfordiscardedplants.Askthenursery ownerormanagertoidentifycullpiles.Examinethemforthepresenceof recentlydiscarded,wiltingordeadgeraniumplants.Discardedhostplants thatarenotcompletelydriedupcanstillbepreparedforsamplesubmission. Documentthesourceofthesample.

Discarded Plants

Potato Cropping Systems


Ralstoniasolanacearumrace3biovar2isreadilycarriedinirrigationwater. Therefore,itsspreadinapotatofieldcanberapidandevidentbylimitedor widespreadwiltingofpotatoplantsinafield.Symptomsmaybewidespread orconcentratedinareasofafieldwherewateraccumulates.
Potato Plants

Conductvisualsurveysofpotatofieldsduringperiodswhentemperatures arehighandsymptomsofbacterialwiltwillbemostobviousifpresent.
EarlySymptomsofbacterialwiltinmaturepotatoplantsthatresultfrom

infectionbyRalstoniasolanacearumrace3biovar2mayinitiallyincludethe following: Wiltingofleavesduringthedaywithrecoveryatnight(Figure 32) Someplantswithonlyoneorafewbranchesexhibitingwiltintheearly stages

FIGURE 3-2 Left: Wilted Potato Plant with Symptoms of Brown Rot, Infected by Ralstonia solanacearum race 3 biovar 2. Right: Healthy Potato Plant [Image courtesy of Centro Internacional de la Papa, Lima, Peru] LateMoreadvancedinfectioncanresultinthefollowingsymptoms:

Entireplantwiltswithoutrecovery. Wiltedleavesdonotdropfromtheplantandcanmaintaintheirnormal greencolorandsize. Infectedfoliageisyellowandstunted.

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

3-11

Chapter 3 Visual Inspection For Detection Survey

Leavesturnbrownanddry. Infectedstemcutincrosssectionshowsglisteningbeadsofdarkgray, slimyooze. Symptomaticstemcutatgroundlevelmayshowawhitishexudateat thecutsurface. Cutstemplacedinwaterproducesvisiblebacterialstreamingfromthe xylem. Continueddevelopmentofthebacterialinfectionwithinthexylem systemeventuallyresultsindeathoftheplant.


Bacterial Ring RotBacterialringrot,Clavibactermichiganensesubsp.

sepedonicus,occursinpotatogrowingareasoftheUnitedStatesandisanother bacterialdiseaseofpotatothatcauseswiltsymptoms. Thesymptomsofbacterialringrotdifferfrombacterialwiltbyoccurring typicallyontheuppermostleavesorflowerontheplantfirstandcausinga slightrollingatthemarginswithsomelossofcolor.Astheinfection progresses,leaftissuebetweenveinsturnsyellow.Eventually,entireleaves andstemsbecomeyellow,brittle,anddie.Infectioncanbelimitedtooneor twostemsonahill. Whilethesymptomologyofthesetwobacterialdiseasesofpotatomaybe different,thepathogensmustbediagnosedwithlaboratorytesting. Additionally,inareasofthesouthernUnitedStateswhereRalstonia solanacearumrace1exists,asamplingofwiltedplantspositiveforR. solanacearumwillrequirefurthertestingtotheraceandbiovarlevelat qualified,authorizeddiagnosticlaboratories.
Other DiseasesOtherdiseases,aswellasdrought,cancausesymptomsof

wiltthataresimilar,sodiagnostictestsmustbeperformedonsamplesfrom symptomaticplantsinordertoconfirmthepresenceofRalstoniasolanacearum race3biovar2.SeeIdentificationonpage 41formoreinformation.


Potato Tubers

Intubersforseedorconsumption,symptomsofRalstoniasolanacearumrace3 biovar2infectionincludethefollowing: Grayishbrowndiscolorationcanbeseenthroughthetuberperiderm. Bacterialoozeaccumulatesaroundthetubereyesoratthestolonend. Soiladherestoooze. Inmoreadvancedlevelsofinfection,squeezingtubermaycausemore bacterialoozetobeexuded(Figure 33). Distinctgrayishbrownvascularringisvisiblewheninfectedtubersare cutincrosssections;canextendintothepithorcortexofthexylem tissue(Figure 33). Applyingpressuretotuberscutinhalfforcesgrayishwhitedropletsof bacterialslimeoozefromthevascularring.

3-12

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Chapter 3 Visual Inspection For Detection Survey

Infectedtubersleftinthesoilcontinuetodecayandsecondary organismsinvadetobreakthemdown.

FIGURE 3-3 Symptoms of Brown Rot on Potato Tubers Exhibiting External Discoloration and Internal Infection with Ooze Exuding around the Vascular Ring of a Tuber cut in Cross-section [Image courtesy of Centro Internacional de la Papa, Lima, Peru]

Tomatoes and Other Solanaceous Crops


Inafield,wiltingandothersymptomsmayappearinseveralplantsorall plantsinarowinafurrowirrigatedcrop.Wiltingmayalsoappear concentratedinplantsinlowareasofthefieldwherewatercanaccumulate. Thefollowingsymptomscanbefoundontomatoandothersolanaceous plantsinfectedwithRalstoniasolanacearumrace3biovar2: Wiltedleavesdonotdropfromtheplantandcanmaintaintheirnormal greencolorandsize. Infectedfoliagecanturnyellowandstunted. Severewiltcancauseleavestoturnbrownanddry. Infectedstemcutincrosssectioncanshowglisteningbeadsofdark gray,slimyooze. Symptomaticstemcutatgroundlevelmayshowawhitishexudateat thecutsurface. Cutstemplacedinwaterproducesvisiblebacterialstreamingfromthe xyleminfectedstem. Continueddevelopmentofthebacterialinfectionwithinthexylem systemeventuallyresultsindeathoftheplant.

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

3-13

Chapter 3 Visual Inspection For Delimiting Survey

Visual Inspection For Delimiting Survey


Greenhouse and Nursery Crops
Followthesamemethodasvisualinspectionfordetectionsurveys.See GreenhouseandNurseryCropsonpage 310.Laboratorydiagnosis accordingtoprescribedproceduresisnecessarytodeterminethecauseof wiltingsymptomsifbacterialinfectionissuspected.

Potato Cropping Systems


Inspectpotatofieldswhentemperaturesarehigh,whensymptomsof bacterialwiltaremostobviousifpresent.Surveysshouldincludethepositive fieldaswellasadjacentfields,thoseonasharedwatersupply,andfieldsthat havehadcontactwithpotentiallycontaminatedequipment. ThedistributionofRalstoniasolanacearuminapotatofieldcanbespotty. Ralstoniasolanacearumiscommonlyfoundinareasofthefieldwithpoor drainage. Delimitingsurveysshouldincludethefollowing: Visualinspectionofplantsforsymptoms,especiallyinwetareasofthe field Visualinspectionofweedhosts Samplingofsoilandtubersnearinfectedplants Inspectionofplantsneardrainagecanalsorirrigationrigs Tracebackandtraceforwardinvestigationofseedpotatoesusedin indexfield Compilationoffieldhistoriesoftheindexandcontactfields Sendplantandsoilsamplesfortestingtotheappropriatediagnostic laboratory.SeeDiagnosticLaboratoriesonpage D1foralistoflaboratories.
Tuber Sampling

Samplingisnotrequiredforfieldsorfacilitieswithadirectlinktopositive testingseedlotsorfields.Thetubersinthiscategoryareautomatically consideredpositiveandaresubjecttocontrolactionswithouttesting. However,tubersamplingforsubsequenttestingwillberequiredfortubers thatarepotentiallyinfested,includingthefollowingtubers: Inadjacentfields, Onasharedwatersource, Commingledinstoragefacilities,or Connectedbyahistoryofsharedharvestingmachinery.

3-14

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Chapter 3 Data Collection

Eachtuberisasamplingunit.Repeatthesamplingforeachfield,warehouse, orstorageunitatagivenlocation.Collect300samplesinordertodetect Ralstoniasolanacearumata1%infestationlevelwith95%confidence.Collect samplesthroughouttheentirebuildingorstoragearea.Iftubersareinbags, collectthe300samplesfromdifferentbags.Disinfectthesamplesthesamples beforelaboratorytesting.SeeIdentificationonpage 41formore informationonlaboratorytesting.SeePotatoStorageFacilitiesandGrower Fieldsonpage 57formoreinformationonpositivetestingseedlotsorfields.
Delimiting Surveys in a General Growing Area

Constructdelimitingsurveysinanareabasedonknownpositivetesting, associatedpositivetesting,orpotentiallyinfestedareasfrominvestigations ofdistributionchannelsandsharedirrigationwater.However,itmaybe necessarytodorandomsamplesinageneralgrowingareatodetectnew infestationsnotdiscoveredthroughinvestigations. Thedelimitingsurveyinageneralgrowingareacanincluderandom samplingofstoredtubersandfieldsthroughoutageographicalarea,with moreintensivesamplingnearknowninfestations.Asthedistanceawayfrom theepicenterofknowninfestationincreases,decreasetherateofrandom sampling.Basedontheepidemiologyandgrowerpractices,anevaluationof riskandresourcesavailablewillhelpdeterminetheextentoftheserandom samplingsurveys.

Sampling of Water Sources

Testwatersourcesnearthepositivefields,processingplants,orwatersources usedforirrigation,todelimitthespreadofthebacterium. Samplesizeisapproximately0.50quarts;bottlesshouldbesterilized.Collect watersamplesatadepthof12inches.Keepsamplescoolandinadark location.Performtestswithin24hoursofcollection.Forbestresults,conduct samplingwhenwatertemperatureexceeds59oFandpopulationsof bacteriumarehighestinwater.Theseconditionsusuallycoincidewiththe periodJunethroughSeptember.

Tomato and Other Solanaceous Hosts


SeeTomatoesandOtherSolanaceousCropsonpage 313foradescription ofsymptoms.Ingreenhousesorfieldgrowncrops,lookforseverewilting.Ifa wiltedplantdoesnotrecoverafterwatering,collectasampleandhaveit tested.SeeProcedureForSamplingAndIdentificationonpage 43formore information.

Data Collection
Surveyorsvisitingsitestoplaceholdsortakesamplesshouldcollectthe followinginformation: Dateofcollectionorobservations Collectorsname Growersfieldorgreenhouseidentificationnumbers
May 12, 2008 Emergency and Domestic Programs Ralstonia solanacearum r 3 b 2 3-15

Chapter 3 Data Collection

GPScoordinates Varietyofhostplantgrown Methodsofirrigation Historyoffarmmachineryusage Observationsofwiltandanyotherrelevantinformation Intheabsenceofinspectionofficials,takethefollowingactionsimmediatelyif wiltingsymptomsarenoticed: 1. Markthelocation. 2. Removetheplantsfromthebenchorrack,orflaglocationinfield. 3. NotifyastateorPPQinspector. 4. Placethewholeplantinsidetworesealableplasticbags. 5. Labelthesealedbagswiththefollowinginformation: Date Nameofpersonresponsible Locationofsamplecollection 6. Keepbaggedplantscoolorrefrigerateduntiltheinspectorarrives.Do notfreezethesample.

3-16

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

4
Ralstonia solanacearum r 3 b 2

Chapter 4
Identification

Contents
Introduction page 41 Authorities page 41 ProcedureForSamplingAndIdentification page 43 DiagnosticScreeningLaboratories page 46 RequiredPermits page 46 DiagnosticTestsToDetermineGenusAndSpecies page 48 Culturing page 49 ConfirmatoryTestingToRaceAndBiovar page 49 PositiveConfirmations page 410

Introduction
UseChapter4IdentificationasaguidetoidentifyRalstoniasolanacearumrace3 biovar2.Accurateidentificationofthispathogenispivotaltoassessingits potentialrisk,developingasurveystrategy,anddeterminingtheleveland mannerofcontrol.

Authorities
PPQNationalIdentificationServicesMolecularDiagnosticsLaboratory (MDL),Beltsville,Maryland,mustpositivelyidentifybacterialinfectionsto thelevelsofraceandbiovarbeforeinitiationofanyprogramquarantine activities.MDLalsohasthenecessaryregistrationsandcontainment approvalstohandleselectagents.Stateandcooperatinguniversitydiagnostic laboratoriesarelimitedtodeterminationsofbacterialinfectionstothelevelof genusandspeciesRalstoniasolanacearum.SeeDiagnosticLaboratorieson page D1foralistoflaboratoriesandtheiraddresses. PPQpermitandregistrationrequirementsforplantdiseasesandlaboratories fallundertwoauthorities: PlantProtectionAct(7CFRPart330)(SeePlantProtectionActon page 42) AgriculturalBioterrorismProtectionActof2002(7CFRPart331)(See AgriculturalBioterrorismProtectionActonpage 42) Laboratoriesreceivingsuspectinfectedplantmaterialorculturesarerequired tohavePPQpermits.Laboratoriespossessing,using,ortransferringselect agentsarerequiredtoberegistered.However,diagnosticlaboratoriesthat identifyselectagentsareexemptfromthisrequirementaslongasthey
May 12, 2008 Emergency and Domestic Programs Identification.fm Ralstonia solanacearum r 3 b 2 4-1

Chapter 4 Authorities

completeAPHISCentersforDiseaseControlandPrevention(CDC)Form4 anddestroyortransferinfectedmaterialtoalaboratoryregisteredwiththe APHISSelectAgentProgramwithinthemandatorysevendays.APHISCDC Form4isavailableattheWebsiteoftheAPHISAgriculturalSelectAgent Program.


Address APHISAgricultural Select Agent Program Web site: http://www.aphis.usda.gov/programs/ag_selectagent/ index.html Telephone: 301-734-5960

Plant Protection Act


PlantProtectionActpermitrequirementsapplytoallplantpestsandinfected plantmaterial,includingdiagnosticsamples,regardlessoftheirquarantine status.Ifanymaterialisshippedinterstate,thereceivinglaboratorymusthave apermit.Forfurtherguidanceonpermittingofplantpestmaterial,consult PPQPermitServicesorvisittheWebsiteofthePPQPermitsProgram.
Address PPQPermits Program Web site: http://www.aphis.usda.gov/plant_health/permits/ Telephone: 301-734-8758

Agricultural Bioterrorism Protection Act


FederalregulationonAgriculturalBioterrorismProtectionActof2002(7CFR Part331)specifiesrequirementsforpossession,use,andtransferoforganisms listedasselectagentsandtoxins.Onceanunregistereddiagnosticlaboratory identifiesaselectagent,theymusttakethefollowingstepsimmediately: 1. NotifystaffatAPHISAgricultureSelectAgentProgram. 2. CompleteandsubmitAPHISCDCForm4within24hours. 3. Destroyortransfertheagenttoaregisteredentitywithinsevendays. IncompliancewiththeAct,ifadiagnosticlaboratoryheldbackpartofa screenedsampleforvoucherpurposesandthatsample(forwardedtoMDL) camebackaspositiveforaselectagent,thediagnosticlaboratoryisrequired tonotifytheAPHISAgriculturalSelectAgentProgramimmediately. Ifthedeterminationoftheunregisteredlaboratoryistodestroythesample, thismusttakeplacewithinsevendaysofresultsnotificationandaPPQofficer mustwitnessthedestructionofthesampleonorbeforethesevendayperiod expires.Clarificationofthisandotherinformationrelatedtoadherencetothe selectagentregulationsisavailableattheWebsiteoftheAPHISAgricultural SelectAgentProgram.
Address APHISAgricultural Select Agent Program Web site: http://www.aphis.usda.gov/programs/ag_selectagent/ index.html Telephone: 301-734-5960

4-2

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Chapter 4 Procedure For Sampling And Identification

Procedure For Sampling And Identification


Somepreidentificationandscreeningcanbeperformedbyfieldpersonnel assignedtotheprogram.Onlyplantsexhibitingwiltingsymptomsaretobe sampledandsubmitted.SeeVisualInspectionForDetectionSurveyon page 310andComparisonofSymptomsonpage F1formoreinformation. Otherbacterialdiseasescanalsocausewiltingsymptoms,soscreeningby diagnosticlaboratoriesisanecessaryandrequiredstep.Thesemustbestate, cooperatinguniversity,NationalPlantDiagnosisNetworklaboratories,or recognizedpermittedprivatelaboratories.SeeDiagnosticLaboratorieson page D1formoreinformation.

Screening Nursery and Field Hosts


Forsamplingofplantsshowingwiltingsymptoms,thewholeplantmustbe collectedincludingroots,preferablywithoutsoil.Barerootplantsareideal. Sincethepathogenisconcentratedinthelowerstem,thediseasemaynotbe detectedfromsamplesifonlyleaforpartialstemsamplesaretaken. 1. Placetheplantsampleinaresealablebagandsealit. 2. Useapermanentmarkertorecordthesamplenumberonthebag.See Labeling,Numbering,andRecordKeepingbelowformore informationonsamplenumbers. 3. Placethebaginsideasecondresealablebag,alongwithacompleted QuestionnaireForNurseryAndPotatoCroppingSystemOwnersOr Managersonpage B5.Fillintheformascompletelyaspossible. 4. Ifplantsinsoilaresubmitted,placeaseparateplasticbagaroundthe potwitharubberbandaroundtheplantbasetorestrictsoilspillageand contaminationoftheplanttissue. 5. Ifsoilisleftover,disposeofitproperly. 6. Placedoublebaggedsamplesinasturdycardboardouterboxwith insulationtopreventmovementwithintheboxduringshipping. Entiresamplesofplantsshouldbesubmitted,notsubsamples.Samplesmust includelowerstems;excludedetachedleavesorpetioles.Samplesthatare deadorfermenteduponarrivalcannotbetestedandwillberejected.

Labeling, Numbering, and Record Keeping


Generatesamplenumbersatthesamplingsite.Recordthesamplenumberon thefollowingforms,andincludeacopyofeachformwitheachsample: PPQForm391SpecimensForDeterminationonpage B2marked Urgent QuestionnaireForNurseryAndPotatoCroppingSystemOwnersOr Managersonpage B5

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

4-3

Chapter 4 Procedure For Sampling And Identification

Foreachsample,assignandrecordauniqueidentificationnumberinthe followingformat:
Example XX-ABC-0001 where XX is your two-letter state code; ABC is a three letter, state assigned facility code; and 0001 is the sample number for that facility.

Useauniquesamplenumberforeachsample.Keepalogofassignedsample numbers. IfthediagnosticscreeninglaboratoryisapartoftheNationalPlantDiagnostic Network(NPDN),samplesmaybesubmittedusingaNPDNnumberformat. Ifabarcodedsamplenumbersystemisavailable,besuretoincludethe barcodeinthesampleonthePPQForm391.SeeTable D1onpage D10for alistoflaboratoriesintheNPDN. Inspectorsmustprovidethesampledatewithallrelevantcollection informationtotheirStatePlantRegulatoryOfficialorStatePlantHealth Directorassoonaspossible.Thisinformationshouldbecommunicated withinastateandwiththeregionalofficeprogramcontact.Ifasample trackingdatabaseisavailableatthetimeofthedetection,entercollection informationinthesystemassoonaspossible.

Storage and Shipping


Samplesmustbeshippedtoapproveddiagnosticscreeninglaboratoriesas soonaspossibletopreventdeteriorationofplanttissue.SeeDiagnostic Laboratoriesonpage D1foralistoflaboratoriesandtheiraddresses. Ifstorageisnecessary,placebaggedsamplesinacoollocationuntilreadyfor shipping.Donotplacesamplesinafreezer.Samplesmaybeheldina standardrefrigeratorat39oForatroomtemperatureiflessthanorequalto 60oF. Shipsamplesbyovernightdelivery.Icepacksareunnecessary.

PPQ Form 391


Diagnosticscreeninglaboratoriesmustwritetheirdeterminationsforeach sampleonthePPQForm391withthenameandphonenumberofthe responsiblediagnostician.Keepacopyandfollowthesamesample packaginginstructionsasabove. NotethefollowingadditionalinformationonthePPQForm391: NameoftestkitusedtomaketheRalstoniasolanacearumdiagnosis IndicationthattheRalstoniasolanacearumtestwasstronglypositiveor weaklypositive,etc.(Ifpossible,aprintoutofadigitalimageofthe stickorplateshouldaccompanythesampleandpaperwork.) Nameofspecificplantpartthattestedpositive(Symptomaticorlower stemareashouldbetheonlytissuetested.)

4-4

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Chapter 4 Procedure For Sampling And Identification

PleasenotifyPPQNationalIdentificationServicesMolecularDiagnostics Laboratory(MDL)thatyouaresendingplantsampleswithsuspected Ralstoniasolanacearum.(Iffaxing,includeallpertinentinformationandcontact nameandnumber).

