NPDN NewsVolume 5 Issue 11, November 2010

Issue Highlights:•Duponcheliafovealis

• Bacterialcankerofkiwifruit

• Diagnostictip:loop-mediatedisothermalamplification

• NPPLAP’sP.ramorumproficiencypanel

• PDIS-newfeatures

• InRegionalNews:redpalmweevil,brownmarmoratedstinkbugandpeachfruitfly

National Institute of Food and Agriculture

1Volume5Issue11

Enhancements to the National RepositoryMikeHillandEileenLuke,PurdueUniversity,CERIS

AttheNPDNIT/Diagnosticians’jointworkgroupmeetinginOctober

threenewproposedenhancementswerepresented.Thefirstenhancement

isanexpansionofthefirstsubmitterbystatereport.Thisreportrepresentsthefirsttimeapest/pathogenhasbeen

reportedinastatetotheNPDNNationalrepository.Currentlythesystememailssixdifferentreportsdependinguponroleaccesstoregionalandnationalprogramleaderstaffdailybasedonthepreviousday’sactivities.Anonlinereportfeatureisalsoavailableforpastdates.TheenhancementwillprovideasimilarreporttotheSPRO’sanddiagnosticianswiththeirspecificstateaccess.Theenhancementwillshowalldiagnosesfromyourstateandalldiagnosesyourlaboratoryprocessedregardlessofstateoforigin.Thefirstreleaseofthisenhancement,withonlinereportcapability,willbeattheendof2010andtheemailfeaturewillbeimplementedinthefirstquarterof2011.

Thesecondenhancementisthepestgroupingfeature.Thisitemwillsupportgroupingbyspecies,entrieswithoutcommonnames,andabettermeanstoaddresssynonyms.Thepest/pathogendictionarywillstillbeavailableinitsclassicformandnosoftwarechangeswillberequiredfordataentryonthepartofthelabmanagementsystems.Mostofthesoftwareforthisefforthasbeendeveloped,butassistancefromasubgroupoftheDatabaseProgramAreaCommitteeisrequiredtoaccuratelydeterminethelistofuniquescientificnamesandtodobetatestingpriortoits

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NationalNews cont...

targetedimplementationdateofMarch31,2011.

ThefinalfeatureisthePest/Hostindexreportwhichshowsconfirmedpest/pathogensandthe

hoststheyhavebeenfoundon.ThisdataindexispresentedasadiagnostictooltoNPDNmemberlaboratoriesonlywitharequirementtochecktheagreementboxtomaintainconfidentialityofthedatawithintheNPDNcommunityandappropriatestateplantregulatoryofficials.Thereportisabletosearchbybothpestandhosttoboththegenusandspecieslevel.Nolocationortimeofoccurrenceareprovidedthusnotbreechingconfidentialityissueswhilestillprovidingusefulinformationtothediagnosticians.Thisfeaturewillbeimplementedattheendof2010.QuestionsaboutthesenewfeaturesoranyotheritemsabouttheNationalRepositoryshouldbesenttoMikeHillmhill@ceris.purdue.eduorEileenLukelukee@purdue.edu.

Thirteen New State Detections of Duponchelia fovealisOnMay18,2010,theCanadianFoodInspectionAgency(CFIA)notifiedAPHISaboutaninterceptionofDuponcheliafovealislarvaeinanurseryshipmentfromSanDiegoCounty,California.OnJuly16,2010,APHISconfirmedadetectionatagreenhouseinVista,California.Trace-forwardinformationprovidedbytheaffectednurseryresultedintheexpansionofdetectionsurveysin26countieswithinCalifornia.

DuringthemonthsofSeptemberandOctober,APHISandStateDepartmentsofAgriculturehaveconfirmedthepresenceofDuponcheliafovealisinportionsoftwelveadditionalstatesincluding;Alabama,Arizona,Colorado,

Florida,Georgia,Mississippi,NorthCarolina,Oklahoma,Oregon,SouthCarolina,Texas,andWashington.

Atypeofmoth,DuponcheliafovealisoriginatedinsouthernEuropeandNorthAfricaandhasawiderangeofhostplants,includingpeppers,pomegranates,aquaticplants,beddingplantsandnurserystock.ReadthefullSPROletterhere.

