council direct ec juli control ralstonia solanacearum smith yabuuchi al council european union regard treati establish european commun articl thereof regard propos commiss regard opinion european parliament regard opinion econom social committe harm organ ralstonia solanacearum smith yabuuchi al previous pseudomona solanacearum smith smith ralstonia solanacearum smith yabuuchi al gener accept organ present direct account scientif develop potato tomato product occupi import place commun agricultur potato tomato yield constantli threaten harm organ protect potato tomato cultiv harm organ product capac maintain agricultur product increas protect measur prevent introduct harm organ territori member state limit effect organ control simultan method commun prevent spread harm organ potato tomato ralstonia solanacearum smith yabuuchi al pathogen agent potato brown rot diseas bacteri wilt potato tomato diseas outbreak caus pathogen occur part commun limit sourc infect exist consider risk potato tomato cultiv commun effect measur respect crop locat organ determin distribut prevent occur spread found prevent spread control aim erad order ensur measur commun member state addit addit stricter measur provid hindranc movement potato tomato commun laid council direct eec decemb protect measur introduct commun organ harm plant plant product spread commun measur notifi member state commiss measur account systemat offici survei locat pathogen survei includ inspect procedur environment circumst diseas remain latent unobserv grow crop potato store potato tuber includ sampl test procedur spread pathogen grow crop import factor pathogen spread surfac water wild solanac plant irrig potato tomato crop contamin water appear pose risk infect crop pathogen exist winter sown volunt potato tomato plant sourc infect carri season pathogen spread contamin potato contact infect potato contact plant harvest handl equip transport storag contain contamin organ previou contact infect potato spread pathogen reduc prevent decontamin object contamin seed potato pose major risk spread pathogen similarli latent infect seed potato pose major risk spread pathogen prevent seed potato produc offici approv programm seed potato test found free infect current knowledg biologi epidemiolog ralstonia solanacearum smith yabuuchi al european condit incomplet anticip review measur propos season similarli improv test procedur anticip light research sensit specif test method order select standardis optimum test method determin detail gener measur stricter addit measur member state prevent introduct pathogen territori desir member state cooper close commiss stand committe plant health hereinaft refer committe adopt direct articl direct concern measur member state ralstonia solanacearum smith yabuuchi al previous pseudomona solanacearum smith smith hereinaft refer organ order respect host plant organ list annex section hereinaft refer list plant materi locat determin distribut prevent occurr spread found prevent spread control aim erad articl member state conduct annual systemat offici survei organ list plant materi origin territori order identifi sourc contamin threaten product list plant materi member state carri risk assess risk spread organ identifi assess product area list plant materi conduct target offici survei organ plant list plant materi includ wild solanac host plant surfac water irrig sprai list plant materi liquid wast discharg industri process packag premis handl list plant materi irrig sprai list plant materi extent target survei determin risk identifi member state conduct offici survei organ materi grow medium soil solid wast industri process packag premis offici survei provid paragraph carri list plant materi detail set point section ii annex host plant list plant materi water includ liquid wast accord method sampl subject offici offici supervis laboratori test materi accord method survei detail inspect procedur number origin stratif time collect sampl decid respons offici bodi mean direct eec base sound scientif statist principl biologi organ take account member state concern product system list plant materi host plant organ detail result offici survei provid paragraph notifi year member state commiss accord provis point section ii annex notif submit june potato farm save seed notif submit septemb detail result crop relat previou year product detail notif submit committe provis adopt accord procedur laid articl direct eec method survei laboratori test provid paragraph subparagraph provis adopt accord procedur laid articl direct eec method survei provid paragraph subparagraph detail survei provid paragraph subparagraph view ensur compar level assur member state articl member state ensur suspect occurr confirm presenc organ territori report respons offici bodi articl case suspect occurr respons offici bodi member state concern ensur complet offici offici supervis laboratori test list plant materi relev method set annex ii accord condit point annex iii case offici approv method order confirm refut suspect occurr case confirm requir laid point annex iii appli pend confirm refut suspect occurr paragraph case suspect occurr diagnost symptom diseas caus organ posit result rapid screen test annex ii section point section ii obtain ii posit result screen test annex ii section point section iii obtain respons offici bodi member state relat product prohibit movement plant tuber crop lot consign sampl control provid establish identifi risk organ spread step trace origin suspect occurr introduc addit precautionari measur base level estim risk relat product list plant materi movement seed potato lot refer point produc place product sampl refer point order prevent spread organ case suspect occurr risk contamin list plant materi surfac water member state member state suspect occurr report immedi notifi risk identifi detail suspect occurr member state concern whall cooper member state member state notifi introduc precautionari measur accord paragraph action accord paragraph provis adopt accord procedur laid articl direct eec measur refer paragraph articl offici offici supervis laboratori test list plant materi relev method set annex ii case offici approv method confirm presenc organ sampl pursuant direct respons offici bodi member