NATIONAL INSTITUTE OF BIOLOGY

Prof. dr. Maja RavnikarProf. dr. Maja Ravnikar

Department of Plant Physiologyand Biotechnology

LABORATORY PROCEDURESDIAGNOSTIC SUPPORT

MainMain pointspoints

IntroductionIntroduction ofof ourour DepartmentDepartmentDescriptonDescripton ofof servicesservices andandphytodiagnosticphytodiagnostic activitiesactivitiesTestingTesting ofof potatopotato

IntroductionIntroduction ofof ourour DepartmentDepartment

6 6 authorisedauthorised laboratorieslaboratories in in publicpublic institutionsinstitutionsCoordinationCoordination mustmust bebe carefullycarefully undertakenundertakenAvtivitiesAvtivities are are plannedplanned withwith annualannual programmeprogrammeandand contractcontractaltogetheraltogether 5000 5000 samplessamples perper yearyearNot ideal Not ideal butbut rationalrational

NIB NIB closeclose connectionconnection ofof researchresearch andand servicesservicesdiagnostitiansdiagnostitians are are involvedinvolved or or workwork in in closeclose connectionconnection withwithresearchersresearchers, , shareshare thethe equipmentequipment, , diagnosticdiagnostic problemsproblems are are quicklyquickly solvedsolved

NIB is NIB is governmentalgovernmentalinstitutioninstitution, , ourourDepartamentDepartament is is authorizedauthorizedbyby PhytosanitaryPhytosanitaryAdministrationAdministration ((MinistryMinistry ofofAgricultureAgriculture, , ForestryForestry andandFoodFood) ) forfor diagnosisdiagnosis ofofbacteriabacteria, , phytoplasmasphytoplasmasandand virusesviruses andand otherotherscientificscientific andand technicaltechnicalassistanceassistance

NATIONAL INSTITUTE OF BIOLOGY

13 13 outout ofof 2255 personspersons workwork in in phytodiagnosisphytodiagnosis::88 Ph.D., Ph.D., 33 technical assistants,technical assistants,6 6 researchersresearchers8 Ph.D. students, 8 Ph.D. students,

Laboratories for:Laboratories for:molecular biologymolecular biologyvirologyvirologyelectron microscopyelectron microscopybacteriologybacteriologybiochemistrybiochemistryplant tissue culturesplant tissue culturesinstrumental CENTRE PLANTAinstrumental CENTRE PLANTA::RealtimeRealtime PCR, PCR, growthgrowth chamberschambers,,quarantinequarantine greenhousegreenhouse,,EM, EM, MicroarrayMicroarray facilitiesfacilities

Department of Plant Physiology and Biotechnology

Organization of the work in Organization of the work in laboratorieslaboratories

InstructionsInstructions forfor safety safety workworkTThe principles of good laboratory practice are introducing he principles of good laboratory practice are introducing for for last 7last 7 years. years. For For phytodiagnosticphytodiagnostic –– sloveneslovene regulationregulation, , preparedprepared bybyPhytosanitaryPhytosanitary administrationadministrationOurOur DepartmentDepartment is is workingworking in in accordanceaccordance withwith ISO ISO 17025 17025 andand for GMO for GMO –– accreditationaccreditation accordingaccording to ISO to ISO 17025 17025 waswas addoptedaddopted in 2003in 2003. . ExternalExternal auditaudit forfor ISO 9001 is ISO 9001 is planedplaned forfor NIB in 2004NIB in 2004BIA LIMS BIA LIMS computercomputerisedised systemsystem forfor reception of samples, reception of samples, registration, results report,registration, results report, etcetc..workwork withwith datadata ofof ourour clientsclients are are highlyhighly confidentialconfidential

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Projects:Projects:

ScientificScientific ProgramProgram foundedfounded byby MMinistryinistry ofof EducationEducation, , ScienceScience andand SportSport, , otherother researchresearch projectprojectss15 contracts (applied projects)15 contracts (applied projects)6 international projects 6 international projects (2 EU, FAO UNIDO, PHARE)(2 EU, FAO UNIDO, PHARE)international cointernational co--operation on nonoperation on non--official basisofficial basis

AppliedApplied workwork ((collaborativecollaborative researchresearch, , servicesservices, , educationeducation ofof personelpersonel):):

ServicesServices andand researchresearch forfor more more thanthan 50 50 companiescompanies (M(M--KŽK, KŽK, LekLek, , KrkaKrka, , SemenarnaSemenarna, Omega, Bia, Omega, Bia SeparationsSeparations, , BIA, BIA, foodfood producersproducers andand importersimporters))governmentalgovernmental institutionsinstitutions ((MMAFF, MESPAFF, MESP andandinspecinspectoratestorates

Research work

1. Physiology of plant – pathogen interactions2. Transformation of plants and studiesof plant secondary methabolites3. Biology of plant pathogens4. Intruduction of new molecularmethods in diagnosis5. New methods in GMO testing