Federal Order for Pseudomonas syringae pv. actinidiae, Bacterial Canker of KiwifruitAFederalOrderwasissuedonNovember10,2010,topreventtheintroductionanddisseminationofabacterialcankerofkiwifruit(Pseudomonassyringaepv.actinidiae)intotheUnitedStates.APHISwillprohibitimportationsofActinidiaspp.plantsforplanting(includingpollenbutexcludingfruitandseed)hostsofP.syringaepv.actinidiae(bacterialcankerofkiwifruit)fromallcountries.

RecentscientificliteratureidentifiesPseudomonassyringaepv.actinidiaeasadestructiveplantpathogenthatisseriouslyaffectingorchardsofkiwiplantsaroundtheworld.ThediseaseisrapidlyspreadingthoughAsiaandEurope.InItaly,itisestimatedthattheeconomiclosses(includingimpactontrade)duetoP.syringaepv.actinidiaehavereached2millionEuros.

Pseudomonassyringaepv.actinidiaeisnotknowntooccurintheUnitedStates.Plantpathogens,includingvirusesandviroids,areextremelydifficulttodetectduringaportofentryinspectionofthehostplants,particularlyintheabsenceofsymptoms.Thepathogenisnotknowntobeseedorfruittransmitted.

ClickheretoreadtheofficialSPROletterandFederalOrder.

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Diagnostic Updates

Loop-mediated Isothermal Amplification (LAMP) as a Diagnostic ToolMelodiePutnam,OregonState

University,DepartmentofBotanyandPlantPathology

AsadiagnosticianIamalwayslookingforassaysthatarerapid,

accurate,easytouse,andinexpensive.OnceIfoundoutitispossibletohavethespecificityofPCRwithouttheneedforthermalcyclers,gels,orexpensiveandshort-livedfluorescentprobesusingthemethodknownasLAMP,Iwasdefinitelyinterested.Loop-mediatedisothermalamplification(LAMP)takesplaceinasinglemicrofugetubeandrequiresonlyawaterbathorheatblock.Resultsareevaluatedvisuallywithoutextensivemanipulationofproduct.Sincethemethodistheequivalentofamusclecarasfarasspeed(fromafewcopiesofDNAto109,optimally,inlessthananhour)andishighlyspecific,itseemstohavethequalitiesthatwouldbeextremelyusefulinadiagnosticlaboratory.WehavedevelopedanassayfordetectionofRhodococcusfasciansusingthismethod(Serdani,etal.,2009),arecurrentlyusingLAMPforfireblightdetection(Templeetal.,2008),andhavebeenveryhappywithbothtests.Theassaycanbeusedonhosttissueinadditiontopurecultures,anditlivesuptotheclaimsforspeedandselectivity.Thefollowinginformationisprovidedtopiqueyourinterestindevelopingyourownassays.

LAMPwasdevelopedbyTsugunoriNotomiofEikenChemicalCompanyandcolleaguesattheUniversityofTokyoandOsakaUniversityMedicalSchool.Theirseminalpaperwaspublishedovertenyearsago(Notomietal,2000),andthemethodhassincebeenadapted

forusewithfungi(Tomlinson,etal,2010),nematodes(Kikuchi,etal.,2009),andviruses(Fukutaetal.2003)inadditiontophytobacteria.TheprocesstakesplaceinamicrofugetubecontainingasmallamountofextractedsampleDNAandfourprimersthatarespecifictosixregionsofyourtargetDNA.Astrand-displacingpolymeraseisusedat60-65°C(thespecifictemperatureuseddependsonsystemoptimization)andaseriesofloopedstructuresresult.TherathercomplexprocessofamplificationandthestructureformationisdetailedattheEikenwebsite,whichalsoincludesananimationofthereaction(http://loopamp.eiken.co.jp/e/lamp/index.html).Asaby-productofamplification,magnesiumpyrophosphateaccumulatesinproportiontotheamountofthespecificproductformed,andcanbeseenasturbidityinthereactiontube.Ifitisdifficulttoseethisturbidity,aDNAbindingdyemaybeused,whichwillintegratewiththeamplifiedproduct.Thisdye-DNAcomplexwillfluoresceunderUVlight,whichmakesiteasiertodistinguishbetweenpositiveandnegativereactions.