state regard sound scientif principl biologi organ product market process system host plant organ member state list plant materi establish investig determin extent primari sourc contamin accord provis annex iv test accord articl clonal relat seed potato stock ii design contamin list plant materi consign lot sampl machineri vehicl vessel store unit thereof object includ packag materi contact list plant materi sampl design contamin field unit protect crop product place product list plant materi harvest sampl sampl grow season design contamin field place product unit protect crop product sampl iii determin accord provis point annex extent probabl contamin pre post harvest contact product irrig sprai link clonal relationship design contamin iv demarc zone basi design contamin point ii determin extent probabl contamin point iii spread organ accord provis point annex crop host plant mention point product list plant materi identifi risk establish investig accord point ii design contamin host plant organ sampl iii determin probabl contamin demarc zone accord point iii iv relat product list plant materi surfac water includ liquid wast discharg industri process packag premis handl list plant materi wild solanac host plant product list plant materi identifi risk irrig sprai flood surfac water establish investig includ offici survei time sampl surfac water present wild solanac host plant establish extent contamin ii design contamin surfac water sampl extent basi investig point iii determin probabl contamin demarc zone basi design contamin point ii spread organ take account provis point ii annex member state immedi notifi member state commiss accord provis point annex contamin design paragraph ii ii detail zone demarc paragraph iv applic paragraph iii detail notif paragraph submit committe member state time submit commiss addit notif set point annex detail notif subparagraph immedi submit member committe result notif paragraph element mention member state detail notif establish investig accord paragraph applic paragraph action accord paragraph articl member state prescrib list plant materi design contamin articl ii plant control approv respons offici bodi subject provis point annex vi establish identifi risk organ spread member state prescrib list plant materi determin contamin articl iii iii includ list plant materi risk identifi produc place product determin contamin articl iii plant control respons offici bodi put dispos point annex vi establish identifi risk organ spread member state prescrib machineri vehicl vessel store unit thereof object includ packag materi design contamin articl ii determin contamin articl iii iii destroi decontamin method point annex vi decontamin object longer consid contamin prejudic measur implement paragraph member state prescrib zone demarc articl iv iii seri measur point annex vi implement detail measur notifi annual member state commiss detail notif submit committe articl member state prescrib seed potato meet requir direct eec deriv direct line potato materi obtain offici approv programm found free organ offici offici supervis test relev method set annex ii aforesaid test carri member state case confirm find organ seed potato product test earlier propag includ initi clonal select systemat test basic seed potato clone ii case establish clonal relationship test basic seed potato clone earlier propag includ initi clonal select case plant initi clonal select repres sampl basic seed potato earlier propag provis adopt accord procedur laid articl direct eec detail rule applic paragraph subparagraph point rule repres sampl provid paragraph subparagraph point articl member state ban hold handl organ articl prejudic provis direct eec member state authoris derog measur refer articl direct accord provis laid direct ec trial scientif purpos work variet select articl member state adopt relat product addit stricter measur requir combat organ prevent spread complianc provis direct eec detail measur notifi member state commiss detail notif submit committe articl amend annex direct made light develop scientif technic knowledg adopt accord procedur laid articl direct eec case method laid annex ii measur paragraph annex vi direct report prepar commiss review method measur light experi gain report submit committe januari articl member state bring forc law regul administr provis compli direct effect august forthwith inform commiss thereof member state adopt measur refer direct accompani refer occas offici public method make refer adopt member state member state immedi commun commiss main provis domest law adopt field govern direct commiss inform member state thereof articl direct enter forc dai public offici journal european commun articl direct address member state brussel juli council presid molter oj oj oj oj oj direct amend commiss direct ec oj oj direct amend commiss direct ec oj annex section list host plant ralstonia solanacearum smith yabuuchi al refer articl gt tabl gt section ii survei offici survei refer articl base biologi organ product system member state concern compris case potato time visual inspect grow crop sampl seed potato grow season store sampl subject offici offici supervis visual inspect cut tuber case seed potato potato offici offici supervis laboratori test method set annex ii ii case tomato visual inspect time grow crop plant intend replant profession notif offici survei refer articl includ case survei potato estim total area grown hectar seed potato stratif seed categori ware region number time sampl test number visual inspect field number size sampl visual inspect tuber ii case survei grow crop plant tomato intend replant profession estim total number plant number visual inspect iii case survei host plant potato tomato includ wild solanac host plant speci number time sampl area river sampl method analysi iv case survei water liquid wast discharg industri process packag premis number time sampl area river locat premis sampl method analysi annex ii test scheme diagnosi detect identif ralstonia solanacearum smith yabuuchi al scope test scheme present scheme describ procedur involv diagnosi brown rot potato tuber bacteri wilt potato tomato plant ii detect ralstonia solanacearum sampl potato tuber iii identif ralstonia solanacearum appendic detail