Saturday Delivery
OnlywithspecificpermissionfromAPHISPPQheadquartersshould samplesbesentonFridaysbyFedEx.Althoughitispossibletoarrange SaturdaydeliverybyovernightcarrierstoMDL,oftensamplesarenot deliveredevenwithspecialarrangementwithFedEx.However,MDLwill operateonSaturdayifalargeprogramisbeingconductedrequiring additionalhoursofoperation.IfyouhaveobtainedpermissionfromAPHIS PPQheadquarterstosendsamplesforSaturdaydelivery,pleasesendthe FedExtrackingnumbertoMDLviaemailtoMaryPalmsoshecannotifyher localFedexofficetoauthorizeSaturdaydelivery.
Address Dr. Mary E. Palm USDAAPHISPPQNational Identification Services Molecular Diagnostics Laboratory BARC-East, Bldg. 580 Powder Mill Rd. Beltsville, MD 20705 T: 301-504-7154 or 504-5700 ext. 327 F: 301-504-6124 Email: mary.palm@aphis.usda.gov

Notification of State Officials of Submissions and Results


NotifytheStatePlantHealthDirectorandStatePlantRegulatoryOfficialsin thesamplestateoforiginandfaxthePPQregionalofficeofanysample forwardinginformation,completeddocumentation,includingovernight freighttrackinginformation.Onceresultsareknown,stateswillbenotifiedby thePPQregionalofficeoftheresults. PleasedonotcallMDLtogetsampleresults.Theinformationwillbereported totheregionsandstatesfromheadquartersassoonastheyareavailable.

Sample Processing Time


Growersandcooperatorsneedtobeawarethatsampleprocessingrequires48 hourstothreeweeksdependingonthesamplecondition,numberofsamples thatrequireprocessing,andtheeasewithwhichdiagnostictestscanbe performed.Thisisinadditiontothetimeittakestoprocessandforward samplesfromtheintermediatestateandcooperatinguniversitydiagnostic laboratories.

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

4-5

Chapter 4 Diagnostic Screening Laboratories

Diagnostic Screening Laboratories


TodetermineifaninfectionisduetoRalstoniasolanacearum,samplesofplants mustbesubmittedthroughnormalregulatorynetworks.Theselabswill performdiagnosticteststothegenusandspecieslevel.Theywillnot determinetheraceandbiovar.SeeDeterminationOfGenusAndSpecieson page D2tofindtheappropriatelaboratory. Diagnosticscreeninglaboratoriesreceivingsamplesaretocommunicatethe dateofreceiptwiththeirStatePlantRegulatoryOfficialorStatePlantHealth Director.Allrelevantsampleinformationandthediagnosticlaboratorys speciesdeterminationmustbecommunicatedassoonaspossiblewithina stateandwiththePPQregionalofficeprogramcontact. Ifasampletrackingsystemisused,ataminimumenterthedatereceivedin thesystem,testresults,thedetermination,dateidentificationmade,andwho madetheidentification.

Alternative Laboratory
LaboratorieswithintheNationalPlantDiagnosticNetworkcanscreento genusandspeciesforstatesthatprefernottoorcannotscreentheirown samples.AnysampletestingpositiveforRalstoniasolanacearumwillbe forwardedtoPPQNationalIdentificationServicesMolecularDiagnostics Laboratory(MDL)inBeltsville,Maryland,forconfirmatorytesting.SeePlant DiagnosticNetworksandPrivateLaboratoriesonpage D10toidentifythe regionallaboratoryforyourstate. Statesthatcannotscreentheirownsamples,orhavenotbeenabletomake arrangementswithanotherpermittedlaboratoryoutofstate,canutilize Agdia,Inc.Thisprivatelaboratoryhasthenecessarypermitsand authorizationstoscreentospeciesbutnotraceorbiovar.Samplestesting positiveforRalstoniasolanacearumwillbeforwardedtoMDLforconfirmatory testing.
Address Agdia, Inc. 30380 County Road 6 Elkhart, IN 46514 T: (574) 264-2014, or 1-800-62-AGDIA Web site: http://www.agdia.com

Otherprivatelaboratorieswillbeidentifiedaspermitsareapproved.

Required Permits
Ifastate,university,NationalPlantDiagnosticNetwork(NPDN),orprivate laboratoryperformsaPCRassayanddetectsapresumptivepositiveoutside thenationalsurveyprocessoutlinedhere,theymustabidebythe

4-6

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Chapter 4 Required Permits

requirementsundertheAgriculturalBioterrorismProtectionActof2002(7CFR 331).Formoreinformation,seeAgriculturalBioterrorismProtectionActon page 42. Diagnosticscreeninglaboratoriesreceivingsamplesaretocommunicatethe dateofreceiptwiththeirStatePlantRegulatoryOfficialand/orStatePlant HealthDirector.Allrelevantsampleinformation,andthediagnostic laboratorysdeterminations,mustbecommunicatedassoonaspossible withinastateandwiththePPQregionalofficeprogramcontact. AnylaboratorythatdiagnosessamplestoRalstoniasolanacearumrace3biovar 2isrequired,undertheAgriculturalBioterrorismProtectionActof2002,to immediatelynotifytheAPHISSelectAgentProgramregardlessofwhethera PlantPestPermitisrequired.
Address APHISSelect Agent Program 4700 River Road, Unit 2 Riverdale, MD 20737 Email: Agricultural.Select.Agent.Program@aphis.usda.gov Telephone: 301-734-5960 Fax: 301-734-3652

Thelaboratorypossessingthesamplehassevendaysinwhichtodestroythe sample.However,theymustgivePPQtheopportunitytowitnessthe destruction.Inaddition,thelaboratorydirectormustcompleteAPHISForm 240.SeeAgriculturalBioterrorismProtectionActonpage 42formore information. Forfurtherguidanceonpermittingofplantpestmaterial,consultthePPQ PermitsWebsite.


Address PPQ Permits Telephone: 301-734-7211, 6828 or 5055 Web site: http://www.aphis.usda.gov/permits/ppq_epermits.shtml

Intrastate Transfer
Forinspectorsshippingplantpestsamplematerialtootherlabstotestforthe presenceofRalstoniasolanacearumidentificationtospeciesonly,notbiovar andracenopermitisrequiredforshipmentwithinthestate.Persons shippingplantmaterialtoadiagnosticlaboratoryintheirownstatedonot needtoobtainaPPQpermitundereitherthePlantProtectionAct(Regulations at7CFRPart330)orPPQForm526. WithinstatetransfersofknownRalstoniasolanacearumrace3biovar2are regulatedundertheAgriculturalBioterrorismProtectionActof2002 (Regulationsat7CFRPart331).However,thesubmissionofunknown samplesorthoseonlyidentifiedtoR.solanacearumnottoraceand biovararenotregulatedunderthisAct.

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

4-7

Chapter 4 Diagnostic Tests To Determine Genus And Species

Interstate Transfer
Stateswithouttheirowndepartmentofagricultureorcooperatinguniversity laboratoryforscreeningtogenusandspeciesmayshipacrossstatelinesto useanotherstateslaboratory. Whenshippingacrossstatelines,theoriginatingstatemustassurethatthe laboratoryinthedestinationstatehasthenecessaryplantpestpermitto receiveinterstatesamples.ApplyforthepermitbycompletingPPQForm523 EmergencyActionNotificationonpage B7. ForthepurposeoftheAgriculturalBioterrorismProtectionActof2002 registrationsarenotnecessary.Aswithintrastatesubmittedsamplesto diagnosticlabs,interstateshipmentofunknownpotentiallyinfectedplantsis notsubjecttoregulationsunderthisAct.

Diagnostic Tests To Determine Genus And Species


Serologicaltestsareavailabletoscreenforthisorganismtothegenusand specieslevelatdiagnosticlaboratories.Specialpermitsarenotneededby theselabstodothistestunlesstheyarereceivingsamplesfromanotherstate. ThethreeserologicaltestsvalidatedandrecognizedbythePPQNational IdentificationServicesNationalPlantGermplasmandBiotechnology Laboratory(NPGBL)arelistedbelow.SeeDiagnosticValidationsand IsolationMethodsonpage C1formoreinformationonthesekits.

Validated Screening Test Kits


TheNationalPlantGermplasmandBiotechnologyLaboratory(NPGBL)in Beltsville,Maryland,evaluatedthefollowingthreerapidserologicaltestsfor detectionofRalstoniasolanacearum.Noneoftheserologicaltestsevaluatedwill determinetheorganismtoraceorbiovar.
Address Rs ImmunoStrip Test Agdia, Inc. 30380 County Road 6 Elkhart, IN 46514 Web site: http://www.agdia.com T: 800-622-4342 F: 219-264-2153 Potato Brown Rot PocketDiagnostic Central Science Laboratory (CSL) Sand Hutton, York, YO41 1LZ Web site: http://www.csl.gov.uk T: 44 1904 462600 F: 44 1904 46211

Address

4-8

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Chapter 4 Culturing

Address

Ralstonia solanacearum SPOTCHECK LF Adgen, Ltd. Nellies Gate, AYR Scotland, KA6 5AW Web site: http://www.adgen.co.uk T: 44 1292 525275 F: 44 1292 5255477

ELISA Test Kits


ELISA(EnzymeLinkedImmunosorbantAssay)kits,testedandvalidatedby NPGBL,Beltsville,Maryland,willalsomakediagnosestothelevelofgenus andspecies.SeeDiagnosticValidationsandIsolationMethodsonpage C1 tolearnmoreaboutthekits.

Water Testing
MethodsoftestingwaterforthepresenceofRalstoniasolanacearumrace3 biovar2havebeendevelopedbyNPGBL,Beltsville,Maryland,butprotocols haveyettobeincludedintheGuidelines.

Culturing
Diagnosticlaboratoriescanforwardculturesafterscreeningtogenusand specieshasbeenperformed.SeeDiagnosticValidationsandIsolation Methodsonpage C1forrecommendedmethodsofculturing.Whensending culturesforraceandbiovardetermination,thesendermustassurethat packagingissecureaswithplantsamples.

Confirmatory Testing To Race And Biovar


OncetheplantmaterialhasbeenscreenedandisknowntocontainRalstonia solanacearum,forwardthesampleorcultureassoonaspossiblebyovernight carriertothePPQNationalIdentificationServicesMolecularDiagnostics Laboratory,Beltsville,Maryland,forconfirmationtoraceandbiovar. MDLhastheproperpermitstotestforthepresenceofRalstoniasolanacearum race3,biovar2.Dr.MaryPalm,MDLDirector,hasthenecessary authorizationstoreceivesamplessubmittedfordiagnosticstotheraceand biovarlevel.Includethenameofthekitusedforthedetectionandspecific descriptiveinformationonthetestresults(i.e.,intensityofthetestband,OD oftheELISAwell,ordigitalimagesofthetestresult).
Important Entire samples of suspect Ralstonia solanacearum positives should be submitted to Molecular Diagnostics Laboratory (MDL)not sub-samples. Samples must include lower stems. Samples should not consist of detached leaves or detached petioles. If samples are necrotic (brown) or fermented upon arrival at MDL, they will be rejected.

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

4-9

Chapter 4 Positive Confirmations

Address

Dr. Mary E. Palm USDAAPHISPPQNational Identification Services Molecular Diagnostics Laboratory BARC-East, Bldg. 580 Powder Mill Rd. Beltsville, MD 20705 T: 301-504-7154 or 504-5700 ext. 327 F: 301-504-6124 Email: mary.palm@aphis.usda.gov

Positive Confirmations
IncompliancewiththeAgriculturalBioterrorismProtectionActof2002(7CFR Part331)ifadiagnosticlaboratoryheldbackpartofascreenedsampleor cultureforvoucherpurposesandthesampleforwardedtothePPQNational IdentificationServicesMolecularDiagnosticsLaboratory(MDL),Beltsville, Maryland,camebackaspositiveforRalstoniasolanacearumrace3biovar2,the diagnosticlaboratoryisrequiredtonotifyPPQPermitsthatthesampleexists. Destructionmusttakeplacewithinsevendaysofnotificationthatthesample ispositiveforR.solanacearumrace3biovar2andaPPQrepresentativemust havetheopportunitytowitnessthedestructionofthesampleorculture withinthattimeperiod.Theresponsiblelaboratorymanager/plant pathologistmustalsocompletetheAPHISForm2040.Theformisavailableat thePermitsWebsite.
Address PPQPermits Telephone: 301-734-7211, 6828 or 5055 Web site: http://www.aphis.usda.gov/permits/ppq_epermits.shtml

MDLhasthenecessarypermitsandregistrationstopossessthisselectagent, andisalsorequiredbylawtoreportthepositiveconfirmationwithin24hours ofdetection.

4-10

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

5
Ralstonia solanacearum r 3 b 2

Chapter 5
Regulatory Procedures

Contents
Introduction page 51 InstructionsToOfficers page 51 OverviewOfRegulatoryProgramAfterAUnitedStatesDetection page 53 FurtherInvestigationsAtNurseriesWithSuspectGeraniums page 56 PlacingHolds page 57 RecordKeeping page 58 PlantstoHold page 59 IssuingPPQForm523EmergencyActionNotification page 511 GeraniumAndPotatoVolunteersAndCullPiles page 512 HarvestingEquipment,StorageAreas,AndConveyances page 512 RegulatoryRequirementsOfSeedProducers page 512 RegulatoryRequirementsForProcessors page 512 RegulatedArticles page 513 RemovingAreasFromQuarantine page 514 RequirementsForPotatoCroppingSystemsUnderQuarantine page 515 UseOfChemicals page 518

Introduction
UseChapter5RegulatoryProceduresasaguidetoproceduresthatmustbe followedwhensurveyingorcontrollingtheplantpathogenRalstonia solanacearumrace3biovar2.

Instructions To Officers
Agriculturalofficersmustfollowinstructionsforregulatorycontrolmeasures, treatmentsorotherprocedureswhenauthorizingthemovementofregulated articles.Afullunderstandingoftheinstructionsandproceduresisessential whenexplainingprocedurestopersonsinterestedinmovingarticlesaffected bythequarantineandregulations.Onlyauthorizedtreatmentsmaybeused inaccordancewithlabelingrestrictions.Duringallnurseryvisits,please assurethatpropersanitationproceduresarefollowed.SeeSurveyProcedures onpage 31andLawsPertainingtoPestControlonpage 61formore information.

1
May 12, 2008 Emergency and Domestic Programs Regulatory Procedures.fm Ralstonia solanacearum r 3 b 2 5-1

Chapter 5 Instructions To Officers

Quarantine Actions Required


Geraniums

Afterinvestigationsareperformedandadditionalconsultationsmadeif necessarytheunitofheldsuspectgeraniumsandpotentiallyinfectedplants associatedwiththemmustbedestroyedordisposedof.Inaddition,thearea mustbedisinfected.SeeDisinfectionProceduresonpage 64formore information Ifapropertyhaspositivetestingorpositiveassociatedfields,allplant materialthatcanbereasonablyremovedfromthefieldmustbedestroyedor disposedof.Thisincludescullpilesandotherplantdebris. Leavethefieldfallowfortwoyears,andirrigatetopromotevolunteer sprouting.Duringthetwoyears,thevolunteercropmustbesampled,tested, anddiskedunderduringthegrowingseasoneveryfourweekstoeliminate hostmaterial.Weedhostsinthefieldandalongtheedgesmustbesprayed withefficaciouslabeledherbicidestoeliminatethemfromthearea. Inthefollowingtwoyears,plantfieldswithnonhostcrops;irrigateto promotevolunteerpotatosprouting,diskinganyvolunteerpotatoesas sproutingoccurs.Fieldsmustbetestedsemiannuallyforfouryearsafteran initialpositivefind.SeeSurveyProceduresonpage 31formore information. Furthermore,noseedproductioncanoccurinthefieldforatleastfiveyears afterdetectionofRalstoniasolanacearumrace3biovar2.Fieldswithsusceptible hosts(potatoesortomatoes)mustbesampledfortwoseasonspriortoany newseedproductionontheproperty. Fieldsadjacenttopositivetestingorassociatedfields,orthoseonashared watersupply,maynotgrowhostcropsfortwoyears,norseedpotatoesfor twoyears,andmusttestandcontrolanyvolunteerpotatoorweedhosts. Maintainstrictsanitationofallvehiclesenteringandleavingtheinfested fields. Storagefacilitiesonpropertieswithpositivetestingtubersmustbeheldin quarantineuntilallpotentiallyinfestedtubersaretestedandeitherfoundto benegativeordestroyed.Thestoragefacilitiesmustthenbeproperlycleaned anddisinfectedwithapproveddisinfectantsinstrictaccordancewithlabeling. SeeDisinfectantsonpage G1formoreinformation.

Potato Cropping Systems

Emergency Quarantine Action

Ifnecessary,theDeputyAdministratorwillissuealetterdirectingPPQfield officestoinitiatespecificactionsunderthePlantProtectionActof2000until emergencyregulationscanbepublishedintheFederalRegister. ThePlantProtectionActof2000providesforauthorityforemergency quarantineaction.Thisprovisionisforinterstateregulatoryactiononly; intrastateregulatoryactionisprovidedunderstateauthority.However,ifthe SecretaryofAgriculturedeterminesthatanextraordinaryemergencyexists

5-2

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Chapter 5 Overview Of Regulatory Program After A United States Detection

andthatthemeasurestakenbythestateareinadequate,USDAcantake intrastateregulatoryactionprovidedthatthegovernorofthestatehasbeen consultedandanoticehasbeenpublishedintheFederalRegister.Ifintrastate actioncannotorwillnotbetakenbyastate,thePPQmayfinditnecessaryto quarantineanentirestate.


Access to Private Property

AGeneralMemorandumofUnderstanding(GMU)betweenstatesand PPQexistsforeachstate.Incertaincircumstances,theGMUmayfacilitate accesstoprivateproperty,intheabsenceoflandownerpermission,byPPQ Officersinconjunctionwithstateinspectorstoplacefacilitiesunder notificationandwitnessactionsspecifiedinthePPQForm523Emergency ActionNotification(EAN).CheckwithyourSPHDforclarification.

Regulatory Records
Maintainstandardizedregulatoryrecordsanddatabase(s)insufficientdetail tocarryoutaneffective,efficient,andresponsibleregulatoryprogram.

Overview Of Regulatory Program After A United States Detection


IfRalstoniasolanacearumrace3biovar2isdetectedongreenhousegeraniums orpotatoesintheUnitedStates,followthisprocedure: 1. Determinetheoriginoftheinfection. 2. Surveytheextentofdistributionofpotentiallyinfectedgeraniumplants orpotatoes.

Determining Origin of Infections


Geraniums
Important There is no treatment for plants infected by Ralstonia solanacearum race 3 biovar 2. All greenhouse plants determined to be regulated and associated potting medium will be destroyed and areas sanitized before facilities can be released from quarantine. If new information indicates a more widespread infestation problem than originally assessed, or new information becomes available, these regulatory guidelines are subject to change.

Conductatracebackinvestigationtodeterminetheforeignsourceofinfection andtheextentofdistributionofpotentiallyinfectedplantsintheUnited Statesfromthatsource.Conductatraceforwardinvestigationtodetermineif potentiallyinfectedplantmaterialhadbeendistributedtootherretail locations. InvestigationsatUnitedStatesgeraniumpropagationfacilitiesandtheir customerswillbeconducted.Resultsofinvestigationswillallow implementionofaprogramofholdingvarietiesorshipmentsthatoriginated ataparticularforeignfacilityduringashippingperiod.Theirrigation methods,handling,andsanitationpracticesofeachnurserywillhelpassessif additionalassociatedplantmaterialisatriskofinfection.Information

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

5-3

Chapter 5 Overview Of Regulatory Program After A United States Detection

gatheredintheinvestigationportionwillbecriticaltodeterminingwhich additionalplantstoholdandsubsequentactionstaken.Whereguidanceis needed,regionalcontactswillbeconsulted.


Potato Cropping Systems
Important There are no treatments for plants or tubers infested by Ralstonia solanacearum race 3 biovar 2. Dispose of all positive testing and positive associated host plant material. If possible, test potentially infested material and assess the risk. All associated harvest, sorting, grading and storage equipment, machinery, structures, and areas involved in any phase of harvesting, sorting, cleaning, grading and storage, will be cleaned and disinfected before facilities can be released from quarantine. Fields where infested material was grown will be assessed for potential quarantine actions. Once the extent of infestation is determined for a growing area, establish quarantine boundaries with growers, seed companies, storage facilities, processors, and distributors coming under restrictions determined by regulation. If new information indicates a more widespread infestation problem than originally assessed, or other information becomes available, these regulatory guidelines are subject to change.

Conductatracebackinvestigationtodeterminethesourceofinfectedtubers andtheextentofdistributionofseedtubersintheUnitedStatesfromthat source.Conductatraceforwardinvestigationtodetermineifpotentially infectedtubershavebeendistributedtogrowersorprocessingplantswhere processedmaterialandwatermaybereleasedtotheenvironment.See TracebackAndTraceForwardSurveyonpage 37formoreinformation. Informationgatheredintheinvestigationwillbecriticaltodeterminingwhich additionaltuberlotsandpropertiestohold,aswellaswhichactionstotake. Consultwithregionalcontactsifguidanceisneeded.

Distribution of Lists of Positive and Suspect Properties


Geraniums

Alistofnurseryfacilitieswillbecompiledofgeraniumvarietiesand/or shippingwindowsthathavebeen associated with positively tested, foreign origin geraniums. Theselistswillbedistributedbystatetothefieldoffices,andare nottobesharedwithindividualsoutsideourregulatorycooperators. Companynamesandlocationsontheselistsarestrictlyconfidential,andany distributionoflistsbeyondappropriateregulatoryagencycontactsis prohibited. Whennotifyingnurseriesonthelist,besuretoidentifyyourselfasaUSDAor stateregulatoryofficialconductinganinvestigationofnurseriesthatmay havereceivedRalstoniasolanacearuminfectedmaterial.Speaktothenursery ownerormanager. Itisimportanttounderstandthatnurseryfacilitieshandlingforeign geraniumsareofseveraltypes.Companiesshipcuttingsandcallusedplants throughPPQPlantInspectionstations.Theclearedmaterialistheneither senttorootingstationsortodirectshipcustomerswhicharewholesalers.