ForourLAMPassayforRhodococcusfascians,weuseeithertheFastSpinorQuiagenkitstoextractDNAwhenstartingwithplantmaterial,orifusingapureculture,simplyboilthebacteriabrieflytoextractDNA.Analiquotisthenaddedtothereactionmixture,thetubesareincubatedat60°C,andvisualizationiswithPicoGreen(orthefluorescentdyeofyourchoice).Theentireprocessis

Positive LAMP reactions are shown in the two tubes on the left. The tube on the right is a negative reaction. The image was taken under UV illumination and fluorescence is due to addition of PicoGreen to all tubes.

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completedinlessthan3hours.Wehavefoundtheprocesstobefast,accurate,andverysimpletouse.However,thereactionproductsareextremelystableandcancontaminatethelaboratoryinapersistentmannerifyouarenotcarefulwhenopeningthetubestoaddthedye.ThisisbestdoneinaroomseparatefromwheretheDNAextractionoccursandwherethereactionmixtureisprepared.

Therearewebsitesthatcanhelpyoutodesignyourownprimers,oneofwhichisathttp://primerexplorer.jp/elamp4.0.0/index.html,andexcellenttechnicalinformationattheEikenwebsite:http://loopamp.eiken.co.jp/e/lamp/index.html.

Referencescited:FukutaS.,Kato,S.,Yoshida,K.Mizukami,Y.,Ishida,A.,Ueda,J.,Kanbe,M.,andIshimoto,Y.2003.Detectionoftomatoyellowleafcurlvirusbyloop-mediatedisothermalamplificationreaction.JournalofVirologicalMethods112(1-2):35-40.doi:10.1016/S0166-0934(03)00187-3Notomi,T.,Okayama,H.,Masubuchi,H.,Yonekawa,T.,Watanabe,K.,Amino,N.andHase,T.2000.Loop-mediatedisothermalamplificationofDNA.NucleicAcidsResearch28(12).doi:10.1093/nar/28.12.e63.Serdani,M.,Curtic,M.,andPutnam,M.L.2009.Loop-mediatedisothermalamplification(LAMP)forrapiddetectionofRhodococcusfasciansonornamentals.Phytopathology99:S117.Temple,T.N.,Stockwell,V.O.andJohnson,K.B.2008.DevelopmentofarapiddetectionmethodforErwiniaamylovorabyloop-mediatedisothermalamplification(LAMP)ActaHort.(ISHS)793:497-503.Tomlinson,J.A.,Dickinson,M.J.,andBoonham,N.2010.DetectionofBotrytiscinereabyloop-mediatedisothermalamplification.LettersinAppliedMicrobiologyDec;51(6):650-7.doi:10.1111/j.1472-765X.2010.02949.x.

National Plant Protection Laboratory Accreditation Program and 2011 P. ramorum Proficiency Test PanelPatrickJ.Shiel,USDA-APHIS-PPQ-CPHST

TheNationalPlantProtectionLaboratoryAccreditationProgram(NPPLAP)evaluateslaboratoriesusingmoleculardiagnosticsonbehalfofUSDA-PPQregulatoryprogramstoensuretheircapabilityformakingaccuratediagnosticdeterminations.InadditiontoensuringlabcapabilitywithinPPQandotheragenciesintheUSDA,NPPLAPengagestheNationalPlantDiagnosticNetwork(NPDN)inthisprocesstoincreasediagnosticcapacityandproficiency.ThegoalofthisprogramistoestablishastateofreadinesswhenneededbyPPQinemergencysituations.NPPLAPalsofosterstheadoptionofpracticesthatpromotecontinuousimprovementandaccreditationstandardssuitableforusebyplantdiagnosticlabsandservestodevelopfunctionalQualityAssuranceprograms.NPPLAPbeganastheProvisionalApprovalprocessfortheUSDAPhytophthoraramorumEmergencyProgramandcurrentlyaccreditslaboratoriestodiagnosePhytophthoraramorumandtheHuanglongbing(CitrusGreening)pathogen,withover30labsparticipatinginoneorbothprograms.TheNPPLAPwebsiteis:www.aphis.usda.gov/plant_health/cphst/npplap.shtml.ThiswebsitedescribestheprogramindetailandcontainslinkstoInspectionChecklistsandotherNPPLAP-relateddocuments.