provid prepar test materi growth media buffer solut reagent section applic test scheme diagnosi brown rot potato tuber bacteri wilt potato tomato plant test procedur intend potato tuber symptom typic suspect brown rot potato tomato plant symptom typic suspect bacteri wilt involv rapid screen test isol pathogen infect vascular tissu diagnost media case posit result identif cultur ralstonia solanacearum flow chart diagram present gt refer graphic gt flowchart refer gt start graphic gt descript symptom provid section ii rapid screen test facilit presumpt diagnosi test stream test vascular stem tissu section ii test poli hydroxybutyr granul section ii test section iii elisa test section iii pcr test section iii isol pathogen plant materi typic symptom dilut plate straightforward cultur fail advanc stage infect saprophyt bacteria grow diseas tissu outgrow inhibit pathogen isol medium isol test neg diseas symptom typic isol repeat prefer select plate test reliabl identif pure cultur ralstonia solanacearum achiev test list section ii combin pathogen test section ii strain characteris option recommend case gt end graphic gt detect identif ralstonia solanacearum sampl potato tuber procedur intend detect latent infect potato tuber prefer screen test posit complement isol pathogen case isol typic coloni identif pure cultur ralstonia solanacearum flow chart diagram present gt refer graphic gt flowchart refer gt start graphic gt sampl size standard size tuber procedur appli conveni sampl tuber screen test singl test suffici sensit reliabl detect ralstonia solanacearum sampl test recommend test prefer base biolog principl immunofluoresc test test establish screen test advantag test fulli develop valid test statutori bacteria clavibact michiganensi subsp sepedonicu read paramet method sensit test sensit threshold cell ml potato extract pellet critic factor reliabl test result qualiti antiserum antiserum high titr minimum crude antiserum accept test carri antiserum titr dilut titr indirect method prefer direct method appli test level sensit specif equival indirect method test advantag subject interpret cell stain morpholog fluoresc intens provid inform reaction specif cross reaction serolog relat bacteria soil potato tissu cell morpholog ralstonia solanacearum common test sole screen test case cross reaction suspect addit screen test base biolog principl case select plate test select plate modifi smsa medium test methodolog method sensit select test ralstonia solanacearum result dai sampl prepar pathogen obtain directli cultur readili identifi full exploit potenti test requir care prepar heel end avoid secondari bacteria potato tuber competitor ralstonia solanacearum medium affect develop pathogen strain grow poorli compon medium affect target organ care requir differenti ralstonia solanacearum bacteria develop medium select plate sole screen test provid case inhibit ralstonia solanacearum bacteria plate suspect neg test result obtain sampl retest test confirm refut diagnosi case test test elisa test elisa test gener sensit test sensit threshold cell ml potato extract pellet test cheap fast gener vulner fals posit cross reaction fals neg result inhibit phenol molecul potato extract requir antiserum specif extrem high elisa test singl screen test pcr test pcr potenti sensit detect test readili inhibit plant tuber extract compon result fals neg potato cultivar inhibitor remov inhibitor inhibit reduc dilut popul ralstonia solanacearum dilut great care step sampl test prepar prevent contamin result fals posit test fals posit aris sequenc homolog organ reason direct pcr sole screen test enrich test incub potato extract pellet sampl semi select broth modifi smsa broth multipl ralstonia solanacearum import dilut potenti inhibitor elisa pcr test ralstonia solanacearum enrich broth detect elisa pcr recommend direct plate made enrich broth enrich method test includ good potenti rel lack experi sole detect method bioassai test bioassai test isol ralstonia solanacearum potato extract pellet select enrich host plant tomato plant eggplant test requir optim incub condit method bacteria inhibitori ralstonia solanacearum smsa medium interfer test confirm test reliabl identif pure cultur ralstonia solanacearum achiev test list section ii combin pathogen test section ii strain characteris option recommend case gt end graphic gt section ii diagnosi brown rot potato tuber bacteri wilt potato tomato plant symptom symptom potato potato plant earli stage infect wilt leav top plant high temperatur dai recoveri night wilt rapidli irrevers result death plant vascular tissu transvers cut stem wilt plant brown milki ooz exud cut surfac easili express squeez cut stem vertic water thread slime stream vascular bundl potato tuber potato tuber cut transvers close heel stolon end earli stage infect glassi yellow light brown discolor vascular ring pale cream ooz emerg spontan minut gentl pressur appli thumb skin cut surfac vascular discolor distinct brown necrosi extend parenchymat tissu advanc stage infect break outward heel end ey result reddish brown slightli sunken lesion skin bacteria ooz caus soil particl adher symptom tomato tomato plant visibl symptom flaccid appear youngest leav favour environment condit pathogen soil temperatur satur humid epinasti wilt side plant dai lead total plant collaps favour condit soil temperatur larg number adventit root develop stem observ greasi cord stem evid necrosi vascular system stem cut crosswis discolour brown vascular tissu stem exud drop white yellowish bacteri ooz rapid screen test rapid screen test facilit presumpt diagnosi test stem stream test presenc ralstonia solanacearum wilt potato tomato stem assess simpl presumpt test cut stem soil level place cut surfac beaker water shortli thread bacteri slime stream spontan vascular bundl bacteria caus vascular infect potato tomato plant show phenomenon detect poli hydroxybutyr phb granul phb granul cell ralstonia solanacearum visualis stain nile blue sudan black prepar smear ooz suspend tissu microscop slide prepar smear hour cultur ypga spa appendix prepar posit control smear biovar race strain consid neg control smear heterolog strain dry pass lower surfac slide time rapidli flame smear fix nile blue test flood fix smear aqueou solut