5-4

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Chapter 5 Overview Of Regulatory Program After A United States Detection

Bothtypesoffacilitiesgrowtheplantsforaperiodoftime(usually5weeks) beforebeingdistributedtootherentities.Rootingstations,undercontract withthecompany,thenshiporderstoavarietyofcustomertypesincluding wholesalersandbothlargeandsmallretailcustomers.Directshipfacilities canhavetheirownnetworkofretailstoresorsellplantstoothercustomers. Handlingandculturalpracticesvaryatallstepsinthedistribution. Duringtheinvestigation,requestthatthenurseryownerormanagerproduce anytags,invoices,shippinglistsassociatedwiththepositivetestedsuspect plantsonthedistributedlist.Makecopiesoftheserecordsandlocatethe suspectplantsstillonthepremises.Takephotographsoftheareawhere suspectgeraniumsarelocatedandanygreenhouseconditionsincludingthe proximityofneighboringplantsthatmayindicatebroadercontamination. Makearecordofthenumberofplants,type,andlocationincludingan accountingofsuspectplantsfromthatshipmentnotonthepremises. Tracebackinformationgatheredfromplanttagsandinvoicescanbeusedto determinetheoriginoftheplants.Withtimelysubmittedrecordsfromplant distributioncompaniessuchasrootingstationsorwholesalegrowers, PPQEmergencyandDomesticProgramsstaffcangeneratelistsofnurseries withimplicatedplantvarieties.Considerableeffortshavebeenmadebythe geraniumindustrytousebarcodingforplantsandcuttingstrackingthatmay haveuseintheseinvestigations. Thisinformationcanbeusedtodeterminetheextentofdistributionofsuspect geraniumsatvariouslocationsintheUnitedStates.Coordinatewithyour statecontactsandregionalofficetogatherthisinformation.Additional resourcesmaybeneededtoquicklygatherthisinformationandanalyzeitin ordertoholdsuspectgeraniumshipmentsandpotentiallyinfectedplants.
Potato Cropping Systems

Asaresultoftracebackortraceforwardinvestigations,alistoffacilities associatedwithfieldgrownpotatoestestingpositiveforRalstoniasolanacearum race3biovar2willbecompiled.Theselistswillbedistributedbystatetothe fieldoffices,andarenottobesharedwithindividualsoutsideourregulatory cooperators.Growernamesandfieldlocationsontheselistsarestrictly confidential,andanydistributionoflistsbeyondappropriateregulatory agencycontactsisprohibited. Thelistswillincludethreecategories:


Positive TestingSeedorwarepotatoes,infields,orasharvested,orin

storage,atshippingpoints,orinprocessingfacilitiesthatareconfirmed positiveforRalstoniasolanacearumrace3biovar2.
Positive AssociatedSeedorwarepotatoes,infields,orasharvested,orin

storage,atshippingpoints,orinprocessingfacilitiesthataredirectlylinkedto positivetestingforRalstoniasolanacearumrace3biovar2.Directlinksinclude butarenotlimitedtothefollowingtypesofpotatoes: Plantedinfieldswithsameseedlotsaspositivetestedlots,

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

5-5

Chapter 5 Further Investigations At Nurseries With Suspect Geraniums

Commingledinthesamestoragebinorareawithpositivetested,and IrrigatedfromR.solanacearumrace3biovar2confirmedcontaminated watersources.


Potentially InfestedSeedorwarepotatoes,infields,orasharvested,orin

storage,atshippingpoints,orinprocessingfacilitiesthatareindirectlylinked topositivetesting,forRalstoniasolanacearumrace3biovar2.Indirectlinks include,butarenotlimitedtothefollowing: Seedlotsfromthesamecompany,orstoredonthesamepropertyas positivetestinglots Hostsinfieldsthatsharethesamewatersupplyaspositivetestingfields Infieldsadjacenttopositivetestingfields Fieldsthathaveahistoryofusingharvestingequipmentwithpositive testingfields

Further Investigations At Nurseries With Suspect Geraniums


OncealistofnurseriesinthestateisgeneratedaftertheinitialUnitedStates detection,itwillbenecessarytovisitthelocationstoassesstheextentofplant distributionandnurseryirrigationandsanitationpractices.Various greenhousepracticeswillpreventorfacilitatethespreadofbacteriaof Ralstoniasolanacearumrace3biovar2.Informationmustbegatheredto determinewhatadditionalpotentiallyinfectedplantsorareasmaybeatrisk forcarryingthepathogen.Formoreinformation,seePlantstoHoldonpage 59. Examinethetypeofirrigationsystemandpracticesusedatthenursery: Areplantsunderasubirrigationsystemwherewaterfloodsthepots andpenetratesfrombelow? Doyounoticehanginggeraniumsfromsuspectshipments? Wereotherplants(hostsorotherwise)undertheirdripline? Arecuttingsofplantsbeingmadetopropagatemoreplants? Doworkersexhibitsanitaryhandlingofplantmaterial? Thiskindofobservationalinformationshouldbedocumentedbythe inspector.Inaddition,interviewthenurseryowner/managerandaskifthey haveseenwiltinggeraniumsorotherhostsduringtheseasoninquestion.Ask ifanywiltingplantshavebeendiscardedandifso,where?Photographsofthe facilityandwrittenobservationscanbeusefulintheinvestigationphase. Askthenurseryowner/managertocompleteandsignQuestionnairefor NurseryOwnersOrManagersonpage A1.

5-6

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Chapter 5 Placing Holds

Theinformationgatheredintheabovequestionsanddocumentationwill assistindeterminingifadditionalplantsmustbeheld.Consultwithyour regionalcontactswhentherearequestionsconcerningaparticularfacility practicethatmayaffectthenumberofadditionalpotentiallyinfectedplants heldandtheextenttowhichthatpracticeisused.

Placing Holds
Nurseries
Afteradestinationnurseryisidentifiedashavingreceivedplantsfrom anothersourceassociatedwithpositivetestinggeraniums,itisimportantto immediatelyplaceholdsonallgeraniumvarietiesnamedondistributedlists andinitiallyanyotherhostsplantsinthefacility.Iftheextentofpotentially infectedplantsatthatfacilitycanbeascertainedatthattime,thosenot determinedtobepotentiallycontaminatedcanbeimmediatelyreleased. Aholdisinterpretedastheprohibitionofmovementofthoseplantsfromthe propertyuntilfurtherevaluationscanbemadeandcontrolactionscanbe takenonsuspectgeraniumsandotherpotentiallyinfectedplants. Afterinvestigationsaremade,unlessgeneralcontaminationissuspected,the holdisnottobeinterpretedto: Prohibitthemovementofallplantsinagreenhouse; Thatplantsnotheldcannotbecaredforinanormalandsanitary manner;and Prohibittherelocationofsuspectgeraniumshipmentsandpotentially infectedplantstoasegregatedareaawayfromotherplants;ifthe nurseryowner/managerwishestomoveheldplants,assesstheriskof suchmovementandkeeprecordsofwhererelocatedplantsaremoved.

Potato Storage Facilities and Grower Fields


Afterafacilityisidentifiedashavingreceivedpositivetestingtubers(i.e., suspecttubers),itisimportanttoimmediatelyplaceholdsonpropertieswith suspecttubersandpotentiallyinfestedtubersthatarelistedastheresultof investigationsordeterminedpositiveintesting.Ifholdswereplaced previously,andtheextentofpotentiallyinfestedtubersatthatpropertycanbe ascertainedafterathoroughinvestigation,thoselotsorpropertiesdetermined nottobeatriskorpotentiallyinfectedcanbeimmediatelyreleased.This couldresultfromnegativetestingresultsornewinformationdiscovered duringinvestigations.Aholdisinterpretedastheprohibitionofmovementof plantmaterialortubersfromthepropertyuntilfurtherevaluationscanbe madeandcontrolactionscanbetakenonsuspecttubersandotherpotentially infectedplantmaterial.

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

5-7

Chapter 5 Record Keeping

Afterinvestigationsaremade,unlessgeneralcontaminationissuspected,the holdisnottobeinterpretedto: Prohibitthemovementofalltubersinafacilityorproperty;and Prohibittherelocationofsuspecttubershipmentsandpotentially infectedtuberstoasegregatedareaawayfromotherplants.Ifthe facilityowner/managerwishestomoveheldtuberstoanotherareaon thesameproperty,assesstheriskofsuchmovementandkeeprecordsof whererelocatedtubersareandwheretheyhavebeen.

Record Keeping
Geraniums
Recordkeepinganddocumentationisimportantforanyholdsand subsequentactionstaken.Relyonshippingrecordsandinformationprovided bythenurseryowner/managerforhowmanyplantsremain,howplantshave movedwithinthenursery,destinationofplantssold,andculturalpractices employed. Keepadetailedaccountingofthenumbersandtypesofeachplantvariety heldand/ordestroyedincontrolactions.Consultamasterlistofvarieties distributedwiththelistsoffacilities.Drawmapsofthegreenhouselayoutto locatedsuspectgeraniums,otherpotentiallyinfectedplants,andwaterrunoff areasincludingrecirculatingpondlocations.Takephotographsofthefacility layout,geraniumplacement,wateringmethod,materialsandmethodsused, plantlabeling,andanyothersituationsthatmaybeusefulfordocumentation andanalysis.

Potato Cropping Systems


Forpositivepotatoes,relyonshippingrecordsandinformationprovidedby thegrower,farmmanagerorfacilitymanagerforhowmanytubersremain, howplantmaterialandtubershavemovedwithinthefacility,destinationof tuberssold,originofpotatoseed,andculturalandsanitationpractices employed. Keepadetailedaccountingofthenumbersandtypesoftuberorplant materialheldand/ordestroyedincontrolactions.Consultamasterlistof varietiesdistributedwiththelistsoffacilities.Drawmapsofthefacilitylayout tolocatedsuspecttubers,otherpotentiallyinfectedlots,cullpiles,andwater runoffareas.Takephotographsofthefacilitylayout,wateringandsanitation methods,materialsandmethodsused,labeling,andanyothersituationthat maybeusefulfordocumentationandanalysis. KeepallwrittenrecordsfiledwithPPQForm523EmergencyAction Notification(EAN)copies,copiesofsamplesubmissionforms, documentationofcontrolactivities,andrelatedstateissueddocumentsif available.SeePPQForm523EmergencyActionNotificationonpage B7.

5-8

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Chapter 5 Plants to Hold

Plants to Hold
CompletePPQForm523EmergencyActionNotificationonpage B7to placeholdsatdestinationnurseriesforthefollowingclassesofplants:

Suspect Geraniums
Variety

Dependingontheparticularprogramdirections,aparticularvarietyor varietiesofgeraniumplantsmaybeconsideredsuspectbecausetheytested positiveandtracebackinvestigationstoaparticularforeignfacility determinedtheiroverallinfectionrisk.Thevarietylevelholdcanbemost appropriateatrootingstationsordirectshippedgrowersthatarelarge operationswhosepracticeistokeepvarietiesseparate. Dependingonprogramdirectionsandpracticesatapreviousfacilityinthe distribution,alevelofsuspectgeraniumstoholdmayincludetheshipmentof allhostplantsreceivedfromthatfacility. Releaseallplantsthatarenotconsideredpotentiallyinfected.Thefollowing categoriesaresubjecttohold.

Shipment

Potentially Infected Plants

High RiskResultsofinvestigationsatnurseryfacilities,andanswerstothe

questionnaire,willdeterminethelevelofadditionalholdsofpotentially infectedplants.Afterinvestigationsaremade,someoftheseconditionswill requireindividualjudgmentandconsultationwithyourregionaloffice.The categoriesthatdefinehigherriskforpotentiallyinfectingplantsinclude: PlantsbeneaththedriplineofsuspectgeraniumsBecauseRalstonia solanacearumbacteriaareeasilyshedinwater,plantsdirectlybelow hanginggeraniums,regardlessoftheplantspecies,areathighriskof soilcontaminationandmustbeincluded.Thisincludeshostsand nonhosts.Thisisalsotrueforplantsstoredunderbencheswithsuspect geraniums. PlantsinthesamepotswithsuspectgeraniumsInfectedgeraniumsin thesamepotswithotherplantscaneasilyinfectthemthroughwater, soil,orrootcontact.Thisincludeshostsandnonhosts. PlantpropagationfromsuspectgeraniumshasoccurredAnyplants propagatedfromsuspectgeraniumsareathighriskforinfection. PlantsonasharedirrigationsystemwithsuspectgeraniumsThemost efficientmethodofspreadforR.solanacearumisthroughwaterthat cameincontactwithinfectedplants.Itisthereforenecessarytoholdall

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

5-9

Chapter 5 Plants to Hold

plants(hostsandnonhosts)thatareonirrigationsystemsthatallows forwaterflowfromoneplanttothenext.Thesearevariousirrigation situationstolookfor: Subirrigation,ebbandflow,orfloodirrigation:potssitinapanand areirrigatedbyfloodingthepaninvariousways. Backflowprevention:nearlyallirrigationsystemshavecheckvalves toasasafeguardtopreventcontaminatedwaterfrombackingup intothegeneralwatersupply.Ifsystemslackbackflowprevention, thereisahighriskofgeneralcontaminationthroughoutthesystem frominfectedmaterial. Plantsinfacilitieswheresanitaryculturalpracticesarenotinplace Usinggraftingknivesformakingcuttingsorgroomingplantswithout disinfectionbetweenvarietiesisahighriskfactorintransmittingthe pathogentootherhostplantlots. Plantsplacedontheground,onplasticsheetingorothermaterialthat allowspuddlingbetweenplantsBecausethepathogenspreadsby water,thereisariskthatpuddlingbetweenplantswillcauseuptakeof bacteriabyhealthplantsinthevicinityofinfected.Theextenttowhich thisoccursneedstobeassessedbyobservationandfurtherevaluations ormonitoringmaybenecessary.
Reduced RiskThefollowinggreenhouseculturalpracticesareconsidered

notashighofriskforspreadingthepathogentootherplants.Evaluationscan bemadebytheinspectorastothedegreetowhichsuchpracticesmaytake placebasedonobservationsandresponsestothequestionnaire.Itis recommendedthattheinspectorconsultwiththesupervisor,SPHD,SPROor regionalofficeifthereisreasontobelievetheextentofthesepracticespresents agreaterriskandthereforerequiresadditionaldestructionofplants.Insome cases,afterconsultation,thedegreetowhichtheseriskfactorsarepracticedat thefacilitymaydeterminetheneedforwiderdestructionofpotentially infectedplants,however,inmostcases,thesenurserieswithplantsinthese reducedriskcategoriesmaybeheldandsubjecttofollowupinspectionsto inspectforwiltsymptoms. PlantsthatwerenotsegregatedfromsuspectgeraniumsRecent informationshowsthatleaftoleafcontactbetweengeraniumsisnotan effectivemethodofpathogenspread,sothereislittletonoriskofplants inthesameproximityofinfectedplantsbecomingcontaminatedby contact.Anexceptiontothismightbeplantsimmediatelyadjacentto infectedplantsthatwerewateredbyhand(usingahoseora waterwand)wheresplashmaytransferthepathogentothesoilin adjacentplants.Generally,dripirrigationandmistsystemsdonot createariskofsplashtoadjacentplants.Whenindoubt,consultyour SPHD,SPRO,andregionalcontact. Plantsthatwerepinched,deleafed,disbuddedbyhandThismethod ofgroomingpresentslessriskofspreadtononinfectedhostsplants thanbytheuseofcuttingtools,butsomedegreeofsanitationbetween

5-10

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Chapter 5 Issuing PPQ Form 523 Emergency Action Notification

varietiesisnecessary.Anexceptiontothisisiflittletonosanitary practicesareinplaceforworkerswhoperformplantgroomingbyhand andsanitationofhandsshouldtakeplaceatleastbetweenvarieties.

Suspect Potato Cropping Systems


Onceinitialdetectionsareconfirmed,regulatedarticlesincludethefollowing: Hostplantmaterialincludingtubersandplants Soil Harvestingequipmentincludingtrucks,harvesters,trailers,machinery, tools,fieldboxes Workerfootwearorimplements Transportconveyances(ifassociatedwith,orsuspectedofbeing potentiallyinfested) Storagefacilities(ifassociatedwith,orsuspectedofbeingpotentially infested) Negativetestingresultsmaydeterminecertainpotentialinfestedarticlestobe unregulated.OtherhostsofRalstoniasolanacearumrace3biovar2mayoccurin apotatoareathatisknowntobefromaninfestedarea.Thecultivatedhosts aretoberegulatedinamannersimilartopotato.SeeHostsonpage 26for moreinformation.

Grower Requirements Under Quarantine


Propertieswithpositivetesting,orpositiveassociatedfields,willberequired todestroyand/ordisposeofallplantmaterialthatcanbereasonablyremoved fromthefield.Thisincludescullpilesandotherplantdebris. Thefieldwillbeleftfallowfortwoyears,andirrigatedtopromotevolunteer sprouting.Duringthesetwoyears,inthegrowingseason,thevolunteercrop willbesampled,tested,anddiskedundereveryfourweekstoeliminatehost material.Weedhostsinthefieldandalongtheedgeswillbesprayedwith efficaciouslabeledherbicidestoeliminatethemfromthearea.

Issuing PPQ Form 523 Emergency Action Notification


PPQForm523EmergencyActionNotificationonpage B7(EAN)isissued toholdallhostplantsatfacilitiesthathavethesuspectgeraniumorpotato varietiesandpotentiallyinfectedplantsortubersconnectedtopositive confirmations.Onceaninvestigationdeterminesthereareplantsthatarenot suspectgeraniumsorpotentiallyinfectedpotatotubers,theymaybereleased anddocumentedontheEAN. TheEANmayalsobeissuedtoholdpotatoorgeraniumplantswhenwiltis discoveredpendingpositiveidentification.Whenadecisiontodestroyplants ismade,orinthecaseofsubmittedsamplesoncepositiveconfirmationis
May 12, 2008 Emergency and Domestic Programs Ralstonia solanacearum r 3 b 2 5-11

Chapter 5 Geranium And Potato Volunteers And Cull Piles

received,thesameEANforwhichtheplantsareonholdisusedtodocument anyactionstakensuchasdestructionanddisinfection.Ifvarietiesor shipmentsaretobeheldasseparateunits,itisadvisabletoissueseparate EANsforeachheldunitofsuspectgeraniumsorpotatoesandpotentially infectedplantsassociatedwiththatunit. ElectronicEANscanbeused.SeePPQForm523EmergencyAction Notificationonpage B7forinstructions.EANsareissuedunderthe authoritygivenin7CFR330.Statesshouldissuetheirownholdorders paralleltotheEANtoassureplantscannotmoveintrastate.
Address http://www.aphis.usda.gov/library/forms/

Geranium And Potato Volunteers And Cull Piles


Fieldswithvolunteerpotatoesmaycomeunderregulationsinagenerally infectedgrowingareamayrequiresamplingandtestingiftheyareconsidered potentiallyinfested. Cullpilesmaybefoundnexttofieldsorgreenhouses,onotherfarmproperty areas,ornearstoragefacilitiesorprocessors.Thesearepotentialreservoirsof thepathogenandareregulateduntiltherisklevelisdeterminedby investigationsand/ortesting.

Harvesting Equipment, Storage Areas, And Conveyances


Potatoharvestingequipmentincludingtrucks,harvesters,trailers,machinery, tools,fieldboxes,workerfootwearorimplements,transportconveyances,and storagefacilitiesaresubjecttocleaninganddisinfectionifpositiveassociated.

Regulatory Requirements Of Seed Producers


Seedtuberproducersmustdestroyand/ordisposeofallpositivetestedand positiveassociatedlots.Theymusttestalllotsintheirinventoryforthe presenceofRalstoniasolanacearumrace3biovar2.Theymustfurthercleanand disinfectallequipment,storageareas,andconveyancesusedintheirbusiness enterprise.RegulartestingforR.solanacearumrace3biovar2mustbecomea partoftheirseedcertificationprogram.

Regulatory Requirements For Processors


Companiesthatmayhavereceivedpotatoesfrompositiveharvestedfieldsare subjecttoholds,inspection,testing,andpossibledisinfectionrequirements. Processorscanincludefacilitiesthatwashpotatoesfortableconsumption (warepotatoes),producepotatochips,fries,orstarch.Inallcases,thefacilities

5-12

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Chapter 5 Regulated Articles

receivedpositivetestingorpositiveassociatedstock,oriftestsarepositive, thewaterandequipmentusedinprocessingandthewasteproductsfrom processingwillrequireregulatoryaction.Placeholdsonfacilities,water sources,highriskplantmaterialfromprocessinguntilassessmentsaremade todeterminewhichpartsofthefacilitywillrequiredisinfection,water treatment,andproperdisposalofhighriskwastematerial. Processorreceivingpositivetestingorpositiveassociatedpotatoesmust destroyordisposeofallofthosepotatoesintheirinventory.Allstorageareas, processingequipment,andconveyancesmustbedisinfected.Allwater sourcesorstoragepondsrecirculatedforprocessingmustbetestedand decontaminatediffoundtobepositive.