VisittheNPDNdiagnosticwebsiteatwww.npdn.org/diagnosticstoreadthe2011Phytophthoraramorumpanelinformationletterandtodownloadaformforrequestingpanels.Thedocumentscanbefoundundertheheading“ProvisionallyApprovedLaboratories.”

PAC Highlights

P R O G R A M A R E A C O M M I T T E E S

Volume5Issue11 5

VisittheNPDNhomepageatwww.npdn.orgformoreinformationonspecificProgramAreaCommittees.Login and password required

Announcements~Membershipinformation~Committeereportsandmeetingminutes~DocumentsandSOPs

TheDiagnosticsCommitteeheldaconferencecallonNovember4,2010andthefollowingagendaitemswerediscussed:

•IT-DiagnosticiansMeeting/OpsComoverview•2011NPDNWorkshop(s)Update•2012FutureWorkshop

•SOPUpdates•SurgeCapacityAdobeConnectTraining

Pleaserefertothewebsite,www.npdn.org/diagnostics,forcompleteminutesofthismeeting.ThenextconferencecallwillbeheldonThursday,December9,2010.

Diagnostics CommitteeAnneVitoreli,CommitteeChair,UniversityofFlorida,DepartmentofPlantPathology

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TheExerciseCommitteeconductedaconferencecallonNovember16,2010andthefollowingagendaitemswerediscussed:

•WrapupfromAugust&Septemberexercises•StatusoffutureAPHIS-PPQExercises

•StatusofStateExercises•Other

ThenextconferencecallisscheduledforTuesday,December21,2010.

Exercise CommitteeSharonDobesh,ProgramAreaManager/CommitteeChair,KansasStateUniversity,DepartmentofPlantPathology

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TheEpidemiologyCommitteeheldaconferencecallonNovember3,2010andthefollowingagendaitemswerediscussed:

•FollowupontheNPDNIT-Diagnostician-EpidemiologymeetinginPhoenixOct.12-13

•Updateondataanalysis•Updateondatasharing

•Updateonwebtoolsandproducts•Otheritems

ThenextEpidemiologyconferencecallisscheduledforDecember3,2010.ForcompleteminutesofthismeetingvisittheNPDNepidemiologypageatwww.npdn.org/epidemiology.

Epidemiology CommitteeCarlaThomas,ProgramAreaManager/CommitteeChair,UniversityofCaliforniaatDavis,DepartmentofPlantPathology

P R O G R A M A R E A C O M M I T T E E SPAC Highlights

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Followingthelastnewsletter,theNationalDatabaseSubcommitteeheldaconferencecallonNovember2,2010.ThesubcommitteecontinuestoworkonreviewingtheextensiveNPDNpestlists.Theagendaincluded:

•Discussionofchangesubmissions

•FollowupontheIT/Diagnostician/Epidemiologymeetingandenhancementstothesoftware

•ChangestotheUploadguidelines•DiscussionoftheAbioticPestList

ThenextconferencecallwillbeheldonDecember15,2010.

National Database CommitteeNancyGregory,CommitteeChair,UniversityofDelaware,DepartmentofPlantandSoilSciences

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TheTrainingandEducationCommitteeheldaconferencecallonNovember15,2010andthefollowingagendaitemswerediscussedonthecall:

•ReviewcommitteeforNPDNmodules(Amanda)•StrategiestobroadenFirstDetectortraining•FirstDetectorawardforthe2011nationalmeeting(Sharon)

•StateaccreditationforeLearningmodules•PosterforNDPN2011nationalmeeting

ThenextconferencecallisscheduledforMonday,December20,2010.