nile blue incub minut drain stain solut wash briefli gentli run tap water remov excess water tissu paper flood smear aqueou acet acid incub minut ambient temperatur wash gentli run tap water blot dry tissu paper remoisten drop water appli coverslip examin stain smear epifluoresc microscop nm oil immers magnif observ bright orang fluoresc phb granul observ normal light ensur granul intracellular cell morpholog typic ralstonia solanacearum sudan black test flood fix smear sudan black solut ethanol incub minut ambient temperatur drain stain solut wash briefli tap water remov excess water remov excess water tissu paper dip smear briefli xylol blot dry tissu paper caution xylol harm product work fume cabinet flood smear aqueou safranin leav second ambient temperatur caution safranin harm product work fume cabinet wash gentli run tap water blot dry tissu paper appli coverslip examin stain smear light microscop transmit light oil immers magnif phb granul cell ralstonia solanacearum stain blue black cell wall stain pink test screen test test section iii elisa test section iii pcr test section iii isol procedur remov ooz section discolour tissu vascular ring potato tuber vascular strand stem potato tomato plant suspend small volum steril distil water mm phosphat buffer leav minut bench prepar seri decim dilut suspens gt num gt gt den gt gt num gt gt den gt consid transfer standard volum suspens dilut gener nutrient medium na ypga spa appendix kelman tetrazolium medium appendix smsa select medium appendix spread streak dilut plate techniqu consid prepar separ plate medium dilut cell suspens cultur virul biovar race strain ralstonia solanacearum posit control incub plate dai incub prolong dai growth slow coloni smsa plate atyp die gener nutrient media virul isol ralstonia solanacearum develop pearli white flat irregular fluidal coloni characterist whorl kelman tetrazolium medium typic coloni virul isol ralstonia solanacearum cream flat irregular fluidal blood red colour whorl centr avirul form ralstonia solanacearum develop butyr deep red coloni smsa medium typic coloni virul isol ralstonia solanacearum milki white flat irregular fluidal blood red colour centr avirul form ralstonia solanacearum develop fluidal coloni complet pink red smsa medium purifi coloni characterist morpholog subcultur gener nutrient medium avoid regular subcultur induc loss virul confirm test identif ralstonia solanacearum identifi pure cultur ralstonia solanacearum procedur nutrit enzymat test note includ control strain test phenotyp properti ralstonia solanacearum univers present absent gt tabl gt media method provid lelliott amp stead test prepar suspens cell ml cultur control strain prepar seri twofold dilut antiserum appli procedur section iii titr cultur equival posit control elisa test prepar suspens gt cell ml cultur control strain appli elisa procedur section iii elisa cultur equival posit control pcr test prepar suspens cell ml cultur control strain appli pcr procedur section iii pcr product cultur size restrict enzym analysi rea pattern posit control fluoresc situ hybridis fish prepar suspens cell ml cultur control strain appli fish procedur van beuningen al oli pcr primer seal al cultur show reaction posit control protein profil denatur cell protein separ polyacrylamid gel electrophoresi page stead fatti acid profil fap grow cultur posit control strain hour trypticas soi agar appli fap procedur jans stead stead profil cultur ident posit control condit characterist fatti acid und strain characteris strain characteris option recommend case biovar determin ralstonia solanacearum separ biovar basi abil produc acid hexos alcohol sugar hayward amp gt tabl gt addit test differenti biovar subphenotyp hayward gt tabl gt race determin race buddenhagen al determin basi pathogen test tomato plant eggplant tobacco plant hypersensit reaction hr test tobacco leav lozano sequeira gt tabl gt race characteris pathogen test tobacco hypersensit test highli reliabl deduc biovar natur host origin cultur character genom fingerprint molecular differenti strain ralstoniasolanacearum complex rflp analysi cook al repetit sequenc pcr rep eric amp box pcr louw al smith al pathogen test test confirm diagnosi ralstonia solanacearum assess virul cultur identifi ralstonia solanacearum prepar inoculum cell ml cultur posit control strain inocul tomato plant eggplant prefer true leaf stage older section iii incub week high rel humid daili water observ wilt epinasti chlorosi stunt isol symptomat plant remov section tissu stem cm inocul point comminut suspend small volum steril distil water mm phosphat buffer plate incub check typic coloni ralstonia solanacearum section iii detect identif ralstonia solanacearum sampl potato tuber note standard sampl size tuber procedur appli conveni sampl tuber prepar sampl test note potato extract pellet obtain procedur detect clavibact michiganensi subsp sepedonicu pre test option consid incub sampl week encourag multipl low ralstoniasolanacearum popul ii wash tuber run water disinfect deterg air dry tuber remov clean disinfect scalpel veget knife skin heel end tuber vascular tissu visibl carefulli cut small conic core mm diamet vascular tissu heel end amount vascular tissu minimum process tuber sampl note visual examin tuber section ii stage set tuber symptom sever rot test separ section ii collect heel end close contain prefer heel end process immedi store hour longer hour process heel end procedur transfer heel end contain add suffici volum macer buffer appendix cover heel end comminut heel end ware blender ultra thurrax complet homogenis achiev avoid excess homogenis macer soak minut ii transfer heel end contain add suffici volum macer buffer cover heel end place contain rotari shaker incub rpm hour hour iii transfer heel end strong dispos macer bag stomach bag dimens mm mm radiat steril crush heel end carefulli tool hammer complet homogenis achiev add suffici volum macer buffer cover heel end macer settl minut extract bacteria process heel end procedur decant macer gentli centrifug tube leav debri contain bag decant macer cloudi centrifug minut temperatur centrifug decant macer supernat centrifug step minut minut temperatur discard supernat disturb pellet ii filter macer filtrat system pore size