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Services:

1. Diagnostics of plant pathogensdevelopment and introducion of detection methodsand procedures for quarantine and economically important plant pathogenic viruses, phytoplasma and bacteria. The detection procedures are designed and organised according to the EPPO recommendations and to EU and Slo directives and rules

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The procedures are designed for each pathogen separately and involvedetection methods such as immunoserological testing

(ELISA, IF, Western blot, aglutination), electron microscopy (EM and IEM), test plants, fatty acid profiling (FAP), molecular biology methods (PCR, RFLP, FISH, Real-time PCR)

FireFire blightblight

ServicesServices::2. Detection of 2. Detection of GMOsGMOs

In In foodfood, , feedfeed andand graingrain samplessamples ofof: RR : RR soyasoya, , corncorn:MON 810, T:MON 810, T--25, 25, BtBt--11, 11, BtBt--176 176 ((oilseedoilseed raperape is is underunder developmentdevelopment).).

3. 3. RealReal--TimeTime PCRPCRVeryVery goodgood methodmethod forfor detectiondetection ofofdifferentdifferent RNA RNA andand DNA in DNA in samplessamples..

ResidualResidual DNA (DNA (pharmacypharmacy, , foodfoodindustryindustry))DetectionDetection ofof microorganismsmicroorganisms((agricultureagriculture))

4.4.MicroarraysMicroarraysResearchResearch focusedfocused towardtoward improvedimprovedindustrialindustrial microorganismsmicroorganisms

TipizationTipization ofof starterstarter culturescultures in in bioprocesesbioprocesesStudyStudy ofof drug drug effectseffects

PHYTODIAGNOSTIC PHYTODIAGNOSTIC ACTIVITIESACTIVITIES

CloseClose connectionconnection to to PhytosanitaryPhytosanitaryAdministrationAdministration andand PhytosanitaryPhytosanitary InspectionInspectionServiceService andand otherother authorisedauthorised institutionsinstitutionsIn last 10 In last 10 yearsyears wewe adoptedadopted methodsmethods forfor more more thanthan 5050 differentdifferent virusesviruses, , bacteriabacteria andandphytoplasmasphytoplasmaswewe confirmedconfirmed withwith laboratorylaboratory methodsmethods more more thanthan 2020 pathogenspathogensaltogetheraltogether wewe are or are or wewe werewere workingworking withwith 8080differentdifferent quarantenequarantene or or economicallyeconomically importantimportantpathogenspathogens

TestingTesting on on regularregular basisbasis ((~~ 40 40 spssps.).)BacteriaBacteria 1414 VirusesViruses 2020 Phytoplasmas5Phytoplasmas5

potatopotato 5 5 RsmRsm,,CmsCms..EspsEsps

tomatotomato andandpepperpepper

4 4 XcvXcv,,CmmCmm,,PssPss,Pst,Pst

33 PcichPcich,,PcorPcor,,EcEc

1313

CucurbitaeCucurbitae spssps, , AlliumAllium, , othersothers

1 1 ErwiniaErwinia spssps 3 3

FruitFruit threesthrees 3 3 EaEa, , XcpXcp,,PssPss 3 3 ESFY,PD,APESFY,PD,AP

grapevinegrapevine 2 2 BN,FDBN,FD

pelargoniumpelargonium 2 2 XcpXcp,Rs,Rs 88

ChrysanthChrysanth,,diffdiff. . ornamentalsornamentals

1 1 EcEc 55

Bacteria confirmed: Erwinia amylovora,Ralstonia s., Xanthomonas and Pseudomonasspp., Xylophyllus ampelinus(import: 40 consigments infected with Cms, or Rs in last 6 years)

Imortant new viruses: RuSPaV1TOSPOVIRUSES: TSWV, INSV, IYSV, CSNVViruses on Allium plants, Fabaceae, pelargonium andothers

We confirmed phytoplasmas:AP (apple proliferation),ESFY (european stone fruitdisease),PD (pear decline), BN (bois noir)

METHODS IN DETECTION OF PLANT METHODS IN DETECTION OF PLANT VIRUSES AND PHYTOPLASMAVIRUSES AND PHYTOPLASMA

VIRUSES:ELISA, test plants, electron microscopy (IS EM), RT-PCR,Real-time PCRPHYTOPLASMAS:ELISA, PCR, nested PCR, RFLP, IF

METHODS IN DETECTION OF METHODS IN DETECTION OF PLANT PATHOGENIC BACTERIAPLANT PATHOGENIC BACTERIA

Most common: IF, media (semiselective), test plants, PCROthers: aglutination, ELISA, FISH, RFLP, biochemical tests, Real time PCR,FAP (other laboratories – PPS, CSL)