Training and Education CommitteeDickHoenisch,CommitteeChair,UniversityofCaliforniaatDavis,DepartmentofPlantPathology

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7Volume5Issue11

PDIS New Features / UpdatesSharmilaDabade,KansasStateUniversity,DepartmentofPlantPathology

ThePDISprogrammingteamwouldliketoannouncetwonewfeatures.

1. SuspectedProblem

Whenusingthe‘SuspectedProblem’fieldonthelabform,PDISwillautomaticallysuggestthepreviouslyusedproblemsfromthatinstitution.Thisfeaturewasaddedsothatyoucanselectfrompreviouslyusedsuspectedproblemsforconsistencyinwordingoftheproblemorissue.Hopefullythiswillreducetheissueofhavinganumberofentriesforthesameissue,suchas“SODSuspect”,“SODSurvey”,or“SuddenOakDeathProblem.”Ifyouhavemultipledataentrypersonnel,thisnewfeatureshouldbenefityou.Youstillhavetheoptionoftypinginanewprobleminyourownwords.

NPDN-USDA APHIS 2010 Fall Training Sessions-BioinformaticsKarenL.Snover-Clift,CornellUniversityandLaureneLevy,USDA-APHIS-PPQ-CHPST-NPGBL

TheNPDNDiagnosticsProgramAreaCommitteeandmembersofUSDA-APHIS-PPQ-CHPST-NationalPlantGermplasmandBiotechnologyLaboratory(NPGBL)hadpreviouslyscheduledaBioinformaticsworkshop

December6-8,2010andDecember8-10,2010.Theworkshopshavebeenpostponedduetolowattendance.Theywillmostlikelybeofferedintheearlyspringof2011alongwithaworkshopforCitrusGreening.IfyouareinterestedinparticipatinginanyoftheseworkshopspleasecontactKarenSnover-Cliftatkls13@cornell.edu.

Bioinformatics Training

Postponed!

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Also,whenyoueditanyexistingimage,youhavetheabilitytoaddoreditatagforthatimage.

2.ImageTags

Diagnosticiansrequestedafeatureforsearchingimagesthatarecontainedwithinsamples.Toaddressthisrequest,anewidentifierfieldcalled‘Tag’hasbeenaddedwhenuploadingsampleimages.Onecanaddthekeywordsinthe‘Tag’field,whichwillgetassociatedwiththatimageandallowyoutosearchusingthetagentered.

9Volume5Issue11

Regional News

Diagnosticiansmayusethisidentifiertosearchforsampleimagesbyselecting‘ImageTag(s)’fromthesearchcriteria.

Brown Marmorated Stink Bug Found in MinnesotaOnNovember22,2010,MinnesotaDepartmentofAgriculture(MDA)announcedthatscientistsfoundanunusualinsectonsomenewequipmentthathadbeendeliveredtotheir

labbuilding.StafffromMDA’sPlantProtectionDivisionconfirmedtheinsectwasthefirstbrownmarmoratedstinkbugfoundinMinnesota.

ClickheretoreadtheannouncementfromMDA.

Elongate Hemlock Scale Found in Maine ForestForthefirsttime,elongatehemlockscalewasfoundinanatural

Photo left: brown marmorated stink bug adult courtesy of David R. Lance, USDA-APHIS-PPQ, Bugwood.org.

Photo right: elongate hemlock scale courtesy of Eric R. Day, Virginia Polytechnic Institute and State University, Bugwood.org.

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forestsettinginKitteryPoint,ME,whichcouldcreateathreattoMaine’sforesthealth.WhileithasbeenfoundinKennebunkportandKennebunkonlandscapehemlock,ithasneverbeenfoundinthewild.

Elongatehemlockscale,aninvasiveinsectfromAsia,canalsoinfestfirtreesandalthoughithasnotyetbeendiscoveredonfirs,itspresencecouldeventuallyimpactthestate’scommercialChristmastreeindustry.

ClickheretoreadtheannouncementattheMaineForestServicewebsite.

Exotic Fruit Fly Found in FloridaFloridaAgricultureandConsumerServices

Commissioner,CharlesH.Bronson,announcedonNovember16,thatapeachfruitfly,Bactrocerazonata,hasbeenfoundinatrapinaguavatreeinMiami-DadeCounty,Florida.TheflywasfoundbyaU.S.DepartmentofAgricultureinspectorduringroutinesurveillanceactivitiesonNovember10,2010.ThiswasthefirstfindforthisspeciesinFloridaandthethethirdexoticfruitflyfindinFloridathisyear.