µm enhanc filtrat vacuum pump collect filtrat centrifug tube wash filter macer buffer centrifug filtrat minut minut temperatur discard supernat distrub pellet resuspend pellet ml pelletbuff appendix divid equal part transfer part microvi microvi test conserv remaind extract test add steril glycerol microvi vortex store week month test antiserum ralstonia solanacearum prefer race biovar determin titr suspens cell ml homolog strain ralstonia solanacearum dilut fluorescein isothiocyan fitc conjug manufactur recommend crude antiserum titr multiwel microscop slide prefer window mm diamet includ fitc conjug control test slide test repeat pb control includ posit cell observ fitc control prepar separ posit control slide suspens cell ml strain race biovar ralstonia solanacearum slide set test prepar test slide procedur pellet rel starch pipett measur standard volum µl mm window diamet scale volum larger window resuspend pellet row window remain row duplic sampl present figur ii pellet prepar decim dilut gt num gt gt den gt gt num gt gt den gt gt num gt gt den gt resuspend pellet pellet buffer pipett measur standard volum µl mm window diamet scale volum larger window resuspend pellet dilut row window remain row duplic sampl present figur droplet dry fix bacteri cell slide heat flame ethanol procedur test slide prepar prepar set twofold dilut antiserum buffer appendix titr gt num gt gt den gt titr gt num gt gt den gt titr titr ii test slide prepar ii prepar work dilut wd antiserum buffer work dilut dilut antiserum optimum specif half titr gt tabl gt gt tabl gt arrang slide damp tissu paper cover test window antiserum dilut appli pb fitc window volum antiserum appli window equival volum extract appli incub cover minut ambient temperatur shake droplet antiserum slide rins slide carefulli buffer wash minut buffer tween subsequ minut buffer appendix carefulli remov excess moistur arrang slide damp tissu paper cover test window fitc window dilut fitc conjug determin titr volum conjug appli window ident volum antiserum appli incub cover minut ambient temperatur shake droplet conjug slide rins wash carefulli remov excess moistur pipett µl phosphat buffer glycerol appendix similar mountant window appli coverslip read test examin test slide epifluoresc microscop filter suitabl excit fitc oil immers magnif scan window diamet angl perimet check posit control slide cell bright fluoresc complet stain note test repeat stain aberr read test slide observ absenc fluoresc cell fitc control window fluoresc cell fitc control specif bind conjug autofluoresc contamin note repeat test observ observ bright fluoresc cell characterist morpholog ralstonia solanacearum test window fluoresc intens equival posit control strain antiserum dilut cell incomplet stain weak fluoresc disregard cell interpret test result interpret test result bright fluoresc cell characterist morpholog found test neg ii bright fluoresc cell characterist morpholog found determin number cell microscop field calcul number cell ml resuspend pellet appendix popul approxim cell ml resuspend pellet consid limit detect test sampl gt cell ml resuspend pellet test consid posit sampl gt cell ml resuspend pellet test consid posit iii larg number gt cell ml incomplet weakli fluoresc cell titr antiserum test carri test base biolog principl repeat test antiserum ten fold dilut pellet elisa test base robinson smith al antiserum ralstonia solanacearum prefer race biovar determin titr suspens cell ml homolog strain ralstonia solanacearum nunc polysorb microtitr plate recommend includ neg potato extract control phosphat buffer salin pb control suspens gt cell ml strain race biovar ralstonia solanacearum posit control test ident manner sampl separ sampl microtitr plate pipett µl resuspend pellet microvi heat minut remov microvi ic add equal volum doubl strength carbon coat buffer appendix vortex appli µl aliquot well microtitr plate incub hour overnight flick extract well wash well time pb tween appendix leav wash solut well minut prepar dilut ralstonia solanacearum antiserum block buffer appendix appli µl antiserum dilut well incub hour flick antiserum well wash well prepar dilut alkalin phosphatas conjug block buffer appli µl conjug dilut well incub hour flick conjug well wash well prepar alkalin phosphatas substrat solut appendix appli µl well incub minut hour dark ambient temperatur read absorb nm interpret elisa test elisa test neg optic densiti od sampl od neg control pcr test base seal al note filterplug pipett tip stage sampl prepar manipul involv pcr prepar suspens cell ml race biovar strain ralstonia solanacearum posit control test ident manner sampl pipett µl resuspend pellet microvi altern transfer µl resuspend pellet microvi µl naoh mix repeatedli invert microvi heat minut remov microvi immedi ic prepar decim dilut gt num gt gt den gt gt num gt gt den gt consid steril distil ultra pure water upw prepar pcr reaction mix appendix steril vial ad compon order gt tabl gt reaction calcul quantiti compon requir number reaction mix compon transfer µl µl mix steril pcr vial vial pcr reaction mix ic µl reaction volum scale compon pcr amplif option puls centrifug vial boil sampl posit control add order µl sampl water control posit control vial pcr reaction mix place vial heat block dna thermal cycler run programm cycl minut denatur templat cycl ii second denatur iii second anneal primer iv second extens copi cycl minut extens cycl vi hold note paramet perkin elmer thermal cycl requir miner oil overlai pcr reaction vial modif durat step ii iii iv amplif profil remov vial thermal cycler analys pcr product immedi store vial dai longer analysi pcr product pcr fragment detect agaros gelelectrophoresi stain ethidium bromid prepar agaros gel gentli bring boil agaros tri acet electrophoresi tae buffer cool molten agaros pour mould electrophoresi unit insert comb solut solidifi remov comb submerg gel tae cover mm buffer place µl droplet load buffer parafilm add µl pcr product sampl posit control water control mix gentl aspir pipett tip load volum modifi fit capac well agaros gel carefulli load well gel includ refer dna marker connect wire power suppli electrophoresi equip run gel cm front track indic cm end gel switch power suppli disconnect wire electrophoresi unit