Regulated Articles
Cultivated Host Plants
Plants,cuttings,orpartsofthefollowinghostsofRalstoniasolanacearumrace3 biovar2areregulatediftestedpositiveandconsideredpotentiallyinfected.
TABLE 5-1 Cultivated Hosts of Ralstonia solanacearum race 3 biovar 2
Geranium Tomato Peppers Eggplant Potato Bean Bittergourd Beet
1

Pelargonium spp.1 Lycopersicon esculentum Capsicum spp. Solanum melongena Solanum tuberosum Phaseolus vulgaris Momoridica charantia Beta vulgaris

See Pelargonium in the Nursery Trade on page E-1

Weed Host Plants


WeedhostsofRalstoniasolanacearumrace3biovar2areoftensymptomlessbut mustbedestroyedingreenhouseswithpositivedetectionsincludinganarea withinonemeteraroundtheoutsideperimeterofthegreenhouse.See Controlonpage 61fordestruction/disposalanddisinfectionprocedures.
TABLE 5-2 Weed Hosts of Ralstonia solanacearum race 3 biovar 2
Black nightshade Climbing nightshade Horsenettle Solanum nigrum Solanum dulcamara Solanum carolinense

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

5-13

Chapter 5 Removing Areas From Quarantine

TABLE 5-2 Weed Hosts of Ralstonia solanacearum race 3 biovar 2


Jimson weed Purslane Mustards Lambsquarters Bittergourd Datura stramonium Portulaca oleracea Brassica spp. Chenopodium album Momoridica charantia

Pots and Media


Potsandsoil,planting,orrootingmedia,thathasbeenincontactwithpositive testedplantshipmentsandotherpotentiallyinfectedplantmaterialarealso regulatedandsubjecttodestruction,disposal,anddisinfectionprocedures (SeeControlonpage 61).

Tools and Equipment


Tools,implements,equipment,benches,andgreenhouses,usedincultivation thatmayhavecontactedpositivetestedplantmaterialaresubjectto disinfection.SeeControlonpage 61formoreinformation. Otherregulatedarticlesincludeanyotherproducts,articles,ormeansof conveyance,ofanycharacterwhatsoever,whenitisdeterminedbyan inspectorthattheypresentahazardofspreadofRalstoniasolanacearumrace3 biovar2andthepersoninpossessionthereofhasbeensonotified.

Removing Areas From Quarantine


PlantsheldcanbereleasedfromPPQForm523EmergencyAction Notification(EAN)conditionsifanyofthefollowingcanbedemonstrated: Nosuspectgeraniumsfromspecifiedsuspectrootinganddistribution facilitieswerereceivedatthedestinationnurseryduringthespecified timeperiods. Nootherhostplantsfromthefacilityhaveshownwiltsymptoms,orif wiltwasdetected,theyweresubsequentlysampled,tested,andfound negativeforRalstoniasolanacearumrace3biovar2. SuspectgeraniumsassociatedwithpositiveconfirmeddetectionsofR. solanacearumrace3biovar2weredisposedofordestroyedalongwith associatedpotentiallyinfectedplants.Thisincludesremovaland destructionofweedhostsandthedisinfectionofnurseryareaswhere destroyedplantswereheldincludingthetoolsandotherequipment thatmayhavecomeincontactwithsuspectgeraniumsandpotentially contaminatedplants.SeeControlonpage 61formoreinformation.

5-14

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Chapter 5 Requirements For Potato Cropping Systems Under Quarantine

Additionally,beforerelease,furthertracebackortraceforwardinvestigations shouldnotindicateariskofdisseminationofRalstoniasolanacearumrace3 biovar2tootherpartsofthenurseryproperty

Requirements For Potato Cropping Systems Under Quarantine


TheinformationinthissectionhasbeensummarizedinTable 53, RequirementsforPotatoCroppingSystemsUnderQuarantine, on page 16.

Properties with Positive Testing or Associated Fields


Ifapropertyhaspositivetestingorpositiveassociatedfields,thendestroyor disposeofallplantmaterialthatcanbereasonablyremovedfromthefield. Thisincludescullpilesandotherplantdebris.Leavethefieldfallowfortwo years,andirrigatetopromotevolunteersprouting.Duringthetwogrowing seasons,thevolunteercropmustbesampled,tested,anddiskedunderevery fourweekstoeliminatehostmaterial.Weedhostsinthefieldandalongthe edgeswillbesprayedwithefficaciouslabeledherbicidestoeliminatethem fromthearea. Fieldsinthefollowingtwoyearsaretobeplantedwithnonhostcropsand irrigatedtopromotevolunteersproutingandvolunteerpotatodestruction. Fieldsmustbetestedsemiannuallyforfouryearsafteraninitialpositivefind. SeeSurveyProceduresonpage 31formoreinformation. Furthermore,noseedproductioncanoccurinfieldforatleast5yearsafter detectionofRalstoniasolanacearumrace3biovar2.Fieldswithsusceptible hosts(potatoesortomatoes)mustbesampledfortwoseasonspriortoany newseedproductionontheproperty.

Fields Adjacent to Positive-Testing or Associated Fields, or Sharing Water


Fieldsadjacenttopositivetestingorassociatedfields,orthoseonashared watersituation,maynotgrowhostcropsfortwoyears,seedpotatoesfortwo years,andmusttestandcontrolanyvolunteerpotatoorweedhosts.Strict sanitationofallvehicleenteringandleavingtheinfestedfieldswillbe maintained.

Storage Facilities
Storagefacilitiesonpropertieswithpositivetestingtubersmustbeheldin quarantineuntilallpotentiallyinfestedtubersaretestedandeitherfoundto benegativeordestroyed.Thestoragefacilitiesmustthenbeproperlycleaned anddisinfectedwithapproveddisinfectantsinstrictaccordancewithlabeling. SeeDisinfectantsonpage G1formoreinformation.

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

5-15

Chapter 5 Requirements For Potato Cropping Systems Under Quarantine

TABLE 5-3 Requirements for Potato Cropping Systems Under Quarantine


And in 1st and 2nd years: Leave the field fallow and irrigate to promote volunteer sprouting. During the growing season of each year: Volunteer crop must be sampled, tested, and disked under every four weeks to eliminate host material. Weed hosts in the field and along the edges must be sprayed with efficacious labeled herbicides to eliminate them from the area. And in 3rd and 4th years: Plant with non-host crops and follow cultural practices from the 1st and 2nd years (i.e., irrigation and destruction of volunteer potatoes). And for at least 5 years after detection: No seed production can occur in field after detection. Fields with susceptible hosts (potatoes or tomatoes) must be sampled for two seasons prior to any new seed production on the property.

If: A property has positive testing or positive associated fields

Then: Destroy or dispose of all plant material that can be reasonably removed from the field, including: Cull piles Other plant debris Storage facilities on the properties must be held in quarantine until all potentially infested tubers are tested and either found to be negative or destroyed. The storage facilities must then be properly cleaned and disinfected with approved disinfectants in strict accordance with labeling. See Disinfectants on page G-1 for more information.

Fields are adjacent to positive testing or associated fields, or are on a shared water situation

Do not grow host crops or seed potatoes, Test and control any volunteer potato or weed hosts. Maintain strict sanitation of all vehicles entering and leaving the infested fields.

Volunteer Potatoes and Cull Piles


Fieldswithvolunteerpotatoesmaycomeunderregulationsinagenerally infectedgrowingareamayrequiresamplingandtestingiftheyareconsidered potentiallyinfested.

5-16

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Chapter 5 Requirements For Potato Cropping Systems Under Quarantine

Cullpilesmaybefoundnexttofields,onotherfarmpropertyareas,ornear storagefacilitiesorprocessors.Thesearepotentialreservoirsofthepathogen andareregulateduntiltherisklevelisdeterminedbyinvestigationsand/or testing.

Harvesting Equipment, Storage Areas, and Conveyances


Potatoharvestingequipmentincludingtrucks,harvesters,trailers,machinery, tools,fieldboxes,workerfootwearorimplements,transportconveyances,and storagefacilities,aresubjecttocleaninganddisinfectionifpositiveassociated.

Regulatory Requirements of Seed Producers


Seedtuberproducersmustdestroyand/ordisposeofallpositivetestedand positiveassociatedlots.Theymusttestalllotsintheirinventoryforthe presenceofRalstoniasolanacearumrace3biovar2.Theymustfurthercleanand disinfectallequipment,storageareas,andconveyancesusedintheirbusiness enterprise.RegulartestingforR.solanacearumrace3biovar2mustbecomea partoftheirseedcertificationprogram.

Regulatory Requirements for Processors


Companiesthatmayhavereceivedpotatoesfrompositiveharvestedfieldsare subjecttoholds,inspection,testing,andpossibledisinfectionrequirements. Processorscanincludefacilitiesthatwashpotatoesfortableconsumption (warepotatoes),orproducepotatochips,fries,orstarch. Inallcases,thefacilitiesreceivedpositivetestingorpositiveassociatedstock, oriftestsarepositive,thewaterandequipmentusedinprocessingandthe wasteproductsfromprocessingwillrequireregulatoryaction.Placeholdson facilities,watersources,highriskplantmaterialfromprocessinguntil assessmentsaremadetodeterminewhichpartsofthefacilitywillrequire disinfection,watertreatment,andproperdisposalofhighriskwastematerial. Processorreceivingpositivetestingorpositiveassociatedpotatoesmust destroyordisposeofallofthosepotatoesintheirinventory.Allstorageareas, processingequipment,andconveyancesmustbedisinfected.Allwater sourcesorstoragepondsrecirculatedforprocessingmustbetestedand decontaminatediffoundtobepositive. Beforereleaseofafacility,PPQOfficersmustconductamonitoringinspection anddocumentontheEANthatnopositivesforRalstoniasolanacearumrace3 biovar2weredetectedor,ifpositivesweredetected,thenalldestruction, disposal,anddisinfectionactionstakenwithdatestaken,location,and witnessedbywhom. Notifynurseryowner/managersthattheirfacilitiesmaybesubjectto additionalmonitoringbystateorfederalofficialsforthepresenceofRalstonia solanacearumrace3biovar2.

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

5-17

Chapter 5 Use Of Chemicals

Ifinvestigationsdeterminethequarantinerestrictionsonpotatofieldsare adheredtoovertheprescribedtimeperiods,actionsaredocumented,and watersourcesaretestedandfreeofinfection,fieldscanbereleasedfrom quarantinerestrictions.Notifygrowersthattheirfieldsmaybesubjectto additionalmonitoringbystateorfederalofficialsforthepresenceofRalstonia solanacearumrace3biovar2.

Use Of Chemicals
ThePPQTreatmentManualandthisGuidelinesidentifytheappropriately labeledchemicals,anddescribethemethodsandratesofapplication. ConcurrencebyPPQisnecessarybeforeusinganyotherchemicalor procedureforregulatorypurposes.

5-18

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

6
Ralstonia solanacearum r 3 b 2

Chapter 6
Control

Contents
Introduction page 61 HandlingOfSamples page 62 PlantDisposalAndDestructionMethods DisinfectionProcedures page 64 ApprovedDisinfectants page 66 page 63

Introduction
UseChapter6ControlasaguidetothecontrolofRalstoniasolanacearumrace3 biovar2.USDAAPHISPlantProtectionandQuarantine(PPQ)developsand makescontrolmeasuresforR.solanacearumrace3biovar2availableto involvedstates.EnvironmentalProtectionAgency(EPA)approvedand labeledtreatmentswillberecommendedwhenavailable.

Laws Pertaining to Pest Control


TheFederalInsecticide,Fungicide,andRodenticideAct(FIFRA)authorizes theEnvironmentalProtectionAgency(EPA)toregulatepesticides.All personsusingandapplyingpesticidesshouldunderstandthelawspertaining topesticideuseandapplication.ThefollowingareprovisionsofFIFRAthat aremostpertinenttoemergencypestcontrolprograms: Restrictedusepesticidesmustbeappliedbyacertifiedapplicator. Useofanypesticideinconsistentwiththelabelisprohibited. Violationscanresultinheavyfinesorimprisonment. Statescanregisterpesticidesonalimitedbasisforspeciallocalneeds accordingtothefollowingSections: Section2(ee)DisinfectionsconductedagainstthetargetpestRalstonia solanacearummustuseaFIFRASection2eeuntilthatnameisspecifically addedtothelabel.Thisdesignationisnecessaryifthesynonym Pseudomonaswasusedonthelabelsofrecommendedproducts. Pseudomonasisthegenusnamethatwasusedpreviously,andis synonymouswithRalstonia. Section18EPAadministratorscanexemptfederalorstateagencies fromFIFRAifitisdeterminedthatemergencyconditionsexistthat requiresuchexemptions.

1
May 12, 2008 Emergency and Domestic Programs Control Procedures.fm Ralstonia solanacearum r 3 b 2 6-1

Chapter 6 Handling Of Samples

Section24Astatecanprovideregistrationforadditionalusesof federallyregisteredpesticidesformulatedfordistributionanduse withinthatstatetomeetspeciallocalneedsinaccordancewiththe purposesofthisact. Foradditionalinformationconcerningexemptions,seetheEmergency ProgramsManual,Section14.ContactEnvironmentalServicesstafftoassure thatanypesticidebeingconsideredaspartofaneradicationprogram conformstopesticideuserequirements.Obtainallrequiredenvironmental documentationbeforebeginning.SeeEnvironmentalComplianceon page A1formoreinformation.

Control Records
Programpersonnelmustmaintainrecordsandmaps,notingthelocationsof alldetections,thenumberandtypeofplantssubjectedtocontrolactions,and thematerialsandformulationsusedineachtreatedarea.Attachall documentationandreceiptstotheofficecopyofthePPQForm523 EmergencyActionNotification.SeePPQForm523EmergencyAction Notificationonpage B7formoreinformation.

Control Decisions and Oversight


Important All quarantine actions related to destruction are to be witnessed, supervised, and documented by a PPQ officer whenever possible. Because Ralstonia solanacearum race 3 biovar 2 is listed as a select agent under the Agriculture Bioterrorism Protection Act of 2000, proper supervision and documentation of destruction of infected plant material is critical. If a PPQ officer is not available, a state cooperating inspector can witness and document the disposal. Disinfections can be witnessed and documented by PPQ officers or state cooperators.

Handling Of Samples
Placeallpotentiallyinfectedplantsindoubleplasticbagsandsealfordisposal ordestruction,includingthefollowing: Shipmentsofsuspectgeraniums AssociatedpotatoplantsortubersthathavetestedpositiveforRalstonia solanacearumrace3biovar2 Pots,directlyassociatedsoilorpottingmedia,andanyotherdisposable itemincontactwiththeplantsorsoil Hostweedsinpositivetestedfacilitiesmustalsoberemovedandcorrectly disposedofordestroyed.SeePlantDisposalAndDestructionMethodson page 63formoreinformation Maintainanaccuratecountofeachtype(variety,etc.)ofplantortubers baggedfordestructionandrecordthisinformation.

6-2

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Chapter 6 Plant Disposal And Destruction Methods

Iflargeinventoriesmustbedestroyed,theuseofplasticbagsmaynotbe reasonable.Alternativesmayincludeadumpsterwithdoublelayersofplastic liningthatcanbefoldedoverthetopandsealedtopreventdebrisfrom escapingduringtransportorstorage.ContactyourregionalofficeorPPQ Headquartersforadditionalguidance. Allbenchareaswhereplantswereheld,andotherareasatriskforexposureto infectedplantmaterial,shouldbedisinfectedaccordingtoprescribed disinfectionprocedures.SeeDisinfectantsonpage G1formore information.

Plant Disposal And Destruction Methods


Incinerationandsteamsterilizationarethepreferredmethodsofdisposal, however,theseoptionsmaynotbepracticalforlargeamountsofwaste.In thesecases,useanapprovedlandfill.

Burning or Incineration
Allplantmaterialandmediamustbeincineratedorburnedtothepointof ash.Ifplasticpotsarenotacceptedattheincinerator,removethemand properlydisinfectorsendtoalandfill.

Steam Treatment Under Pressure


Useanautoclave,orfollowtheguidelinesinthePPQTreatmentManual.

Soil Fumigation
Soilfumigationwithanapprovedfumigantsuchasmethylbromideor metamsodiumatthelabeledratewillgreatlyreducepopulationsofthe bacteriaininfestedfields.However,fieldswithhostplantresidueincluding tubers,roots,andstems,maystillharboradequateamountsofRalstonia solanacearumforuptotwoyears.Beforefumigation,removeanddestroyas muchhostplantmaterialfromthefieldsaspossible. Fumigationofthesoilmaynottotallyeliminatethepathogenduetothe presenceofbacterialreservoirsinburiedplanthostresidueatthetimeof fumigation.ContinuedmonitoringofthesoilforthepresenceofRalstonia solanacearumrace3biovar2willberequired.

Approved Landfill
Anapprovedlandfillisastatelicensedmunicipalorprivatefacilitythatis managedunderstateregulationtomeetconditionsthatwouldprevent potentialpollutantsfromleachingintogroundwater.

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

6-3

Chapter 6 Disinfection Procedures

SeeDisposalMemorandumonpage B9foraUSDAMemorandumfor Recordconcerninglandfillsthatmaybeusedtoalleviateconcernsoflocal officials,landfilloperators,orthepublic.


Important This plant pathogen is not considered a hazardous waste, biomedical waste, epizootic, or any other substance of concern for human healthit is an agricultural pathogen only. Proper disposal is required because there is a remote possibility that it could reach ground water for eventual uptake from wells used for irrigation of host crops. The concentration of the bacteria will be minimal relative to the volume of material disposed of, so the risk is low if the landfill has normal safeguards in place.

Theregulatoryofficialthatwitnessesthedisposalofdoublebaggedmaterial fromnurseriesorpotatocroppingsystemsmustassurethematerialisburied undertwoormorefeetofsoil.Notifythelandfillinadvancetoarrangefora holetobeduginwhichthematerialcanbedisposedandcoveredwithsoil.If diggingaholeisimpracticalforalandfilloperation,anotheroptionistocover theplantmaterialwithalternativedailycoverorcompostedgreenwaste normallyusedtocoverrefuseatlandfills.Theseoperationsmustbe witnessed,supervised,anddocumentedbyaplantregulatoryofficial. Thequantitiesofpotatohostmaterialorderedfordestructionmaybe impracticalforthedisposaloptionsprescribed.Compostingwilllikelyheat thetemperatureofhostmaterialtosufficienttemperaturesnecessarytokill thepathogen.Theparametersofacomposingdisposalmethodmustbe proposedbyaRalstoniatechnicalgroup.Theefficacyofcompostingmustbe testedbyCPHSTpriortoimplementationasaprogrammethod.Caremustbe takentocontrolwaterrunofffromcomposingoperationsifimplemented.

Disinfection Procedures
Usethefollowingprocedurestodisinfectallpotentiallycontaminated surfaces(benches,flats,walkways,footbaths,drainageareasunderbenches) andequipmentwithinaninfectedgreenhouseorareaincontactwithinfected material: 1. Cleanawayanysoilormedia. 2. Sprayandsoaktothepointofrunoffwithapproveddisinfectantsorby pressurizedsteamcleaningtoraisethesurfacetemperatureto212oF. Formoreinformation,consultthefollowingsources: ApprovedDisinfectantsonpage 66 Disinfectantsonpage G1 PPQTreatmentManual

6-4

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Chapter 6 Disinfection Procedures

Tools and Equipment


Usethefollowingprocedurestodisinfecttoolsandequipmentthatmayhave comeincontactwithinfectedplantsorcontaminatedsoil: 1. Cleanawayanysoilormedia. 2. Applyapproveddisinfectantstothepointofrunoff,orthoroughlywash withpressurizedsteamtoraisethetemperatureto212oF. SeePPQTreatmentManualformoreinformation.

Exposed Irrigation Systems


Drainandcleanthefollowingsystems: Recirculatingirrigationsystem Subirrigationsystem Anysystemthatdoesnotpreventbackflowofwaterfrominfected greenhouses Cleanallparts,sumps,andpumps,withapproveddisinfectantsolutions.To beeffectiveagainstRalstoniasolanacearumrace3biovar2,thesystemsmust haveozonationwith0.4ppmresidualO3for4minuteswithUVlightofat least300j/m2at>50%transmission(PPQ,2004).

Holding Ponds
Whenholdingpondshavebecomecontaminatedfromirrigationsystem runoff,contactyourregionalPPQofficeandStateaboutpossible environmentalconsiderationsandtreatmentoptions.

Outdoor Soil or Holding Areas


Whenoutdoorsoilorholdingareashavebecomecontaminatedduringplant storageorrunoff,contactyourregionalofficeandStateaboutpossible environmentalconsiderationandtreatmentoptions.