Thepeachfruitflyattacksmanydifferentfruits,vegetablesandnuts,andisconsideredoneofthemostseriousoftheworld’sfruitflypestsduetoitspotentialforeconomicharm.

ClickheretoreadtheannoucementfromtheFloridaDepartmentofAgricultureandConsumerServices.Factsheetsandotherinformationcanbefoundatwww.fl-dpi.com/enpp/ento/peach-fruit-fly.html.

First U.S. Detection of Red Palm Weevil in CaliforniaOnNovember12,APHIS-PPQannouncedthatAPHISconfirmedthedetectionofadeadredpalmweevil(RPW),Rhynchophorusferrugineus,onOctober15,2010.AlocalarboristreportedthefindinatreestumpinaresidentialareaofLagunaBeach,OrangeCounty,California.ThisisthefirstdetectionofRPWintheUnitedStates.

APHISisworkingcloselywiththeCaliforniaDepartmentofFoodandAgriculture(CDFA)todeterminethesourceandtheextentofanypotentialinfestation.OnOctober26,CDFA

inspectorsfoundasingleliveadultRPWinacanarypalmtreeapproximately100metersawayfromtheoriginaldetectioninLagunaBeach.Inspectorswerealertedtothepresenceofseveralcocoonsaroundthetree.

ReadthefullPPQannouncementhereorvisittheCaliforniaDepartmentofFoodandAgriculturewebsiteatwww.cdfa.ca.gov/phpps/rpw/index.htmlandtheCenterforInvasiveSpeciesResearchwebsiteathttp://cisr.ucr.edu/red_palm_weevil.htmlformoreinformationandexcellentphotos.

Photo courtesy of Plant Pest Diagnostics Center, CDFA.

11Volume5Issue11

RachelMcCarthy,EditorNEPDN

CornellUniversity

UpcomingEvents

JobOpportunities

National EventsDecember1-3,2010NationalCAPSMeetingKansasCity,MO

December12-15,2010ESAAnnualMeetingSanDiego,CA

January25-28,2011NucleicAcid-BasedPathogenWorkshopLexington,KY

November6-9,2011NPDNNationalMeetingSanFrancisco,CA

Regional EventsFebruary22-24,2011NEPDNMeetingNewHaven,CT

March21-23,2011SoilbornePlantPathogensandCaliforniaNematologyWorkshopDavis,CA

AssociatePlantPathologist(Diagnostician)withtheCaliforniaDepartmentofFoodandAgriculture,PlantPestDiagnosticsCenter.Clickheretoviewthefulljobannouncement.

TheIdahoStateDepartmentofAgricultureiscurrentlyrecruitingforanAgricultureProgramManager-Entomologist.ThispositionwillbeposteduntilDecember15,2010.Clickheretoreviewthejobannouncement.

TheNatureConservancywillbepostingajobdescriptionforaForestHealthProgramDirector.TheDirectoroftheForestHealthProgramhelpscoordinateandguidetheContinentalDialogueonNon-nativeInsectsandDiseases,overseesmultipleoutreachandpolicycampaignsfocusingonpreventionofintroductionandspreadofinvasivepestsandpathogens,andsupervisesseveralcontractorsandfourstaff.

ThestaffoftheForestHealthprogramwouldliketoinviteanyoneinterestedinthispositiontoplacethemselvesonalistof“interestedparties.”ThepeopleontheinterestedpartieslistwilldirectlyreceiveanemailnoticewhenthejobhasofficiallyopenedonTNC’shiringsite(www.nature.org/careers/).Toaddyourselftothelistofinterestedparties:

EmailSarahVolkman,svolkman@tnc.orgInthesubjectlinewrite“addtoFHProgramDirectorinterestedpartieslist”Includeinyouremail,yourname&yourpreferredemailaddress.Pleasedonotsendanattachedcoverletterorresume;thesewillnotbereadorusedaspartoftheapplicationprocess.