carefulli remov gel soak ethidium bromid solut minut note wear dispos glove time handl ethidium bromid power mutagen destain distil water minut visualis amplifi dna fragment uv transillumin pcr product ralstonia solanacearum primer set oli bp length check dna marker posit control note water control neg case posit repeat test photograph gel perman record requir confirm authent amplifi fragment restrict enzym analysi rea restrict enzym analysi rea transfer µl pcr product microvi add µl enzym buffer µl restrict enzym ava ii mix gentl aspir pipett tip drop remain wall vial puls spin microcentrifug incub hour analys digest pcr fragment agaros gelelectrophoresi interpret pcr test result pcr test neg characterist bp fragment detect fragment detect posit control strain ralstonia solanacearum pcr test posit bp fragment detect rea analysi amplifi fragment ident posit control strain ralstonia solanacearum select plate test base elphinston al perform test dilut plate techniqu prepar decim dilut gt num gt gt den gt gt num gt gt den gt consid resuspend pellet pelletbuff pipett measur standard volum µl resuspend pellet dilut modifi smsa select medium appendix spread glass rod surfac medium consid perform dilut streak µl loop resuspend pellet flame loop streak ii transfer measur standard volum µl resuspend pellet modifi smsa select medium spread glass rod surfac medium streak rod flame modifi smsa plate appli dilut plate techniqu suspens cell ml virul race biovar strain ralstonia solanacearum posit control set separ modifi smsa plate incub plate start read plate dai neg incub dai coloni virul isol ralstonia solanacearum milki white flat irregular fluidal blood red color centr show intern streak whorl purifi coloni characterist morpholog subcultur gener nutrient medium appendix identifi pure cultur section ii confirm ralstonia solanacearum cultur pathogen test section ii interpret select plate test result select plate test neg bacteri coloni isol dai coloni characterist ralstonia solanacearum isol provid inhibit suspect coloni bacteria coloni characterist ralstonia solanacearum found posit control select plate test posit coloni characterist ralstonia solanacearum isol bioassai test base jans test plant suscept tomato eggplant seedl true leaf stage sampl water testplant hour inocul distribut µl resuspend pellet testplant inocul stem cotyledon place inocul techniqu seedl suspens cell ml virul biovar race strain ralstonia solanacearum posit control pelletbuff neg control separ posit control plant plant avoid cross contamin grow test plant week high rel humid daili water observ wilt epinasti chlorosi stunt isol infect plant section ii identifi pure cultur characterist morpholog section ii confirm ralstonia solanacearum cultur pathogen test section ii consid check absenc infect batch test plant show indic infect remov test plant cm section stem cm inocul point homogenis tissu macer buffer perform dilut plate section iii posit identifi pure cultur characterist morpholog section ii confirm ralstonia solanacearum cultur pathogen test section ii interpret bioassai test result bioassai test neg test plant infect ralstonia solanacearum provid ralstonia solanacearum detect posit control bioassai test posit test plant infect ralstonia solanacearum enrich test base elphinston al transfer µl resuspend pellet ml modifi smsa broth appendix incub hour case longer hour cap tube loos fit aerat tighten cap vortex aliquot test section elisa test section pcr test section pathogen test refer section ii appendix nutrient media isol cultur ralstonia solanacearum nutrient agar na gt tabl gt prepar litr volum medium litr flask dissolv ingredi sterilis autoclav minut cool pour plate yeast pepton glucos agar ypga gt tabl gt prepar litr volum medium litr flask dissolv ingredi sterilis autoclav minut cool pour plate sucros pepton agar spa gt tabl gt prepar litr volum medium litr flask dissolv ingredi adjust ph sterilis autoclav minut cool pour plate kelman tetrazolium medium gt tabl gt prepar litr volum medium litr flask dissolv ingredi sterilis autoclav minut cool add filter sterilis aqueou solut triphenyl tetrazolium chlorid sigma obtain final concentr mg litr pour plate appendix materi sampl prepar macer buffer mm phophat buffer ph buffer tissu macer gt tabl gt dissolv ingredi check ph aliquot consid sterilis autoclav minut addit polyvinylpyrrolidon mwt pvp recommend perform direct pcr test reduc incid amplif inhibit aromat molecul extract addit defloccul antifoam agent anti oxid recommend ware blender ultra turrax homogenis procedur macer potato tissu core gt tabl gt autoclav separ add desir concentr pelletbuff mm phosphat buffer ph buffer resuspens dilut potato heel pellet gt tabl gt dissolv ingredi check ph aliquot consid sterilis autoclav minut appendix materi test buffer mm phosphat buffer salin pb ph buffer dilut antisera gt tabl gt dissolv ingredi check ph aliquot consid sterilis autoclav minut buffer tween buffer wash slide add tween buffer phosphat buffer glycerol ph buffer mountant fluid window slide enhanc fluoresc gt tabl gt appendix determin contamin level test surfac area window multispot slide gt num gt gt den gt gt tabl gt surfac area object field gt num gt gt den gt gt tabl gt calcul direct measur formula gt num gt gt den gt gt tabl gt amp radic gt num gt gt den gt amp radic gt num gt gt num gt gt den gt gt den gt gt num gt gt den gt gk count number typic fluoresc cell field calcul number typic fluoresc cell window gt num gt gt den gt calcul number typic fluoresc cell ml pellet gt num gt gt den gt gt tabl gt appendix materi elisa test doubl strength carbon coat buffer ph gt tabl gt dissolv ingredi check ph aliquot consid sterilis autoclav minut sodium sulphit final concentr ad anti oxid extract high fraction aromat molecul phosphat buffer salin pb ph gt tabl gt dissolv ingredi check ph aliquot consid sterilis autoclav minut phosphat buffer salin tween pb gt tabl gt block antibodi buffer freshli prepar gt tabl gt alkalin phosphatas substrat solut ph gt tabl gt mix adjust ph concentr hci make litr distil water add mgcl dissolv phosphatas substrat mg tablet sigma ml solut appendix materi pcr test oligonucleotid primer sequenc gt tabl gt materi seal al appendix materi select plate enrich test smsa select