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

6-5

Chapter 6 Approved Disinfectants

Approved Disinfectants
UsetheapproveddisinfectantsinTable 61todisinfectalltools,benches, walkways,surfaces,andgravelbedsunderbenches,incontactwith potentiallyinfectedplantmaterialafterremovalofsoilormedia.Besurethat thelabelspecifiesRalstoniaorPseudomonas.CheckwithStatelabeling requirements.SeeDisinfectantsonpage G1formoreinformation
TABLE 6-1 Approved Disinfectants
Product Quaternary Ammonia Physan 20 Green Shield Maquat 615-HD or 615-LR ZeroTol

Notes Several quaternary ammonia (20% ammonium chloride) products are approved for greenhouse use. Physan 20 is not approved for greenhouse where food crops are grown. It is not approved for use in California Green Shield is approved for most uses. In California, use the special formulation Green ShieldCA. None ZeroTol is 27% hydrogen dioxide. It is not a quaternary ammonia, and has rates for disinfection including surfaces with soil or media contamination that cannot be cleaned. This product is also approved for use in disinfection of contaminated irrigation systems.

6-6

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

7
Ralstonia solanacearum r 3 b 2

Chapter 7
Environmental Compliance

Contents
Introduction page 71 Disclaimer page 71 NationalEnvironmentalPolicyAct page 72 EndangeredSpeciesAct page 72 FederalInsecticide,fungicide,AndrodenticideAct OtherLaws page 73 EnvironmentalMonitoring page 73

page 73

Introduction
UseChapter7EnvironmentalComplianceasaguidetoenvironmental regulationspertinenttotheplantpathogenRalstoniasolanacearumrace3 biovar2ingeraniums,potatoes,andothersolanaceoushosts. Akeyelementindesigningaprogramoranemergencyresponseis consultationwithEnvironmentalServices(ES),aunitofAPHISPolicyand ProgramDevelopmentstaff.ESperformsthefollowingfunctions: Preparesenvironmentaldocumentationsuchasenvironmentalimpact statementsandenvironmentalassessmentstoaidinmakingdecisions Providesconsultationonthetopicofendangeredspecies CoordinatespesticideregistrationandapprovalsforAPHISprograms, ensuringthatregistrationsandapprovalsmeettheneedsofprograms andconformtopesticideuserequirements Inaddition,PPQsEnvironmentalComplianceteamassistsESinthe developmentofrequireddocumentationandimplementsanyenvironmental monitoringthatmayberequiredofprogramactivities.RefertoResourceson page A1foradditionalinformation.

Disclaimer
AllusesofpesticidesmustberegisteredorapprovedbyappropriateFederal, State,and/orTribalagenciesbeforetheycanbeapplied.Theinformation providedonpesticidelabelsmaynotreflectalloftheactualinformation, includingprecautionsandinstructionsforuse,whichyouarerequiredto followinyourspecificstateorlocality.Itistheresponsibilityofpersons
May 12, 2008 Emergency and Domestic Programs Env.fm Ralstonia solanacearum r 3 b 2

7-1

Chapter 7 National Environmental Policy Act

intendingtouseapesticidetoabidebythelabel,includinglabelingthathas beenapprovedfortheparticularstateorlocalityinwhichthechemicalistobe used,andtocomplywithallfederal,state,tribal,andlocallawsand regulationsrelatingtotheuseofthepesticide.StaffwithinAPHISprograms areresponsiblefortheircompliancewithapplicableenvironmental regulations,whichoftenincludemeasuresaboveandbeyondthoselistedon pesticidelabels.

National Environmental Policy Act


TheNationalEnvironmentalPolicyAct(NEPA)requiresthatfederalagencies documentthepotentialadverseeffectsoftheiractions.Theprocessoften requirespublicinput.Theexactnatureofthedocumentationandpublic involvementisdictatedbythepotentialforadverseeffectsandthe significanceofthoseeffects. Itislikelythatmostpestcontrolresponseswillincludeactionsthatneedupto 30daysofpubliccommentpriortoinitiation.Therefore,itisimperativeto involveEnvironmentalServicesandEnvironmentalComplianceearlyinthe planningprocess.Doingsoassurespublicinvolvementandaquickresponse. Dependingontheproposedprogram,NEPArequirementswillbemetwitha categoricalexclusion,environmentalassessment,orenvironmentalimpact statement.SomeprogramscanpreparetheirownNEPAdocumentation. ContactEnvironmentalServicesorEnvironmentalComplianceifyouare unsurewhichdocumentshouldbeprepared,orifyouhavelittleexperience writingsuchdocuments.RefertoResourcesonpage A1forcontact information.

Endangered Species Act


TheEndangeredSpeciesAct(ESA)requiresthatallfederalactions,including emergencyresponses,donotharmfederallyprotectedthreatenedor endangeredspecies.Beforeanactioncanbegin,itmustbedeterminedif protectedspeciesareintheprojectarea.Ifsuchspeciesarepresent,measures mustbeputinplacetoprotectthemfrompotentialadverseeffectsofthe action.SuchworkrequirescoordinationwiththeU.S.FishandWildlife Serviceand/ortheNationalMarineFisheriesService. SeveralmethodsareavailabletoensurecompliancewithESA,buttheexact onechosenisdictatedbythenatureoftheemergency,proposedresponse,and location.Assoonaspossibleintheearlystagesoftheresponse,contactstaffat EnvironmentalServicesorEnvironmentalCompliance,whocanprovidethe necessaryguidanceandsupportinconductingthenecessaryanalysesand developingtherequireddocumentation.RefertoResourcesonpage A1for contactinformation.

7-2

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Chapter 7 Federal Insecticide, fungicide, And rodenticide Act

Federal Insecticide, fungicide, And rodenticide Act


TheFederalInsecticide,Fungicide,andRodenticideAct(FIFRA)requiresthat chemicalsusedforcontrolhaveapprovedlabelsandthatalllabel requirementsarefollowed.Theserequirementscanincludeapplicableuses, maximumapplicationrates,handlinginstructions,andpersonalprotective equipment.Ifnolabelisavailablefortheemergencyinquestion(i.e.,thepest ofconcernisnotlistedasoneforwhichthechemicalmaybeused),itis possibletoobtainanewlabeloralabelexemption.Ifalabelchangeisneeded ornolabelcanbelocatedforyourprogramneeds,immediatelycontact EnvironmentalServices,whocanassistinlabelchangesandemergencyuse exemptions.RefertoResourcesonpage A1forcontactinformation.

Other Laws
TheNationalEnvironmentalPolicyAct,EndangeredSpeciesAct,andthe FederalInsecticide,Fungicide,andRodenticideAct,areofcriticalimportance toallpestcontrolprograms,butotherlawsmayapplydependingonprogram locationsandactivities.TheseincludetheMigratoryBirdTreatyAct,the CoastalZoneManagementAct,andtheBaldandGoldenEagleProtection Act.ByincludingEnvironmentalServicesandEnvironmentalCompliance earlyinprogramplanning,guidancecanbeprovidedonmeetingthe requirementsoftheseandotherlawsthatmayapply.RefertoResourceson page A1forcontactinformation.

Environmental Monitoring
EnvironmentalmonitoringofAPHISpestcontrolactivitiesmayberequired aspartofcompliancewiththeabovelaws,asrequestedbyprogram managers,orassuggestedtoaddressconcernswithcontroversialactivities. Thisisespeciallytrueforlessbenignchemicalcontrolsandaerialapplication ofchemicals. Monitoringmaybeconductedwithregardstoworkerexposure,quality assuranceandcontrol,offsitedeposition,orprogramefficacy.Differenttools andtechniquesareuseddependingonthemonitoringgoals,program chemicals,andcontroltechniques.Environmentalmonitoringiscoordinated byEnvironmentalCompliance(EC).StafffromECwillworkwiththe programmanagertodevelopanenvironmentalmonitoringplan,conduct trainingtoimplementtheplan,providedaytodayguidanceonmonitoring, andprovideaninterpretivereportofmonitoringactivities.

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

7-3

Chapter 7 Environmental Monitoring

7-4

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

Glossary
approvedlandfill.Statelicensedmunicipalorprivatelandfillthatismanaged understateregulationtomeetconditionsthatwouldpreventleachingof potentialpollutantsintogroundwater. biovar.Groupofbacterialstrainsthataredistinguishablefromotherstrainsof thesamespeciesonthebasisoftheirphysiologicalcharacteristics. chlorosis.Yellowingofnormallygreentissueduetochlorophylldestruction ininfectedplants. decontamination.Applicationofanapprovedchemicalorothertreatmentto contaminatedimplements,material,orbuildingsforkillingordeactivatinga pathogen. detectionsurvey.Surveyconductedinanenvironmentallyfavorablearea whereRalstoniasolanacearumrace3biovar2isnotknowntooccur. destinationnursery.Anynurseryreceivingsuspectgeraniumsorsuspect plantsfromarootingstationorothernursery. disposal.Methodusedtoeliminatediseasedplantmaterialormaterial associatedwithdiseasedplantmaterial,usuallyatanapprovedlandfill. ELISA.AcronymforEnzymeLinkedImmunosorbentAssay,aserological laboratorytechniqueusedtodeterminethegenusandspeciesinaRalstonia hostsamplingandtestingprogram;excludesraceandbiovar. EnzymeLinkedImmunosorbentAssay.SeeELISAabove. host.Plantwhichisinvadedbyaparasiteorpathogenandfromwhichit obtainsitsnutrients. incineration.Burningofplantsandassociatedsoilormediathatresultsin theircompletedestruction. infection.Establishmentofaparasiteonorwithinahostplant. monitoringsurvey.Surveyconductedatasitewhereadiseasewasfoundand whereaneradicationprogramisbeingperformed. necrosis.Deadordiscoloredplanttissue.

1
Glossary-1

May 12, 2008 Emergency and Domestic Programs Glossary.fm

Ralstonia solanacearum r 3 b 2

pathogen.Microorganismthatcaninciteadisease. PCR.AcronymforPolymeraseChainReaction,alaboratorytechniquethat amplifiesDNAsequencesinordertodeterminetheraceandbiovarina Ralstoniahostsamplingandtestingprogram. positiveassociated.Seedorwarepotatoes,infields,orasharvested,orin storage,atshippingpoints,orinprocessingfacilitiesthataredirectlylinkedto positivetestingforRalstoniasolanacearumrace3biovar2. positivetesting.Seedorwarepotatoes,infields,orasharvested,orin storage,atshippingpoints,orinprocessingfacilitiesthatareconfirmed positiveforRalstoniasolanacearumrace3biovar2. potentiallyinfestedtubers.Seedorwarepotatoes,infields,orasharvested, orinstorage,atshippingpoints,orinprocessingfacilitiesthatareindirectly linkedtopositivetesting,forRalstoniasolanacearumrace3biovar2. PolymeraseChainReaction.SeePCRabove. potentiallyinfectedplants.Withinthenurseryproperty,theseinclude: Allplantsunderthedriplineofhangingsuspectgeraniums Otherplantsthathavebeenplantedinthesamepotswithsuspect geraniums Allplantsonasharedwaterirrigationsystemwithsuspectgeraniums, includingthefollowing: Ebbandflow Flood Subirrigation Systemsthatlackofbackflowprevention Allhostplantsthatmayhavebeeninfectedbyapositivetestingsuspect geraniumshipmentthroughunsanitarygreenhousepractices,including thefollowing: Failuretodisinfecttools,hands,orequipmentduringgrafting, pruning,debudding Deleafingbetweenvarieties Plantsonthegroundwithanonporoussurfacesuchthat puddlingmayoccurunderplants

Glossary-2

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

potentiallyinfectedplantswithinnurseryproperty.Includesallplantsthat arelocatedunderthedriplineofhangingsuspectgeraniumsoronashared irrigationsystemwithapositivetestingsuspectgeranium.Alsoincludes plantsthatmayhavebeeninfectedbypositivetestingsuspectgeraniums throughunsanitarygreenhousepractices. positivetestinggeraniums.GeraniumsinwhichRalstoniasolanacearumrace3 biovar2hasbeenconfirmedtobepresentbytheUSDAPPQCPHST laboratoryinBeltsville,MD. shipment.Allplantsthatwerepreparedforshipmentandtransportedwith suspectgeraniumsfromacommonsupplier,i.e.,listedontheinvoiceor packinglist. suspecthosts(geraniumsorpotatoes).Geraniums(Pelargoniumspp.)thatare associatedwithpositivetestinggeraniums.Thesemayhaveoriginatedfroma foreigncountry,arootingstation,oranothernurseryandbethesamevariety orinthesameshipmentdependingonthecircumstancessuspecttubers.Also, potatotubersinfieldsorstoragefacilitiesthatareassociatedwithpotato tubersthathavetestedpositiveforRalstoniasolanacearumrace3biovar2. suspectgeraniums.Geraniums(Pelargoniumspp.)thatareassociatedwith positivetestinggeraniums.Thesemaybeaparticularimplicatedvarietyor shipmentthatoriginatedfromarootingstation,directshipfacility,oranother nursery. suspectshipment.Allhostplantsthatwerepreparedforshipmentand transportedwithsuspectgeraniumsfromacommonsupplier(listedonthe invoiceorpackinglist). symptom.Theexternalandinternalreactionsoralterationsofaplantasthe resultofadisease. traceback.Toinvestigatetheoriginofinfestedplantsthroughintermediate stepsincommercialdistributionchannelstotheorigin. traceforward.Toinvestigatewhereinfectedplantsmayhavebeendistributed fromasourcethroughstepsincommercialdistributionchannels.

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

Glossary-3

Glossary-4

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

References
Buddenhagen,I.W.,L.SequeiraandA.Kelman,A.1962.Designationofraces ofPseudomonassolanacearum.Phytopathology52:726. CABI.2003.CropProtectionCompendium.UnitedKingdom:Wallingford: CABInternational.Onlinedatabase.http://www.cabicompendium.org/cpc. AccessedNovember4,2003. CABI.2006.CropProtectionCompendium.UnitedKingdom:Wallingford: CABInternational.Onlinedatabase.http://www.cabicompendium.org/cpc. AccessedJuly2007. Cook,D.R.andL.Sequeira.1994.StrainDifferentiationofPseudomonas solanacearumbyMolecularGeneticMethods.,pages7793.In:A.C.Hayward andG.L.Hartman(editors).BacterialWilt:TheDiseaseanditsCausative Agent,Pseudomonassolanacearum.CABInternational,Wallingford,UK.259pp. Daughtrey,M.March4,2003.SouthernBacterialWilt,causedbyRalstonia solanacearum.In:SummaryofPresentationatSocietyofAmericanFlorists19th AnnualConferenceonInsectandDiseaseManagementonOrnamentals. http://www.greenhouse.cornell.edu/pestsdis/MD.SAFtalk.pdf. DepartmentforEnvironment,FoodandRuralAffairsofUnitedKingdom (DEFRA).2003a.PlantPathology.www.defra.gov.uk/planth/phnews/ openday/brown.pdf.AccessedMarch2003. DepartmentforEnvironment,FoodandRuralAffairsofUnitedKingdom (DEFRA).2003b.PlantPathology.http://www.defra.gov.uk/planth/stake.pdf. AccessedMarch2003. DepartmentforEnvironment,FoodandRuralAffairsofUnitedKingdom (DEFRA).2003c.PlantPathology.http://www.defra.gov.uk/science/LINK/ Publications/Newsletters/AgricultureLINK/AgLINK_Issue3.pdf.Accessed March2003. Denny,T.P.andA.C.Hayward.2001.Ralstonia,pages151174.In:Schaad,N. W.etal.LaboratoryGuidefortheIdentificationofPlantPathogenicBacteria, 3rded.APSPress.Minnesota:St.Paul.373pp. Douglas,S.M.2002.DiseasesofGeranium.TheConnecticutAgricultural ExperimentStation.Connecticut:NewHaven.

1
Monday, May 12, 2008 Emergency and Domestic Programs Ralstonia solanacearum r 3 b 2 References-1

EconomicResearchService(ERS).2003.StatisticalIndicators. USDAEconomicResearchServicePublications.http://www.ers.usda.gov/ publications/. French,E.R.,L.Gutarra,P.AleyandJ.Elphinstone.1995.Culturemediafor Pseudomonassolanacearum:isolation,identificationandmaintenance. Phytopathologia30(3):126130. FrenchE.R.1998.RecentSpreadofBacterialWiltofPotato:Regional PerspectivesandExperiences,LatinAmerica,pages426427.In:Prior,P.,C. AllenandJ.Elphinstone(eds.).BacterialWiltDisease:Molecularand EcologicalAspects.ReportsoftheSecondInternationalBacterialWilt SymposiumheldinGosier,Guadeloupe,France,2227June,1997. SpringerVerlag.BerlinHeidelberg,INRA,Paris.447pp. Garret,L.J.2003(Draft).PotentialLossesofRalstoniasolanacearumrace3, biovar2.USDAAPHISCPHST,Raleigh,NC. Hayward,A.C.1964.CharacteristicsofPseudomonassolanacearum.J.Appl. Bacteriol.2:265277. Hayward,A.C.1991.Biologyandepidemiologyofbacterialwiltcausedby Pseudomonassolanacearum.Annul.Rev.Phytopathol.29:6587. Horita,M.andK.Tsuchiya.2001.GeneticdiversityofJapanesestrainsof Ralstoniasolanacearum.Phytopathology91:399407. Hudelson,B.D.May11,1999.SouthernBacterialWilt.Univ.Wisconsin GardenFacts. Janse,J.D.1991.InfraandintraspecificclassificationofPseudomonas solanacearumstrainsusingwholecellfattyacidanalysis.SystematicandApplied Microbiology14:335345. Janse,J.D.1996.PotatoBrownRotinWesternEuropeHistory,Present OccurrenceandSomeRemarksonPossibleOrigin,EpidemiologyandControl Strategies.Bull.OEPP26:679695. Janse,J.D.,H.E.VanDenBeld,J.Elphinstone,S.Simpkins,N.TijoiTamSin, andV.Vaerenbergh.2003.IntroductiontoEuropeofRalstoniasolanacearum Biovar2/Race3inPelargoniumzonaleCuttings.AmericanPhytopathological Society. Kelman,A.1953.ThebacterialwiltcausedbyPseudomonassolanacearum.A literaturereviewandbibliography.NorthCarolinaAgriculturalExperiment StationTechnicalBulletinNo.99.194pp. Kim,S.H.andT.N.Olson.2003.RalstoniasolanacearumRace3,Biovar2,the CausalAgentofBrownRotofPotato,IdentifiedinPennsylvania,Delaware andConnecticut.PlantDisease(87)4:450.

References-2

Ralstonia solanacearum r 3 b 2

Monday, May 12, 2008 Emergency and Domestic Programs

Lee,et.al.2001.PlantDisease85:261266. Meiners,U.2000.Bacterialringrot:possibilitiesandlimitsofinsurance coverage.http://ibws.nexenservices.com/from_july_%2001.htm. Melton,T.A.,andH.D.Shew.2000.GranvilleWilt.TobaccoDisease InformationNote.TDIN002.NorthCarolinaStateUniversity,Collegeof AgricultureandLifeSciences,PlantPathologyExtension.http:// www.ces.ncsu.edu/depts/pp/notes/Tobacco/tdin002/tdin002.htm. NorthAmericanPlantProtectionOrganisation(NAPPO).2001.Ralstonia (Pseudomonas)solanacearum(E.F.Smith,1896)Yabuuchietal.1995race3 biovar2.PhytosanitaryAlertSystem.http://www.pestalert.org.Accessed March2003. NationalInstituteofAgricultureScienceandTechnology.2003.Research Highlights.http://www.niast.go.kr/english/sub/highlights.htm.AccessedMay 15,2002. Momol,T.,J.Jones,andS.Olson.2003.NewOutbreakofRalstonia solanacearum(race3biovar2)ingeraniumsinUnitedStatesandeffectsof biofumigantsonRalstoniasolanacearum(race1biovar1).UniversityofFlorida PestAlert.http://extlab7.entnem.ufl.edu/PestAlert/tmm0303.htm. Pittman,H.A.1933.BacterialWiltofTomatoesandotherSolanaceousCrops. West.Austral.Dept.Agr.J.(II)10:373374. PlantProtectionandQuarantine(PPQ).2004.MinimumSanitationProtocols ForOffshoreGeraniumCuttingProduction.USDAAPHISPPQ.http:// www.aphis.usda.gov/plant_health/plant_pest_info/ralstonia/downloads/ ralstoniaworkplan.pdf.AccessedAugust20,2007. Prior,P.andH.Steva.1990.CharacteristicsofstrainsofPseudomonas solanacearumfromtheFrenchWestIndies.PlantDisease74(1):1317. Possier,S.,P.VandewalleandJ.Luisetti.1999.GeneticdiversityofAfricanand worldwidestrainsofRalstoniasolanacearumasdeterminedbyPCRrestriction fragmentlengthpolymorphismanalysisofthehrpgeneregion.Appliedand EnvironmentalMicrobiology65:21842194. Poussier,S.,etal.2002.JournalofMicrobiologicalMethods51:349359. Schaad,N.W.,J.B.JonesandW.Chun.2001,3rded.LaboratoryGuideof IdentificationofPlantPathogenicBacteria.Minneapolis:APSPress. Sequeira,L.1994.Epilogue:LifewithaMutableandTreacherousTribe.In: Hayward,A.C.andG.L.Hartman.(eds.).BacterialWilt.TheDiseaseandits CausativeAgent,Pseudomonassolanacearum.Pp.235247.