medium engelbrecht modifi elphinston al delet agar tetrazolium salt basal medium gt tabl gt prepar gt num gt gt den gt litr volum medium litr flask dissolv ingredi check ph adjust ph autoclav ralstonia solanacearum grow medium ph gt sterilis autoclav minut cool add ingredi sigma obtain final concentr gt tabl gt dissolv ingredi ethanol concentr volum medium prepar ingredi polymixin chloramphenicol requir slight warm shake smsa broth elphinston al prepar smsa select medium delet agar dispens ml aliquot ml dispos univers tube refer buddenhagen sequeira kelman descript race pseudomona solanacearum phytopatholog cook elizabeth sequeira genet divers pseudomona solanacearum detect restrict fragment length polymorph dna probe virul hypersensit respon molecular plant microb interact dinesen deboer extract clavibact michiganensi subsp sepedonicu composit sampl potato tuber american potato journal elphinston hennessi wilson stead sensit method detect pseudomona solanacearum smith smith potato tuber extract eppo bulletin engelbrecht modif semi select medium isol quantif pseudomona solanacearum aciar bacteri wilt newslett hayward characterist pseudomona solanacearum journal appli bacteriolog hayward systemat phylogeni pseudomona solanacearum relat bacteria bacteri wilt diseas caus agent pseudomona solanacearum ed hayward hartman cab intern oxford jans detect method pseudomona solanacearum symptomless potato tuber data sensit specif eppo bulletin jans infra intraspecif classif pseudomona solanacearum strain cell fatti acid analysi systemat appli microbiolog kelman relationship pathogen pseudomona solanacearum coloni appear tetrazolium medium phytopatholog lelliot stead method diagnosi bacteri diseas plant preec ed blackwel scientif public oxford pp louw fulbright stephen de bruijn differenti genom structur rep pcr fingerprint rapidli classifi xanthomona campestri pv vesicatoria phytopatholog lozano sequeira differenti race pseudomona solanacearum leaf infiltr techniqu phytopatholog mirza rademak jans akkerman specif ribosom rna target oligonucleotid probe clavibact michiganensi subsp sepedonicu canadian journal microbiolog robinson smith jone elphinston ford product antibodi pseudomona solanacearum caus agent bacteri wilt food agricultur immunolog seal jackson young daniel differenti pseudomona solanacearum syzygii picketti blood diseas bacterium partial rrna sequenc construct oligonucleotid primer sensit detect polymeras chain reaction journal gener microbiolog smith offord holder saddler genet divers burkholderia solanacearum synonym pseudomona solanacearum race kenya appli environment microbiolog stead techniqu detect identifi plant pathogen bacteria techniqu rapid detect plant pathogen ed duncan torranc blackwel scientif public oxford stead group plant pathogen pseudomona spp cellular fatti acid profil intern journal systemat bacteriolog van beuningen derk jans detect identif clavibact michiganensi subsp sepedonicu special attent fluoresc situ hybridis fish rrna target oligonucleotid probe züchtungsforschung annex iii suspect occurr posit result screen test identifi list plant materi relev method set annex ii case offici approv method confirm refut complet method await retent conserv lot part thereof sampl origin packag label remain part sampl remain extract addit prepar materi screen test immunofluoresc slide relev document complet method case confirm organ retent conserv materi paragraph sampl infect tomato eggplant materi inocul tuber plant extract isol cultur organ month notif procedur articl annex iv element investig refer articl includ relev place product grow grown potato clonal relat potato found infect organ grow grown tomato sourc tomato found infect organ grow grown potato tomato offici control suspect occurr organ grow grown potato clonal relat potato grown place product found infest organ grow potato tomato locat neighbourhood infest place product includ place product share product equip facil directli common contractor surfac water irrig sprai sourc confirm suspect infest organ surfac water irrig sprai sourc common place product confirm suspect infest organ flood flood surfac water confirm suspect infest organ ii surfac water irrig sprai flood field place product confirm infest organ annex element determin extent probabl contamin articl iii iii includ relev list plant materi grown place product design contamin articl ii place product product link list plant materi design contamin articl ii includ share product equip facil directli common contractor list plant materi produc place product refer previou indent present place product period list plant materi design contamin articl ii present place product refer indent store handl list plant materi place product machineri vehicl vessel store unit thereof object includ packag materi contact list plant materi design contamin articl ii list plant materi store contact structur object list previou indent prior cleans disinfect structur object result investig test articl case potato tuber plant sister parent clonal relationship case tomato plant sourc list plant materi design contamin articl ii test neg organ appear contamin probabl clonal link place product list plant materi refer previou indent place product list plant materi water irrig sprai design contamin articl ii list plant materi produc field flood surfac water confirm infest organ determin spread articl iv iii includ case articl iv consider element proxim place product grow list plant materi common product seed potato stock place product surfac water irrig sprai list plant materi case risk surfac water run flood place product design contamin articl ii ii case surfac water design contamin articl ii place product produc list plant materi adjac risk flood surfac water design contamin discret irrig basin surfac water design contamin detail notif refer subparagraph articl includ date report suspect occurr articl date sampl confirm presenc organ articl descript element design contamin zone demarc detail addit notif refer subparagraph articl includ potato consign lot design contamin certif prescrib articl direct eec passport number registr number potato produc collect warehous dispatch centr tomato plant consign lot design contamin certif prescrib articl direct eec passport number