Monday, May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

References-3

StatePlantRegulatoryOfficialNotification.2002.Ralstonia2002. USDAAPHISPPQISPM.Vmalik/Mneal:jhm:7348261:02/25/ 02>ralsto~1.doc. Strider,D.L.,R.K.Jones,andR.A.Haygood.1981.SouthernBacterialWiltof GeraniumCausedbyPseudomonassolanacearum.PlantDis.65:5253. Tusiime,G.,E.Adipala,F.Opio,andA.S.Bhagsari.1998.WeedsasLatent HostsofRalstoniasolanacearuminHighlandUganda:Implicationsto DevelopmentofaniNtegratedControlPackageforBacterialWilt,pages 413419.In:Prior,P.,C.Allen,andJ.Elphinstone(editors).BacterialWilt Disease:MolecularandEcologicalAspects.Berlin:SpringerVerlag Publishing;447pp. Wallace,G.B.1934.Reportofthemycologistfor1934.TanganyikaDept.Agr. Ann.Rpt.:9093. Zachos,D.G.1957.ThebrownrotofpotatoesinGreece.AnnalesdelInstitut PhytopathologiqueBenaki,NewSeries1:115117. Zehr,E.I.1969.Studiesofthedistributionandeconomicimportanceof PseudomonassolanacearumincertaincropsinthePhilippines.Philippine Agriculturist53:218223.

References-4

Ralstonia solanacearum r 3 b 2

Monday, May 12, 2008 Emergency and Domestic Programs

A
Ralstonia solanacearum r 3 b 2

Appendix A
Resources
Diagnostic Tools and Equipment
CentralScienceLaboratory SandHutton York YO411LZ UnitedKingdom Telephone:+44(0)1904462000 http://www.csl.gov.uk/ LoeweBiochemicaGmbH Mhlweg2a D82054Sauerlach Germany Telephone:+49810461620 http://www.loeweinfo.com/ NeogenEuropeLimited (AcquiredAdgenLtd.) CunninghamBuilding Auchincruive Ayr,KA65HW Scotland,UK http://www.adgen.co.uk/ AgdiaIncorporated 30380CountyRoad6 Elkhart,Indiana46514USA Telephone:5742642014 Tollfree(USA):8006224342 http://www.agdia.com/ BIOREBAAG STALaboratoriesColorado 1821VistaViewDrive Longmont,CO80504 Telephone:3036516417 http://www.bioreba.ch/ http://www.stalabs.com/

Environmental Compliance
Environmental Monitoring, Categorical Exclusions

USDAAPHISPPQEmergencyand DomesticPrograms EnvironmentalCompliance 4700RiverRoad,Unit150 Riverdale,MD20737 Telephone:3017348247

Environmental Services
FIFRA, ESA, Environmental Assessments

USDAAPHISPolicyandProgram Development EnvironmentalServices 4700RiverRoad,Unit149 Riverdale,MD20737 Telephone:3017348565

1
May 12, 2008 Emergency and Domestic Programs Ralstonia solanacearum r 3 b 2 A-1

Appendix A

A-2

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

B
Ralstonia solanacearum r 3 b 2

Appendix B
Forms and Questionnaires

Contents
PPQForm391SpecimensForDetermination page B2 AddendumtoPPQForm391 page B4 QuestionnaireForNurseryAndPotatoCroppingSystemOwnersOr Managers page B5 PPQForm523EmergencyActionNotification page B7 DisposalMemorandum page B9 PPQForm526PermittoMoveLivePlantPlantsorNoxiousWeeds page B10

1
May 12, 2008 Emergency and Domestic Programs Forms.fm Ralstonia solanacearum r 3 b 2 B-1

Appendix B PPQ Form 391 Specimens For Determination

PPQ Form 391 Specimens For Determination


This report is authorized by law (7 U.S.C. 147a). While you are not required to respond your cooperation is needed to make an accurate record of plant pest conditions.
U.S. DEPARTMENT OF AGRICULTURE ANIMAL AND PLANT HEALTH INSPECTION SERVICE FORM APPROVED OMB NO. 0579-0010 FOR IIBIII USE LOT NO. PRIORITY

See reverse for additional OMB information.

SPECIMENS FOR DETERMINATION


1. COLLECTION NUMBER

Instructions: Type or print information requested. Press hard and print legibly when handwritten. Item 1 - assign number for each collection beginning with year, followed by collectors initials and collectors number. Example (collector, John J. Dingle): 83-JJD-001. Pest Data Section Complete Items 14, 15 and 16 or 19 or 20 and 21 as applicable. Complete Items 17 and 18 if a trap was used. 2. DATE MO DA YR State Cooperator PPQ 3. SUBMITTING AGENCY

Other

4. NAME OF SENDER

5. TYPE OF PROPERTY (Farm, Feedmill, Nursery, etc.)

SENDER AND ORIGIN

6. ADDRESS OF SENDER

INTERCEPTION SITE

7. NAME AND ADDRESS OF PROPERTY OR OWNER

ZIP

COUNTRY/ COUNTY

8. REASON FOR IDENTIFICATION (x ALL Applicable Items)

A.
PURPOSE

Biological Control (Target Pest Name Damaging Crops/Plants

E. F. G. H.

Livestock, Domestic Animal Pest Possible Immigrant (Explain in REMARKS) Survey (Explain in REMARKS) Other (Explain in REMARKS)

B. C. D.

Suspected Pest of Regulatory Concern (Explain in REMARKS) Stored Product Pest

9. IF PROMPT OR URGENT IDENTIFICATION IS REQUESTED, PLEASE PROVIDE A BRIEF EXPLANATION UNDER REMARKS. 10. HOST INFORMATION NAME OF HOST (Scientific name when possible) NUMBER OF ACRES/PLANTS 11. QUANTITY OF HOST PLANTS AFFECTED (Insert figure and Number indicate

HOST DATA

Percent):
12. PLANT DISTRIBUTION 13. PLANT PARTS AFFECTED

LIMITED SCATTERED WIDESPREAD


14. PEST DISTRIBUTION

Leaves, Upper Surface Leaves, Lower Surface Petiole Stem 15.


NUMBER SUBMITTED

Trunk/Bark Branches Growing Tips Roots

Bulbs, Tubers, Corms Buds Flowers Fruits or Nuts NEMATODES

Seeds

INSECTS
PUPAE ADULTS

MOLLUSKS
EGGS NYMPHS JUVS. CYSTS

PEST DATA

FEW COMMON ABUNDANT EXTREME


16. SAMPLING METHOD

LARVAE

CAST SKINS

ALIVE DEAD
17. TYPE OF TRAP AND LURE 18. TRAP NUMBER

19. PLANT PATHOLOGY PLANT SYMPTOMS (X one and describe symptoms)

ISOLATED
20. WEED DENSITY

GENERAL
21. WEED GROWTH STAGE

FEW
22. REMARKS

SPOTTY

GENERAL

SEEDLING

VEGETATIVE

FLOWERING/FRUITING

MATURE

23. TENTATIVE DETERMINATION 24. DETERMINATION AND NOTES (Not for Field Use)

FOR IIBIII USE


DATE RECEIVED

NO. LABEL SORTED PREPARED DATE ACCEPTED SIGNATURE DATE RR

PPQ FORM 391 (AUG 02) PART 1 PPQ

Previous editions are obsolete.

This is a 6-Part form. Copies must be disseminated as follows:


PART 2 RETURN TO SUBMITTER AFTER IDENTIFICATION PART 5 INTERMEDIATE IDENTIFIER PART 3 IIBIII OR FINAL IDENTIFIER PART 6 RETAINED BY SUBMITTER PART 4 INTERMEDIATE IDENTIFIER

FIGURE B-4 Example of PPQ Form 391 [side 1]

B-2

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Appendix B PPQ Form 391 Specimens For Determination

OMB Information According to the Paperwork Reduction Act of 1995, no persons are required to respond to a collection of information unless it displays a valid OMB control number. The valid OMB control number for this information collection is 0579-0010. The time required to complete this information collection is estimated to average .25 hours per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information.

Instructions
Use PPQ Form 391, Specimens for Determination, for domestic collections (warehouse inspections, local and individual collecting, special survey programs, export certification). BLOCK INSTRUCTIONS 1. Assign a number for each collection beginning the year, followed by the collectors initials and collectors number EXAMPLE
In 2001, Brian K. Long collected his first specimen for determination of the year. His first collection number is 01-BLK-001

2. Enter the collection number 2 3 4 5 6 7 8A-8L 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Enter date Check block to indicate Agency submitting specimens for identification Enter name of sender Enter type of property specimen obtained from (farm, nursery, feedmill, etc.) Enter address Enter name and address of property owner Check all appropriate blocks Leave Blank Enter scientific name of host, if possible Enter quantity of host and plants affected Check block to indicate distribution of plant Check appropriate blocks to indicate plant parts affected Check block to indicate pest distribution Check appropriate block to indicate type of specimen Enter number specimens submitted under appropriate column Enter sampling method Enter type of trap and lure Enter trap number Enter X in block to indicate isolated or general plant symptoms Enter X in appropriate block for weed density Enter X in appropriate block for weed growth stage Provide a brief explanation if Prompt or URGENT identification is requested Enter a tentative determination if you made one Leave blank

Distribution of PPQ Form 391


Distribute PPQ Form 391 as follows: 1. Send Original along with the sample to your Area Identifier. 2. Retain and file a copy for your records.

FIGURE B-5 Example of PPQ Form 391 [side 2]

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

B-3

Appendix B Addendum to PPQ Form 391

Addendum to PPQ Form 391


Onaseparatepieceofpaper,recordthefollowinginformation.Placethe paperintheouterdoublebagwiththecompletedPPQ391. 1. Collectiondate. 2. Submissiondate. 3. Samplenumber[StatecodeFacilitycode00000]. 4. Nameandaddressofnursery. 5. Contactname. 6. Telephonenumber. 7. Emailaddress. 8. Samplerepresentingshipmentinvoicenumber. 9. Weekshipped. 10. Specificnumberandtypeofplantsintheshipment. 11. Locationofshipmentinnursery(benchnumbers,sectionnumbers,etc.). 12. Completenameofplant(genus,species,cultivar,color,anyother). 13. Name,agencyandtelephonenumberofcollector. 14. Labname,telephonenumberandcontactperson.

B-4

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Appendix B Questionnaire For Nursery And Potato Cropping System Owners Or Managers

Questionnaire For Nursery And Potato Cropping System Owners Or Managers


Location
Pleaseprovidethefollowinginformation: 1. Nameofnursery. 2. Address,City,State,ZipCode. 3. Nameofownerandtelephonenumber. 4. Alternatecontactpersonandtelephonenumber. 5. GPScoordinates. 6. Typeoffacility: Geraniumrootingstation Geraniumorpotatodirectshipwholesaler Geranium,potatoseedorpotatotablestockwholesaler Retailer Other

Questions
Pleaseprovidetheinformationrequestedbelow.Youranswerswillhelp agriculturalofficialsdeterminetheextentofcontaminationinyournursery facility.Thankyouforyourcooperation. 1. Indicatethenumbersofsuspectgeraniumplantsbyvarietyand shippingdatereceivedfromtheforeignfacility,rootingstation,or wholesaler.Indicatethenameoftheshipperyoureceivedtheplants from. 2. Thecurrentlocationandnumberofallsuspectgeraniumvarietiesor shipmentsinquestion. 3. Thehistoryofmovementofsuspectgeraniumswithinthefacility. 4. Thehistoryofmovementofsuspectgeraniumsoutofthefacility.If shippedout,whichcustomerdidtheygoto?Indicatethequantityof eachvarietyandthedatesshipped. 5. Theconditionofplantssinceyoureceivedthem.Areplantsshowing wilt?Ifso,indicatethenumberofaffectedplants,andthevarieties. 6. Ifsymptomswereobservedthisseason,wherewithinthefacilitywere thewiltingplantsnoticedandwhichvariety? 7. Ifyouhaddeadorwiltedplants,whatwasthedispositionofdeador culledplantsandsoil/pottingmediaassociatedwiththoseplants?If theyweredisposedof,where?

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

B-5

Appendix B Questionnaire For Nursery And Potato Cropping System Owners Or Managers

8. Whatwasthelocationandnumberofplantsofotherhosts(tomato, eggplant,potato,peppers)inyourfacilityinventorysincereceiving suspectshipments? 9. Whattypeofirrigationsystemdoyouuse?(i.e.,subirrigation,ebband flow,drip,handwatering,etc.)Isbackflowpreventioninplace? 10. Ifflood,subirrigation,orebbandflow,ispracticed,identifythe locationofallplantssharingthesamewatersourcewithsuspect geraniums. 11. Doyoufilterortreatyourirrigationwater?Doyouusewaterfroman outdoorholdingpondforirrigationrecirculationoroverflow? 12. Describethetypeofgreenhousebenchesusedandfloorcomposition. 13. Doyoueverstoreplantsontheground?Ifso,onwhatkindofsurface? 14. Doyoukeepyouvarietiesorshipmentsegregated? 15. Doyouuseaparticulartaggingortrackingsystemforgeraniumplants youdistribute?Ifso,pleasedescribethesystemused. 16. Doyouperformpropagationofgeraniumsfromtheplantsyoureceive fromforeignsources? 17. Whatkindofprotectiondoyournurseryworkersusewhenhandling plants?Howoftendotheywashtheirhandsorcuttingimplements? 18. Doyouuseadisinfectant?Ifso,whatisthenameofthedisinfectant? 19. Isthereastandardgreenhousesanitationprotocoldocumentavailable foryourfacility?Pleaseprovideacopyifso.

SignatureofowneroralternatecontactpersonDate

B-6

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Appendix B PPQ Form 523 Emergency Action Notification

PPQ Form 523 Emergency Action Notification

FIGURE B-6 Example of PPQ Form 526

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

B-7

Appendix B PPQ Form 523 Emergency Action Notification

Instructions
UsePPQForm523EmergencyActionNotificationtoholdsuspectgeraniums andpotentiallyinfectedplantsatnurseriesorotherfacilities.Onlyoneform shouldbeusedtoholdeachlotofsuspectgeraniumsandassociated potentiallyinfectedplantmaterial.Thesameformwillbeusedtodetermine whatisheldorordereddestroyed,andwhatisreleased. Formscanbecompletedbyhand,orusetheelectronicversion.Followthe instructionsbelow.
Address http://www.aphis.usda.gov/library/forms/

Procedure
Block 1EnterthenameoflocationofthenearestPPQoffice. Block 3UnderNameofArticleenterPelargoniumspp.,notGeranium. Block 4Enterthegreenhousenumbersorotherinformationindicatingthe

locationoftheplantsheld.
Block 6Enterthesourcenurseryorforeigncountryshipper. Blocks 7 and 8Completeonlyifthatinformationisknown. Block 12ToplaceplantswithinanurseryonHold,enterfortheRalstonia solanacearumrace3biovar2asthepest.Theauthorityunderwhichactions aretakenis7CFR330andthePlantProtectionAct. Block 15ActionRequiredtextisasfollows: Allgeranium(Pelargoniumspp.)_________________________varieties receivedfrom__________________duringthedates___________________ areprohibitedfrommovementfromthenurserypropertypendingfurther notificationbyUSDAAPHISPPQ.Anyotherplantmaterialreceivedby thosesameshipmentsthatmayhavebeenexposeddirectly,inshippingor sincebeingreceived,bysharedirrigationsystems,orbyunsanitarynursery culturalpracticesarealsosubjecttothishold.Allhostplantsassociated withtheaboveexhibitingsymptomsofwiltmustbereportedimmediately toUSDAAPHISPPQandhelduntilfurthernotice.Nootherpotentialhost materialofRalstoniasolanacearumrace3biovar2mayleavegreenhouses containingsuspectplantmaterialuntilfurtherevaluationscanbemade.The abovelistedplantsandallpotentiallyinfestedmaterialafterfurther investigationsareconductedwillbedestroyedeitherbyincineration,steam sterilization,oranapprovedlandfillinaccordancewithUSDApolicies. AreashousinginfectedmaterialshallbedisinfectedaccordingtoUSDA protocols.

Releasing Plants
Block 16Afterallhostshavebeenheldatanurserywithsuspectgeraniums

andinvestigationshavedeterminedwhichotherplantsarepotentially infectedbecauseofsharedwaterorunsanitarynurserypractices,releaseall otherplantsandmakeanotationinBlock16.

B-8

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Appendix B Disposal Memorandum

Documenting Actions Taken


Block 19Afterplantshavebeendestroyedandtheareasanitized,indicate

themethodsused(forexample:incineration,steamsterilization,ordisposalat approvedlandfill)andthenameofthedisinfectantsused.Indicatewhich articlesweredisinfected.

Disposal Memorandum
SubmittheDisposalMemorandum(Figure B7)onUSDAletterheadif approvedlandfilloperatorsrequiredocumentation.

Memorandum For Record March 28, 2003 Based on a review of available data, consultation with the U.S. Environmental Protection Agency (EPA) Office of Solid Waste Management and the APHIS Safety, Health, Environmental and Security Branch, we have determined that Ralstonia solanacearum race 3 biovar 2 does not present a risk to human health or the environment, with the exception of certain agricultural crops. We have based our conclusion on the following findings: Ralstonia solanacearum race 3 biovar 2 is not a human or animal pathogen EPA reports that Ralstonia solanacearum race 3 biovar 2 is not considered a hazardous or medical waste EPA reports that it can be disposed of in permitted solid waste It poses no threat to ground water There is a potential risk of infection for certain agricultural crops, especially potatoes but also tomatoes and peppers and other solanaceous plants It can be established in the environment through contamination of natural waterways, soil, and host plants In order to prevent the release of Ralstonia solanacearum race 3 biovar 2 to the environment and to protect at-risk crops, APHIS requires the following mitigations in its Ralstonia eradication action plan: 1. Waste (whole plants, plant material, soil and equipment) shall be: double-bagged in securely sealed, leak-proof plastic bags disposed of in a state, local or tribally permitted solid waste landfill covered to a depth of two feet with soil at landfill 2. A federal or state officials shall witness disposal at the landfill to ensure proper handling. Copies of the action plan are available at the APHIS Web site at: http://www.aphis.usda.gov/ ppq/ep/ralstonia/index.html

FIGURE B-7 Example of Disposal Memorandum

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

B-9

Appendix B PPQ Form 526 Permit to Move Live Plant Plants or Noxious Weeds

PPQ Form 526 Permit to Move Live Plant Plants or Noxious Weeds
According to the Paperwork Reduction Act of 1995, no persons are required to respond to a collection of information unless it displays a valid OMB control number. The valid OMB control number for this information is 0579-0054. The time required to complete this information collection is estimated to average 0.17 hours per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. No permit can be issued to move live plant pests or noxious weeds until an application is received (7 CFR 330 (live plant pests) or 7 CFR 360 (noxious weeds)). U.S. DEPARTMENT OF AGRICULTURE ANIMAL AND PLANT HEALTH INSPECTION SERVICE PLANT PROTECTION AND QUARANTINE PERMITS AND RISK ASSESSMENT, UNIT 133 RIVERDALE, MARYLAND 20737

FORM APPROVED OMB NO. 0579-0054

SECTION A - TO BE COMPLETED BY THE APPLICANT


1. NAME, TITLE, AND ADDRESS (Include Zip Code)

APPLICATION FOR PERMIT TO MOVE LIVE PLANT PESTS OR NOXIOUS WEEDS


3. TYPE OF PEST TO BE MOVED

Pathogens

Arthropods

Noxious Weeds

Other (Specify) _____________________________________ This permit does not authorize the introduction, importation, interstate movement, or release into the environment of any genetically engineered organisms or products. A. SCIENTIFIC NAMES OF PESTS TO BE MOVED B.. CLASSIFICATION (Orders, Familites, Races, or Strains)

2. TELEPHONE NO. ( C. LIFE STAGES, IF APPLICABLE

) E. SHIPPED FROM (Country or State) F. ARE PESTS G. ESTABLISHED IN U.S.? MAJOR HOST(S) OF THE PEST

D. NO. OF SPECIMENS OR UNITS

4. 5. 6. 7. WHAT HOST MATERIAL OR SUBSTITUTES WILL ACCOMPANY WHICH PESTS (Indicate by line number)

8. DESTINATION

9. PORT OF ARRIVAL

10. APPROXIMATE DATE OF ARRIVAL OR INTERSTATE MOVEMENT

11. NO. OF SHIPMENTS

12. SUPPLIER

13. METHOD OF SHIPMENT Air Mail Air Freight Baggage Auto

14. INTENDED USE (Be specific, attach outline of intended research)

15. METHODS TO BE USED TO PREVENT PLANT PEST ESCAPE

16. METHOD OF FINAL DISPOSITION

17.

SIGNATURE OF APPLICANT (Must be person named in Item 1) Applicant must be a resident of the U.S.A. I/We agree to comply with the safeguards printed on the reverse of this form, and understand that a permit may be subject to other conditions specified in Section B and C.

18. DATE

WARNING: Any alteration, forgery, or unauthorized use of this document is subject to civil penalties of up to $250,000 (7 U.S.C. s7734(b)) or punishable by a fine of not more than $10,000, or imprisonment of not more than 5 years, or both (18 U.S.C. s1001). SECTION B - TO BE COMPLETED BY STATE OFFICIAL
19. RECOMMENDATION Concur (Approve) (Accept USDA Decision) 21. SIGNATURE 22. TITLE 23. STATE 24. DATE 20. CONDITIONS RECOMMENDED Comments (Disapprove)

SECTION C - TO BE COMPLETED BY FEDERAL OFFICIAL


25. PERMIT NO.

PERMIT
(Permit not valid unless signed by an authorized official of the Animal and Plant Health Inspection Service) Under authority of the Plant Protection Act of 2000, permission is hereby granted to the applicant named above to move the pests described, except as deleted, subject to the conditions stated on, or attached to this application. (See standard conditions on reverse side.)

* For exotic plant pathogens, attach a completed PPQ Form 526-1.


24. SIGNATURE OF PLANT PROTECTION AND QUARANTINE OFFICIAL 25. DATE

26. LABELS ISSUED

27. VALID UNTIL

28. PEST CATEGORY

PPQ FORM 526

(OCT 2001)

Previous editions are obsolete.

FIGURE B-8 Example of PPQ Form 526 [side 1]

B-10

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Appendix B

STANDARD SAFEGUARDS OF PERMIT


1. All pests must be shipped in sturdy, escape-proof containers. 2. Upon receipt of pests, all packing material media, substrate, soil and shipping containers shall be sterilized or destroyed immediately after removing pests. 3. Pests shall be kept only within the laboratory or designated area at the permittee's address. 4. No living pests kept under this permit shall be removed from confined area except by prior approval from State and Federal regulatory officials. 5. Without prior notice and during reasonable hours, authorized PPQ and State regulatory officials shall be allowed to inspect the conditions under which the pests are kept. 6. All pests kept under this permit shall be destroyed at the completion of the intended use, and not later than the expiration date, unless an extension is granted by this issuing office. 7. All necessary precautions must be taken to prevent escape of pests. In the event of an excapte, notify this office.

FIGURE B-9 Example of PPQ Form 526 [side 2]

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

B-11

Appendix B

B-12

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

C
Ralstonia solanacearum r 3 b 2

Appendix C
Diagnostic Validations and Isolation Methods

Contents
Introduction page C1 Nonplate,Rapid,ImmunoassayDetection page C2 ImmunoplateAssays(ELISA) page C3 ComparisonOfNonplateImmunoassayAndPlateImmunoassay page C4 ValidationOfCentralScienceLaboratoryTaqmanAssay page C5 ValidationOfSampleAndDnaPreparationMethods page C5 PreparationOfMediaForIsolation page C6 IsolatingBacteriaFromTissueSamples page C8

Introduction
UseAppendixAtolearnmoreaboutlaboratorytestsusedtodetectthespecies Ralstoniasolanacearum. Validationstudieswereperformedonvariousdiagnostickitsformaking determinationsofRalstoniasolanacearumfromgeraniumsamples.Additional informationonisolatingthebacteriumwasalsoprovided,aswellasspecific mediarecipes. InformationinthisAppendixwasprovidedbytheUSDAAPHISPPQ CPHSTNationalPlantGermplasmandBiotechnologyLaboratory(NPGBL), Beltsville,Maryland.SeeResourcesonpage A1forinformationon contactingmanufacturersofthediagnostickits.
Important All Ralstonia solanacearum diagnostics mentioned below determine species, not biovar or race. Buffers vary by manufacturer and often produce false positive reactions when used with another manufacturers kit. Kit instructions must be followed while performing the tests. For best results, plant samples need to be symptomatic, and include the lower main stem area, or material near base of the first lateral shoot. Negative results do not necessarily indicate Ralstonia solanacearum is absent in the plant.

Status of Commercial Diagnostic Kits


PersonnelatNPGBLcontactedfivecompaniestolearnthestatusoftheir commercialRalstoniasolanacearumdiagnostickits:AdgenLtd.1,Agdia,Inc., BiorebaAG2,CentralScienceLaboratory(CSL),andLoeweBiochemica

1 2

Recently acquired by Neogen Europe Limited. STA Laboratories, Inc., in the United States.

1
C-1

May 12, 2008 Emergency and Domestic Programs Appendix diagnostic.fm

Ralstonia solanacearum r 3 b 2

Appendix C Non-plate, Rapid, Immunoassay Detection

GmbH.Thecompaniesreportednoprotocolordevicechanges,withthe exceptionofAgdia,Inc.Agdia,Inc.hadtwonewkitsreadyforevaluation: ImmunoStripandarapidELISAtest.SeeResourcesonpage A1forcontact informationforallmanufacturers.

Non-plate, Rapid, Immunoassay Detection


Rapiddetectiondevicessuchasstriporsticktestsandlateralflowdevicescan beusedtodetectRalstoniasolanacearuminplantsaporfromisolatedbacteria, accordingtothemanufacturersrecommendations.Testingbacterialcultures, however,wouldrequireplatingonselectivemedia,addingafewdaystothe testingprocedure. Wetestedthenewkit,Agdia,Inc.s,ImmunoStripalongsidetheCSLPocket DiagnosticTMandAdgensSpotCheckLFTMlateralflowdeviceswhichwere bothunchangedfromlastyear(Table C1).TheAgdiaandCSLdevices consistentlydetectedRalstoniasolanacearumat107cfu/mland,inafewtests,at 106cfu/ml.TheAdgenSpotCheckLFTMperformedverywellinsensitivity testing,detectingaslowas104cfu/ml.

Adgen Spot Check LFTM


TheAdgenSpotCheckLFTMforRalstoniasolanacearumproducedlighterband intensitiesforthetestandcontrolbandswhentestinginfectedplanttissue, makingthetestmoredifficulttovisuallyscorecomparedtotheAgdiaand CSLkits.TheAdgenkitinstructstheusertosampleleafmaterial,whereas CSLandAgdiainstructtheusertosamplefromstems.Intestingleafand stemmaterial,theAdgenSpotCheckLFTMproducedafalsenegativeon infectedstemmaterialwhichtestedpositivewithboththeAgdiaandCSL kits.

Agdia ImmunoStrip
TheAgdiadeviceconsistentlydetectedRalstoniasolanacearumat107cfu/ml and,inafewtests,at106cfu/ml.Whentestinginfectedplantmaterial,thenew AgdiaImmunoStripwaseasiertovisuallyscore.Thedarkerbandcolor madelowconcentrationsofRalstoniasolanacearumeasiertoseethaneitherthe CSLorAdgendevices.TheAgdiaImmunoStriptestrequires30minutesto completewhereasboththeCSLandAdgenlateralflowkitsrequireabout3 minutes.Thiskitinstructstheusertosamplefromstems.

C-2

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Appendix C Immunoplate Assays (ELISA)

CSL Pocket DiagnosticTM


TheCSLPocketDiagnosticTMconsistentlydetectedRalstoniasolanacearumat 107cfu/mland,inafewtests,at106cfu/ml.Thiskitinstructstheuserto samplefromstems.
TABLE C-1 Comparison of Non-plate Rapid Immunoassay Devices
Manufacturer Part number Adgen Ltd. Spot Check LFTM Lateral Flow Device 14850 (10 tests) 14851 (100 tests) 23 Macerate by shaking bottle containing ball bearings. Sample information can be written on cassette. Extra fee for brokerage. Agdia, Inc. ImmunoStrip STX 33900 SK 33900 kit 30 Must keep strip upright and slightly immersed for 30 minutes. Tissue samples are ground in special bags. Includes information on testing bacteria. CSL1 Pocket DiagnosticTM for RS Lateral Flow Device 13 Macerate by shaking bottle containing ball bearings. Sample information can be written on cassette.

Incubation2 Comments

1 2

Central Science Laboratory Minutes

Immunoplate Assays (ELISA)


WecomparedEnzymeLinkedImmunosorbentAssay(ELISA)kitsfor Ralstoniasolanacearumfromfourcompanies:AdgenLtd.,Agdia,Inc.,Bioreba AG,andLoeweBiochemicaGmbH.Ofthese,onlythekitfromAgdia,Inc.was modifiedin2003withnewcomponents.Totaltesttimevariesby manufacturer,rangingfrom2hrsto27+hrs.Inadditiontotestingplant samples,bothAgdia,Inc.,andLoeweincludeinstructionontestingbacterial samples. AllfourkitsdetectedRalstoniasolanacearum;however,Agdiaskitperformed thebestbydetectingmorepositiveswithina30minuteperiod,withinthe2 hoursfortheentiretest.

Agdia PathoScreen ELISA


AgdiasPathoScreenELISAkitwasveryeasytouse.Theplatecomesas precoatedstrips,savingtime,labor,andavoidingwaste.Nodilutionswere neededfortheliquidcomponents,whereasotherkitsrequired1to2dilutions perstep,priortodispensing.TheAgdiaELISArequirednoincubator;the entiretestisperformedatroomtemperature.Turnaroundtimeisveryshort, <2hrstotalfromstarttofinalread,significantlylessthantheotherkits. Samplescanbeprocessedquicklyandefficientlywithoutalossofassay sensitivity.Thepositivecontrolreactionisstrong,i.e.:OD=2.30at650nm with15minutesincubationatroomtemperature.
May 12, 2008 Emergency and Domestic Programs Ralstonia solanacearum r 3 b 2 C-3

Appendix C Comparison Of Non-plate Immunoassay And Plate Immunoassay

Comparison Of Non-plate Immunoassay And Plate Immunoassay


Boththerapid,nonplateimmunodetectionsystems(strips,lateralflow devices)andplateimmunodetectionsystems(ELISA)havesimilarreported sensitivity(Table C2).Forexample,AgdiasImmunoStripandELISAtests bothreportadetection(low)limitof105cfu/ml;however,inourtestingthe lowlimitwas106cfu/ml. AnadvantageofusingplateELISAistheabilitytoprocesslargenumbersof samplesinashortperiodoftime.Itiseconomicalandallowsforreplicated testing.ELISAcanbeautomatedifdesired. Incomparison,detectionusingrapid,nonplate,immunoassaysisusefulif youhavefewersamplestotest.Itisdirectandreadytouse.Itrequires significantlylesstime(minutesversushoursordays).Nobufferdilutionsare required.LikeELISA,itdoesnotrequireaspectrophotometerreader.Itcanbe performedonsite.
TABLE C-2 Comparison of Plate Immunodetection Assays
Loewe Biochemica GmbH 07056

Manufacturer

Adgen Ltd. Identikit 1131-09 (pre-coated) > 4802 Antibody pre-coated microplates with removable strips. Extra antibody binding step required for TAS. Several buffer dilutions required; extra fee for brokerage. Incubator required. Lacks instruction for testing bacterial samples.

Agdia, Inc. PSP 33900 (pre-coated)

Bioreba AG 170575 (480) 170572 (960)

Part number Incubation1

150
Antibody pre-coated microplates with removable strips; no buffer dilutions; short incubations. All incubations at room temperature. Very rapid. Includes instruction for testing bacterial samples.

1620
Uncoated; antibody coating step required; 10X and 5X buffers need diluting; overnight incubation step required in step 2. Incubator required. Lacks instruction for testing bacterial samples.

1560
Uncoated; antibody coating step required; buffers must be made by user. Overnight incubation step required. Soaking is required in 3rd & 4th wash in each wash step. Incubator required. Includes instruction for testing bacterial samples.

Comments
1 2 Minutes

Triple Antibody Sandwich [TAS] protocol used with blue substrate

C-4

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Appendix C Validation Of Central Science Laboratory Taqman Assay

Validation Of Central Science Laboratory Taqman Assay


PCRvalidationwasperformedusingthePCRprimerandprobesequences, andprotocolsprovidedbyDr.JohnElphinstone,CentralScienceLaboratory, UK.TherealtimetestisbasedontheRalstoniasolanacearumTaqManassay (CSLSOPNo.PLH7/32)foruseinanABI9700or2400thermalcycler.Thetest wasconvertedwithminormodificationstotwoothermobile,realtimePCR platforms:theIdahoTechnologiesR.A.P.I.D.fieldhardenedthermalcycler, andtheCepheidSmartCycler.TheCSLrealtimePCRprotocolforRalstonia solanacearumdetectionwassuccessfullytransferredandvalidatedineachof thetwomobilePCRmachines.(Protocolstandardoperatingprocedures [SOPs]canbeprovidedseparatelyforthesakeofbrevity.) Whilebothtestsprovidethesamelevelofdetection,theCepheidSmart Cyclerwaseasiertouseoverall.Itrequiredlessspecializedknowledgeand incorporatedOmniMixHS(Cepheidpartnumber9000078),areadytouse, lyophilizedbeadforPCRthatcontainsallreagents.Afterthebeadis hydrated,primer,probe,andDNAareaddedtogetherandthePCRreactionis readytorunonthecycler.Thebeadreducespreparationtimeanddecreases thepossibilityofcontaminationoftestsamples,therebyreducingfalse positiveresults.UseoftheCSLrealtimePCRforRalstoniasolanacearumis recommendedforPCRtestingbecauseoftheeaseofuse,sensitivity, consistency,andreducedpossibilityofcontamination.Furthermoreuseofthe OmniMixbeadsmakePCRtestseasierfornonmolecularclinicians. TheconventionalRalstoniasolanacearumPCRtest(Leeetal.,2001)usedinour lablastseasonremainsprepublicationandisnotavailableforwide distribution.

Validation Of Sample And Dna Preparation Methods


IncombinationwiththeCSLRealtimePCRtest,fourextractionmethods wereevaluated: ModifiedhotalkalineDNAextractionmethod,asusedforClavibacter michiganensissubsp.sepedonicus(Leeetal.,2001) GeneReleaser(BioVentures,Inc.) QiagenDNeasyPlantMiniKit(cat.no.69104and69106) QiagenQIAmpDNAMiniKit(cat.no.51304and51306)asinPoussier etal.(2002) NoPCRproductswereobservedusingthemodifiedhotalkalinemethod. ProductswereobservedusingtheGeneReleaserprotocol,butmanipulation ofthetubesmadethepreparationdifficultandpossiblyproneto

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

C-5

Appendix C Preparation Of Media For Isolation

contaminationinunskilledhands.ThetwoQiagenkitswerethemost consistentofthefourmethodstested,andeitherisrecommendedforusewith thePCRtestsevaluated.

Preparation Of Media For Isolation


SeveralmediahavebeendescribedforisolationofRalstoniasolanacearum. Plantscientistspreferthefollowingmedias.

Selective Medium for Ralstonia solanacearum3


Step 1Prepare1%TZCstocksolution.
How to Prepare 1% TZC Stock Solution (2, 3, 5 Triphenyl Tetrazolium Chloride1 1. Add 1 gram TZC to 100 ml dH2O. 2. Filter solution. 3. Store @ 4C or -20C in dark bottle.
1 Also known as TZC, TTC, TPTZ, or Tetrazolium Red.

Step 2Preparebasalmedium.
How to Prepare Basal Medium 1. Per liter of dH2O add each of the following: 5g Glucose, sucrose or dextrose 10g Peptone 1g Casamino acids 12-18g Agar 2. Adjust pH of to 7.0 with KOH if necessary. 3. Autoclave 20 min. @ 121 C. 4. Cool to 55 C.

Step 3Add5mlof1%TZCstocksolutiontobasalmedium. Step 4Pour~20mlperplate. Step 5Allowtosetupovernight Step 6Incubateat28to30Cfor3648hours. Step 7Identifycolonies. TypicalRalstoniasolanacearumcoloniesonthisselectivemediumproduce irregularshaped,fluidal,mucoid,nonsymmetricalcolonies.Coloniesare typicallywhitewithpink,lightpink,orpaleredcenters.

Semi-selective Medium for Ralstonia solanacearum4


ThismediumwasreportedinSchaadetal.(2001)andFrenchetal.(1995).
3 Also known as TZC medium, Kelmans medium, or TTC medium.

C-6

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Appendix C Isolating Bacteria From Tissue Samples

Step 1Filter10mlPolymyxinBsulfatesolution(8100units/mg). Step 2Filter5ml1%2,3,5TriphenyletrazoliumChloride. Step 3Preparebasalmedium.


How to Prepare Basal Medium 1. Per liter of dH2O add each of the following: 1g Casamino acids 5 ml Glycerol or glucose 10g Bacto Peptone 15-17 g Bacto agar 2. Autoclave 20 min. @ 121 C. 3. Cool to 50 C.

Step 4Addthefollowingstocksolutionsdissolvedin70%ethanol: 0.5ml1%Crystalviolet 2.5ml1%Bacitracin(73000units/g) 0.5ml1%Chloramphenicol(watersoluble) 0.5ml0.1%PenicillinGsodiumsalt Step 5Optional:Add2.5ml1%Cyclohexamidetoinhibitfungi. Step 6Addthefollowingfilteredsolutions(dH20): 10mlPolymyxinBSulfateSolution(8100units/mg) 5ml1%2,3,5TriphenyltetrazoliumChloride Step 7Pour~20ml/plate. Step 8Allowtodryovernight. Step 9Storeat4C. Step 10Incubateat35Cfor48hours. Step 11Identifycolonies. TypicalRalstoniasolanacearumcoloniesonthissemiselectivemediumproduce creamycolonies.RedpigmentedcoloniesarenotRalstoniasolanacearum.

Isolating Bacteria From Tissue Samples


Severalmethodsareavailableforisolatingbacteriafromplants.Threeare describedbelow.IsolationofRalstoniasolanacearumisbestaccomplishedusing symptomatic(i.e.wilting)plants.Itispreferabletousevascular(xylem) tissue,especiallyfromthelowerstemarea.Isolationfromnonsymptomatic plantsismoredifficultduetolowerviable,bacterialpopulations.

Also known as mSMSA.

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

C-7

Appendix C Isolating Bacteria From Tissue Samples

Method A
MethodAwassuppliedbyC.Allen(pers.comm.). Step 1Cutasmalltissuesection(~0.5cmsquare),ideallyfromstemtissue. Surfacesterilizeasectionofstem,ifdesired. Step 2Maceratein<1mlsterilewater. Step 3Allowtosit<15minutes. Step 4Streakseveralplatesdirectly,usingloopfulsofthesuspension.You maywanttousemorethanonetissuesampleandstreakplatesfromeach.

Method B
MethodBwassuppliedbyK.Rane(pers.comm.). Squeezeoutadropofcreamyoozefromasymptomaticpetioleorstemand streakitout.

Method C
MethodCwassuppliedbyS.H.Kim(pers.comm.). Step 1Locatesymptomaticleaves. Step 2Observebacterialstreamingfromleaf,oroozingfromapetioleorstem. Step 3Placeafewpetioleorstemsegments(~3mm),orfinelysliced leafblades,inatesttubecontaining10ml,0.1Mphosphatebuffer(pH7.0). Step 4Wait15minutesandstreakplates.

C-8

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

D
Ralstonia solanacearum r 3 b 2

Appendix D
Diagnostic Laboratories

Contents
Introduction page D1 DeterminationOfRaceAndBiovar page D1 DeterminationOfGenusAndSpecies page D2

Introduction
UseAppendixBtoidentifydiagnosticlaboratories.
Important Laboratories must notify their State Plant Regulatory Official (SPRO) (http://www.aphis.usda.gov/npb/ npbmemb.html#Membership%20Dirctory) and State Plant Health Director (SPHD) (http://www.ceris.purdue.edu/napis/names/sphdXstate.html) when sending samples for race and biovar determination. States sending samples for Ralstonia solanacearum determination to other states must assure that the laboratory receiving samples has a Plant Pest Permit (PPQ Form 526) to receive those samples from out-of-state. Laboratories receiving samples from within their own states do not need permits. For more information, see: http://www.aphis.usda.gov/ppq/ permits/plantpest/index.html.

Important

Determination Of Race And Biovar


Determinationofraceandbiovarrequiresadditionalregistrationsforselect agentsundertheAgriculturalBioterrorismProtectionActof2002.Currently,the onlylaboratorywithproperregistrationstomakethesedeterminationsfor Ralstoniasolanacearumrace3biovar2isthePPQNationalIdentification ServicesMolecularDiagnosticsLaboratoryinBeltsville,MD.See Identificationonpage 41forinformationonpackaginganddocumentation.
Address Dr. Mary E. Palm USDAAPHISPPQNational Identification Services Molecular Diagnostics Laboratory BARC-East, Bldg. 580 Powder Mill Rd. Beltsville, MD 20705 T: 301-504-7154 or 504-5700 ext. 327 F: 301-504-6124 Email: mary.palm@aphis.usda.gov

1
May 12, 2008 Emergency and Domestic Programs Appendix State labs.fm Ralstonia solanacearum r 3 b 2 D-1

Appendix D Determination Of Genus And Species

Determination Of Genus And Species


Thelaboratoriesincludedinthissectionarelimitedtodeterminationofgenus andspecies.