accord list annex part section direct eec varieti categori seed potato stock case inform relat confirm outbreak commiss requir annex vi refer articl provis inciner anim feed heat treatment risk organ surviv deep burial dispos site risk seepag agricultur land contact water sourc irrig agricultur land industri process direct deliveri process plant offici approv wast dispos facil conform provis laid annex vii direct measur provid establish identifi risk organ spread measur immedi notifi commiss member state dispos list plant materi refer articl control respons offici bodi member state concern commun respons offici bodi ensur control time approv respons offici bodi member state potato pack process respect wast dispos facil refer indent potato tuber ware potato intend consumpt pack site wast dispos facil readi direct deliveri repack intend direct deliveri ware potato intend industri process intend direct deliveri process plant wast dispos facil dispos provid establish identifi risk organ spread subject approv respons offici bodi measur immedi notifi commiss member state ii plant part includ stem foliag debri destruct dispos provid establish identifi risk organ spread measur notifi commiss member state method decontamin object refer articl cleans disinfect identifi risk organ spread emploi supervis respons offici bodi member state seri measur implement member state demarc zone establish articl iv iii refer articl includ case place product design contamin articl ii field unit protect crop product design contamin articl ii grow year design contamin measur elimin volunt potato tomato plant host plant organ includ solanac weed plant potato tuber plant tomato plant seed take account biologi organ host plant plant speci brassica identifi risk organ surviv crop identifi risk organ spread potato tomato crop season period preced indent condit field found free volunt potato tomato plant host plant includ solanac weed consecut grow year prior plant case potato offici certifi seed potato plant ware product offici survei includ test detail articl conduct potato tomato crop season succeed refer previou indent rotat cycl case potato offici certifi seed potato plant seed ware product case potato tomato offici survei detail articl conduct ii grow year design contamin measur elimin volunt potato tomato plant host plant organ includ solanac weed field establish maintain year bare fallow cereal risk identifi perman pastur frequent close cut intens graze grass seed product plant succeed year host plant organ identifi risk organ surviv spread potato tomato crop season period preced indent case potato offici certifi seed potato plant seed ware product offici survei includ test detail articl conduct field grow year design contamin potato tuber plant host plant organ plant measur elimin volunt potato tomato plant host plant includ solanac weed case potato tuber offici certifi seed potato plant ware product condit respons offici bodi satisfi risk volunt potato tomato plant host plant organ includ solanac weed elimin grow crop inspect time volunt potato plant test organ addit potato harvest tuber inspect grow year indent case potato offici certifi seed potato plant seed ware product grow year indent case potato offici certifi seed potato seed potato grown offici control offici certifi seed potato plant seed ware product grow year refer previou indent measur elimin volunt potato tomato plant host plant organ includ solanac weed offici survei detail articl conduct case seed potato plant seed product test tuber carri immedi design contamin articl ii subsequ grow year includ permiss potato tomato crop season field design contamin detail paragraph machineri storag facil place product involv potato tomato product cleans disinfect method point offici control irrig sprai programm includ ban thereof introduc order prevent spread organ unit protect crop product design contamin articl ii complet replac grow medium potato tuber plant host plant organ includ tomato plant seed plant unit subject offici supervis measur elimin organ remov host plant materi includ complet chang grow medium cleans disinfect unit equip subsequ grant approv potato tomato product respons offici bodi potato product product offici certifi seed potato mini tuber micro plant deriv test sourc offici control irrig sprai programm includ ban thereof introduc order prevent spread organ demarc zone prejudic measur detail member state immedi grow year design contamin aa case demarc zone determin articl iv ensur supervis respons offici bodi premis grow store handl potato tuber tomato premis oper machineri potato tomato product contract requir cleans disinfect machineri store premis method point requir plant certifi seed seed grown offici control potato crop zone test harvest seed potato crop grown place product determin contamin articl iii requir separ handl harvest potato seed stock ware premis zone conduct offici survei detail articl ab case surfac water design contamin articl ii includ element spread organ accord annex point conduct annual survei time includ sampl surfac water solanac host plant relev water sourc test accord list plant materi relev method set annex ii case offici approv method introduc offici control irrig sprai programm includ ban water design contamin irrig sprai list plant materi host plant order prevent spread organ ban review basi result obtain annual survei case liquid wast discharg contamin introduc offici control dispos wast industri process packag premis handl list plant materi establish programm replac seed potato stock period time annex vii offici approv wast dispos facil refer annex vi paragraph fourth indent conform provis risk spread organ obviat potato tomato process wast includ reject potato peel tomato solid wast potato tomato dispos deep burial dispos site risk seepag agricultur land contact water sourc irrig agricultur land wast convei directli site contain condit risk loss wast inciner ii liquid process wast prior dispos liquid wast suspend solid subject filtrat settlement process remov solid solid dispos set subparagraph liquid wast heat minimum minut prior dispos dispos subject offici approv offici control risk wast contact agricultur land water sourc irrig agricultur land detail thereof notifi member state commiss