D-2

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Appendix D Determination Of Genus And Species

State and Cooperating University Diagnostic Laboratories


TABLE D-1 State and Cooperating University Diagnostic Laboratories
If in this state: Alabama Then contact this laboratory: Dr. Jackie Mullen Extension Plant Pathologist Plant Diagnostic Lab Auburn University Auburn, AL 36849 T: 334-844-5508 jmullen@acesag.auburn.edu GO TO California. Dr. Stephan Vann University of Arkansas Cooperative Extension Service, Plant Diagnostic Clinic 2001 Hwy 70 East Lonoke, AR 72086 T: 501-676-3124 Dr. Dan Opgenorth California Department of Food and Agriculture Plant Pest Diagnostics Laboratory 3294 Meadowview Road Sacramento, CA 95832-1448 T: 916-262-1100 F: 916-262-1190 dopgenor@cdfa.ca.gov Tamela Blunt Center for Crop Biosecurity Colorado State University Fort Collins, CO 80526 T: 970-491-6950 Dr. Sharon Douglas Connecticut Agricultural Experiment Station P. O. Box 1106 New Haven, CT 06504 T: 203-974-8499 sharon.douglas@po.state.ct.us Dr. Bob Mulrooney Department of Plant and Soil Sciences Room 152 Townsend Hall University of Delaware Newark, DE 19717 T: 302-831-4865 F: 302-831-0605 bobmul@udel.edu

Arizona Arkansas

California

Colorado

Connecticut

Delaware

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

D-3

Appendix D Determination Of Genus And Species

TABLE D-1 State and Cooperating University Diagnostic Laboratories


If in this state: Florida Then contact this laboratory: Dr. Tim Schubert FDACS-DPI P. O. Box 147100 Gainesville, FL 37614-7100 T: 352-372-3505 x 143 schubet@doacs.state.fl.us Dr. Jean Woodward University of Georgia Plant Pathology Department 2106 Miller Plant Science Bldg. Athens, GA 30602-7274 T: 706-542-9146 woodwar@uga.edu Liz Vavricka Plant Industry Lab Idaho State Department of Agriculture P. O. Box 790 Boise, ID 83701 T: 208-332-8640 Agdia, Inc. Mike Tiffany 30380 County Road 6 Elkhart, IN 46514 T: 574-264-2014 info@agdia.com Dr. Karen Rane Plant Pest Diagnostic Lab Purdue University 915 W. State Street West Lafayette, IN 47907-2054 T: 765-494-5821 rane@purdue.edu Iowa Paula Flynn ISU Plant Disease Clinic 323 Bessey Hall Iowa State University Ames, Iowa 50011 T: 515-294-0581 Dr. Ned Tisserat Plant Pathology Department Kansas State University Manhattan, Kansas T: 785-532-1387 Dr. John Hartman S-305 Ag Sci Bldg N University of Kentucky Lexington, KY 40546 T: 859-257-5779 jhartman@ca.uky.edu

Georgia

Idaho

Indiana

Kansas

Kentucky

D-4

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Appendix D Determination Of Genus And Species

TABLE D-1 State and Cooperating University Diagnostic Laboratories


If in this state: Louisiana Then contact this laboratory: Dr. Clayton A. Hollier Department of Plant Pathology LSU AgCenter Baton Rouge, LA T: 225-578-2186 Dr. Chris Clark Department of Plant Pathology LSU AgCenter T: 225-578-1381 Dr. Gordon Holcomb Department of Plant Pathology LSU AgCenter T: 225-578-1386 Craig Roussel or Tad Hardy Louisiana Dept. Agriculture and Forestry Baton Rouge, La. T: 225-952-8100 Maine Bruce Watt University of Maine Pest Management Office 491 College Avenue Orono, ME 04473-1295 T: 207-581-3880 bwatt@umext.maine.edu Jennifer Dominiak Maryland Department of Agriculture 50 Harry S. Truman Pkwy. Room 345 Annapolis, Maryland 21401 T: 410-841-5920 dominijd@mda.state.md.us Dr. Robert L. Wick Department of Microbiology Morrill Science Center N203 University of Massachusetts Amherst, MA 01003 T: 413-545-1045 rwick@pltpath.umass.edu Dr. Richard Kaitany Geagley Laboratory Michigan Department of Agriculture 1615 S. Harrison Rd. East Lansing, MI 48823 T: 517-337-5091 kaitanyr@msu.edu

Maryland

Massachusetts

Michigan

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

D-5

Appendix D Determination Of Genus And Species

TABLE D-1 State and Cooperating University Diagnostic Laboratories


If in this state: Minnesota Then contact this laboratory: Gary Horvath Minnesota Department of Agriculture Laboratory Services Division 90 W. Plato Blvd. St. Paul, MN 55107-2094 T: 651-215-9063 F: 651-297-8787 gary.horvath@state.mn.us Dr. Alan Henn, Associate Extension Professor Entomology and Plant Pathology Mail Stop 9655 Mississippi State, MS 39762 T: 662-325-4535 ahenn@ext.msstate.edu Dave Johnson, Plant Pathologist Missouri Department of Agriculture Plant Industries Division 115 Constitution Drive P.O. Box 630 Jefferson City, Missouri 65102 T: 573-751-8319 F: 573-526-7777 Jennifer Chaky Plant Pathology Department 448 Plant Science Building University of Nebraska, Lincoln Lincoln NE 68583 T: 402-472-8725 Shouhua Wang Plant Pathologist Nevada Department of Agriculture Plant Division 350 Capitol Hill Avenue Reno, NV 89502 T: 775-688-1182x246 F: 775-688-1178 shwang@govmail.state.nv.us Cheryl Smith Extension Education, Adjunct Professor University of New Hampshire Spaulding Life Sciences Bldg., Room 242 Durham, NH 03824-3544 T: 603-862-3841 cheryl.smith@unh.edu

Mississippi

Missouri

Nebraska

Nevada

New Hampshire

D-6

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Appendix D Determination Of Genus And Species

TABLE D-1 State and Cooperating University Diagnostic Laboratories


If in this state: New Jersey Then contact this laboratory: Dr. Glenn Freeman New Jersey Department of Agriculture Plant Laboratory Services P. O. Box 330 Trenton, NJ 08625-0330 T: 609-292-5484 glenn.freeman@ag.state.nj.us NMDA Bureau Chief Entomology and Nursery Industries, MSC 3 BA Las Cruces, NM 88003-0005 T: 505-646-3207 Margery Daughtrey Cornell University Long Island Horticultural Research & Extension Center 3059 Sound Ave. Riverhead, NY 11901 T: 631-727-3595 mld9@cornell.edu Karen Snover-Clift Cornell University Department of Plant Pathology 334 Plant Science Bldg. Ithaca, NY 14853 T: 607-255-7850 kls13@cornell.edu North Carolina Tom Creswell NCSU Plant Disease and Insect Clinic William Hall, Room 1104 100 Derieux Hall Campus Box 7211 Raleigh, NC 27695-7211 T: 919-515-3619 F: 919-515-7716 tom_creswell@ncsu.edu GO TO Agdia, Inc. Nancy J. Taylor Extension Associate & Coordinator Plant Pest Diagnostic Clinic 110 Kottman Hall 2021 Coffey Road Columbus, Ohio 43210-1087 T: 614-292-5006 F: 614-292-7162 taylor.8@osu.edu

New Mexico

New York

North Dakota Ohio

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

D-7

Appendix D Determination Of Genus And Species

TABLE D-1 State and Cooperating University Diagnostic Laboratories


If in this state: Oklahoma Then contact this laboratory: Dr. Brian Olson Oklahoma State University Entomology and Plant Pathology 119 NRC Stillwater, OK 74078-3033 T: 405-744-7126 olsonb@okstate.edu Acting Supervisor Plant Health Lab, Plant Health Section Oregon Dept. of Agriculture 635 Capitol St. NE Salem, OR 97301-2532 T: 503-986-4636 or 503-986-4661 F: 503-986-4786 jgriesba@oda.state.or.us Dr. Seong Kim Pennsylvania Dept. of Agriculture Bureau of Plant Industry Plant Pathology Lab 2301 North Cameron Street Harrisburg, PA 17110-9408 T: 717-772.5221 F: 717-705.6518 skim@state.pa.us GO TO Agdia, Inc. GO TO Agdia, Inc. Meg Williamson Clemson University Plant Problem Clinic 171 Old Cherry Road Clemson, SC 29634-0114 T: 864-656-3125 ppclnc@clemson.edu South Dakota South Dakota State University Brookings, SD 57006 T: 605-688-5157 GO TO Agdia, Inc. Dr. Larry Barnes Texas Plant Disease Diagnostic Laboratory 1500 Research Parkway, Suite A-130 College Station, Texas 77845 T: 979-845-8032 barnes@tamu.edu

Oregon

Pennsylvania

Rhode Island South Carolina

Tennessee Texas

D-8

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Appendix D Determination Of Genus And Species

TABLE D-1 State and Cooperating University Diagnostic Laboratories


If in this state: Utah Then contact this laboratory: Scott C. Ockey Plant Disease Diagnostician, Senior Research Associate Utah State University Old Main Hill Logan, UT 84322-5305 T: 435-797-2435 F: 435-797-1575 scotto@ext.usu.edu Washington Building, Room 703 1100 Bank Street Richmond, VA 23219 T: 804-786-3515 F: 804-371-7793 gokeefe@vdacs.state.va.us Scott Pfister Vermont State Plant Pathologist 103 S. Main Street Waterbury, VT 05671-0101 T: 802-828-3481 spfister@agr.state.vt.us Jennifer Falacy Plant Pathology Project Coordinator Plant Pathology Laboratory 3939 Cleveland Avenue S.E. Olympia, WA 978501-4079 F: 360-586-5286 awagner@agr.wa.gov Gary Gibson WVDA, Plant Industries Division 1900 Kanawha Blvd., East Charleston, WV 25305-0191 T: 304-558.2212 ggibson@ag.state.wv.us Annette Phibbs WDATCP, ARM, Plant Industry Division 4702 University Avenue Madison, WI 53705 T: 608-266-7132 F: 608-266-5855 anette.phibbs@datcp.state.wi.us Mr. Gary Franc University of Wyoming Dept. of Plant Pathology P.O. Box 3354 Laramie, Wyoming 82071-3354 T: 307-766-2397

Virginia

Vermont

Washington

West Virginia

Wisconsin

Wyoming

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

D-9

Appendix D Determination Of Genus And Species

Plant Diagnostic Networks and Private Laboratories


DiagnosticscreeningcantakeplaceatalaboratorydesignatedintheNational PlantDiagnosticNetwork,oratAgdia,Inc.Currently,Agdia,Inc.istheonly privatelaboratoryabletoperformthistypeoftesting.UseTable D1on page D10toidentifytheregionallaboratoryforyourstate.
TABLE D-1 : Regional Plant Diagnostic Networks (EPDN) and Private Laboratories
If in this state: Connecticut, Delaware, Maine, Maryland, Massachusetts, New Hampshire, New Jersey, New York, Pennsylvania, Rhode Island, Vermont, West Virginia Then contact this laboratory: Northeast Plant Diagnostic Network (NEPDN) Cornell University Karen L. Snover-Clift Department of Plant Pathology 334 Plant Science Bldg. Ithaca, NY 14853 Great Plains Diagnostic Network (GPDN) Kansas State University Ned Tisserat Department of Plant Pathology 4024 Throckmorton Hall Manhattan, KS 66506 T: (785) 532-1383 tissne@ksu.edu North Central Plant Diagnostic Network (NCPDN) Jan Bryne 114 CIPS Michigan State University East Lansing, MI 48824-1311 T: (517) 355-3504 byrnejm@msu.edu Southern Plant Diagnostic Network (SPDN) Richard Cullen Plant Disease Clinic UF Bldg 78 Mowry Road P.O. Box 110830 Gainesville, FL 32611-0830 T: (352) 392-1795 or 3631 (X 254) Western Plant Diagnostic Network (WPDN) Melodie Putnam 1089 Cordley Hall Corvallis, OR 97331-2903 T: (541) 737-3472 putnamm@science.oregonstate.edu Agdia Inc. 30380 County Road 6 Elkhart, IN 46514 T: (574) 264-2014 or (800) 62-AGDIA Web site: www.agdia.com

Colorado, Kansas, Montana, North Dakota, Oklahoma, South Dakota, northern Texas, Nebraska, Wyoming

Iowa, Michigan, Ohio, Indiana, Illinois, Minnesota, Missouri, Wisconsin

Alabama, Louisiana, southern Texas, Arkansas, Mississippi, Virginia, Florida, North Carolina, Puerto Rico, Georgia, South Carolina, Kentucky, Tennessee

American Samoa, Arizona, California, Hawaii, Idaho, Alaska, Nevada, New Mexico, Oregon, Guam, Utah, Washington

Any state

D-10

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

E
Ralstonia solanacearum r 3 b 2

Appendix E
Pelargonium in the N u r s e r y Tr a d e

Geraniumisacommonnamethatcanrefertoplantspeciesinthegenus PelargoniumandthegenusGeranium.Thecommonnurseryplantisoneof severalPelargoniumspp.listedbelow(Table E1).ThePPQRalstoniaprogram doesnotregulateplantsinthegenusGeranium,whichareprimarilyperennial landscapeplantsnotknowtobehostsofRalstoniasolanacearum.


TABLE E-1 Common Pelargonium in the Nursery Trade
Scientific Name Pelargonium x hortorum Common Name Zonal geranium or Florist's geranium Description Americana geraniums are in this group. This genus and species makes up 7080% of the geraniums sold in the United States each year. Vegetatively propagated and most are tetraploids. A smaller portion of the market. These are diploids and are reproduced via seed. Approximately 1020% of the market; vegetatively propagated. Typically grown in baskets (hanging above other crops). There are also seed propagated lines of this species. This species is grown mainly as a flowering potted crop through florists and upper end retail garden centers. Vegetatively propagated; less than 510% of the market. These are used as pot plants to display in the home and typically are planted out into the garden since they will not flower during the heat of the summer. The foliage and flowers are significantly different from zonal geraniums. With unusual flowers, foliage or scented foliage. Very small market segment of unusual types. Vegetatively propagated. Broad diversity of genetics make up this group and difficult to type to species.

Seed geranium

Pelargonium peltatum

Ivy geranium

Pelargonium domesticum

Regal or Martha Washington geranium

Other Pelargonium spp.

Scented geraniums and other novelty types

May 12, 2008 Emergency and Domestic Programs Appendix geraniums trade.fm

Ralstonia solanacearum r 3 b 2

E-1

Appendix E

E-2

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

F
Ralstonia solanacearum r 3 b 2

Appendix F

Comparison of Symptoms

FIGURE F-1: Left: Early wilting caused by infection with Ralstonia solanacearum race 3 biovar 2 Right: Advanced wilting and abnormal leaf yellowing (chlorosis) caused by infection with R. solanacearum race 3 biovar 2 [Images courtesy of Wisconsin Department of Agriculture, Trade and Consumer Protection]

FIGURE F-2: Left: Abnormal yellowing caused by infection with Ralstonia solanacearum race 3 biovar 2 [Courtesy of the Plant Disease Diagnostics Clinic, University of Wisconsin-Madison, Cooperative Extension Service] Right: Close-up of wilting and necrosis caused by infection with R. solanacearum race 3 biovar 2 [Courtesy of Margery Daughtrey, Cornell University]

1
May 12, 2008 Emergency and Domestic Programs Appendix symptom.fm Ralstonia solanacearum r 3 b 2 F-1

Appendix F

FIGURE F-3: Left: Wilting and mortality of geraniums caused by infection with Ralstonia solanacearum race 3 biovar 2. Right: Mortality (top) and early wilt (right) caused by infection with R. solanacearum race 3 biovar 2

FIGURE F-4: Left: Cross-section of geranium stem with bacterial ooze caused by infection with Ralstonia solanacearum race 3 biovar 2 Right: Bacterial streaming from an infected stem immersed in water caused by infection with R. solanacearum race 3 biovar 2

F-2

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Appendix F

FIGURE F-5: Left: Wilting caused by bacterial blight (Xanthomonas pelargonii) is indistinguishable from Ralstonia solanacearum wilting Right: Bacterial blight (Xanthomonas pelargonii) also causes characteristic spotting of leaf tissue [Images courtesy of M. Daughtrey, Cornell University]

FIGURE F-6: Left: Early wilting symptoms on pepper caused by infection with Ralstonia solanacearum Right: Advanced wilting and necrosis of potato caused by infection with Ralstonia solanacearum [Images courtesy of CABI, 2006]

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

F-3

Appendix F

FIGURE F-7: Left: Early tuber symptoms caused by Ralstonia solanacearum (Easily confused with ring rot caused by Clavibacter michiganense pv. sepidonicus) Right: Advanced tuber symptoms caused by R. solanacearum [Images courtesy CABI, 2006]

FIGURE F-8: Left: Bacterial streaming of infected stem immersed in water caused by infection with Ralstonia solanacearum (RS) [Image courtesy of UGA] Right: Bacterial ooze on tubers infected with R. solanacearum [Image courtesy of EMBRAPA]

F-4

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Appendix F

FIGURE F-9: Left and Right: Advanced ring rot of tuber caused by Clavibacter michiganense pv. sepidonicus [Left image courtesy of CABI, 2006; right image courtesy of CIP]

FIGURE F-10: Left and Right: Intermediate ring rot of tuber caused by Clavibacter michiganense pv sepidonicus [Images courtesy of Canadian Government of British Columbia and Manitoba]

May 12, 2008 Emergency and Domestic Programs

Ralstonia solanacearum r 3 b 2

F-5

Appendix F

F-6

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

G
Ralstonia solanacearum r 3 b 2

Appendix G
Disinfectants
TABLE G-1 : Disinfectants Registered Against Pseudomonas spp. (Ralstonia solanacearum = Pseudomonas solanacearum)
EPA Reg. No. 499-3 68

Trade Name GreenShield

Active Ingredients n-Alkyl dimethyl benzyl ammonium chloride; n-Alkyl dimethyl ethylbenzyl ammonium chloride; same as Physan 20

Use Sites Work area, benches, pots, flats, flower buckets, cutting tools, greenhouse glass, walkways, evaporative coolers, decorative pools, fountains, and water displays Greenhouses, hard surfaces, lawn and turf grass, seedlings, cut flowers, decorative fountains, pools, birdbaths, and plants Greenhouse structures, benches, pots, watering systems, evaporative coolers, storage rooms, ventilation equipment, floors and other equipment Greenhouse hard surfaces, work areas, benches, flower pots, buckets, flats, cutting tools, walkways, garden bird baths, and evaporative coolers Greenhouse hard surfaces, work areas, benches, cutting tools, walkways, garden bird baths, and evaporative coolers Non-porous, inanimate surfaces such as floors, walls, metal surfaces, stainless steel surfaces, plastic surfaces, knobs, handles, and railings

Pertinent Bacteria Pseudomonas spp.(unlabelled)

Physan 20

5536 4-5

Same as Green-Shield

Pseudomonas spp.

Zero TolZT

7029 9-1

Hydrogen dioxide

Pseudomonas spp.

Consan Triple Action 20

5804 4-3

n-Alkyl dimethyl benzyl ammonium chloride; n-Alkyl dimethyl ethylbenzyl ammonium chloride; same as Triathlon Same as Consan Triple Action 20

Triathlon

5804 4-3-59 807

AE-90

4737 1-89

n-Alkyl dimethyl benzyl ammonium chloride; n-Alkyl dimethyl ethylbenzyl ammonium chloride

Pseudomonas aeruginosa

1
G-1

May 12, 2008 Emergency and Domestic Programs Appendix disinfectants.fm

Ralstonia solanacearum r 3 b 2

Appendix G

TABLE G-1 : Disinfectants Registered Against Pseudomonas spp. (Ralstonia solanacearum = Pseudomonas solanacearum)
Lonza S-18 683677 Octyl decyl dimethyl ammonium chloride; Dioctyl dimethyl ammonium chloride; didecyl dimethyl ammonium chloride; n-Alkyl dimethyl benzyl ammonium chloride Octyl decyl dimethyl ammonium chloride; Dioctyl dimethyl ammonium chloride; didecyl dimethyl ammonium chloride; n-Alkyl dimethyl benzyl ammonium chloride Octyl decyl dimethyl ammonium chloride; Dioctyl dimethyl ammonium chloride; didecyl dimethyl ammonium chloride; n-Alkyl dimethyl benzyl ammonium chloride Octyl decyl dimethyl ammonium chloride; Dioctyl dimethyl ammonium chloride; didecyl dimethyl ammonium chloride; n-Alkyl dimethyl benzyl ammonium chloride; same as MAQUAT 615-LR Same as MAQUAT 615-HD Farm, poultry, swine, and mushroom premise sanitation; veterinary practice, animal care, animal laboratory disinfection Pseudomonas aeruginosa and Pseudomonas cepacia

MAQUAT 128-MT

1032 4-112

Outer clothing, field harvesting equipment, walls/floors of coolers, flower buckets, and greenhouse packing areas

Pseudomonas aeruginosa

MAQUAT 64 MN

1032 4-113

Florist shops, wholesale florists, shippers, greenhouse packing areas, flower buckets, floors/walls of coolers, design and packaging benches, and counter tops Greenhouses, hard non porous surfaces (flower buckets, floors, walls, coolers, design, packing benches and counter tops)

Pseudomonas aeruginosa

MAQUAT 615-HD

1032 4-72

Pseudomonas aeruginosa, Xanthomonas axonopodis pv. Citri, and vesicatoria

MAQUAT 615-LR

1032 4-109

Same as MAQUAT 615-HD

Same as MAQUAT 615-HD

G-2

Ralstonia solanacearum r 3 b 2

May 12, 2008 Emergency and Domestic Programs

Das könnte Ihnen auch gefallen