TestingTesting ofof RS RS andand CmsCms

EkstractionEkstraction ofof bacteriabacteria fromfrom 200 200 tuberstubers perper 25t25tFirstFirst screeningscreening test is IF (FISH or PCR test is IF (FISH or PCR withwith newnew EU EU directivedirective))IsolationIsolation::

MediaMediaEnrichmentEnrichment on on hosthost plantsplants

ConfirmationConfirmation teststests::Test Test plantsplantsIFIFPCRPCRFISHFISHOthersOthers: : BiochemisrtyBiochemisrty, , RealReal timetime PCR, FAPPCR, FAP

BROWN ROTBROWN ROT

RING ROTRING ROT

TestingTesting ofof RS RS andand CmsCms

AverageAverage timetime forfor analysisanalysis is 3 is 3 –– 5 5 workingworkingdaysdays, in , in thethe casecase ofof positivepositive resultresult or or firstfirstscreeningscreening test it test it willwill prolongedprolonged forfor Rs Rs forfor 30 30 ––65 65 daysdays andand forfor CmsCms forfor 80 80 –– 120 120 daysdays

AcceptanceAcceptance ofof newnew EU EU directivedirective in 2004 in 2004 willwillenableenable shortershorter responseresponse timetime in in thethe casecase ofofpositivepositive resultresult ofof firstfirst screeningscreening test, test, introducingintroducing additionaladditional screeningscreening methodmethod forforconfirmationconfirmation or or rejectionrejection ofof firstfirst testtest

Cooperation with other phytodiagnostic laboratories:

Phare Twining programes wirh CSL, GB and PPS, NL

• Cooperation in European Plant Protection Organization(EPPO), Panel on Bacteriology

•Cooperation in 6th EU Framework programe PORTCHECK, leading by CSL, GB in phytodiagnostics

•Taking part in ring teste for Cms (ring rot)

• Cooperation with laboratories in Great Britain, Netherlands, Germany, Italy, France, Portugal, USA

•

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To enhance productionand help to our economy

http://www.nib.si

aleksialeksi AlexivirusesAlexivirusesAMVAMVAlfalfaAlfalfa mosaic virusmosaic virus APAPappleappleproliferationproliferation ArMVArMVArabisArabis mosaic virusmosaic virus AYAYasteraster yellowsyellows BCMVBCMVBeanBeancommon mosaic viruscommon mosaic virus BNFBNFboisbois noir noir phytoplasmasphytoplasmas BYMVBYMVBeanBean yellow yellow mosaic virusmosaic virus CaMVCaMVCauliflowerCauliflower mosaic virusmosaic virus CERVCERVCarnCarn etch ring etch ring virusvirus CmmCmmtomatotomato cankerClavibactercankerClavibacter michiganensismichiganensis subspsubsp michiganensismichiganensisCmsCmspotatopotato ring ring rotClavibacterrotClavibacter michiganensismichiganensis subspsubsp. . sepedonicussepedonicusCMVCMVCucumberCucumber mosaic virusmosaic virus CSNVCSNVChrysanthemumChrysanthemum stem stem necrosis virusnecrosis virus CVBCVBChrysanthemumChrysanthemum B B carlaviruscarlavirus EaEaFireFire blightErwiniablightErwiniaamylovoraamylovoraEcEc ErwiniaErwinia carotovoracarotovoraEchryEchry ErwiniaErwiniachrysanthemichrysanthemiESFYESFYeuropeaneuropean stone fruit yellow diseasestone fruit yellow disease FDFDflavescenceflavescencedoreedoree GCLVGCLVGarlicGarlic common latent viruscommon latent virus GFkVGFkVGrapevineGrapevine fleck fleck virusvirus GFLVGFLVGrapevineGrapevine fanleaffanleaf virusvirus GLRaVGLRaV--11Grapevine Grapevine leafrollleafroll--assoc. assoc. virus 1virus 1 GLRaVGLRaV--22Grapevine Grapevine leafrollleafroll--assoc. virus 2assoc. virus 2 GLRaVGLRaV--33Grapevine Grapevine leafrollleafroll--assoc. virus 3assoc. virus 3 GLRaVGLRaV--66Grapevine Grapevine leafrollleafroll--assoc. virus assoc. virus 66 GRSVGRSVGroundnutGroundnut ringspotringspot virusvirus GVAGVAGrapevineGrapevine virus virus AA GVBGVBGrapevineGrapevine virus Bvirus B INSVINSVImpatiensImpatiens necrotic spot virusnecrotic spot virus IYSVIYSVIrisIrisyellow spot virusyellow spot virus LMVLMVLettuceLettuce mosaic virusmosaic virus LYSVLYSVLeekLeek yellow stripe yellow stripe virusvirus MBSMBSmaizemaize bushy stuntbushy stunt MWMVMWMVMoroccanMoroccan watermelon mosaic watermelon mosaic virusvirus OYDVOYDVOnionOnion yellow dwarf virusyellow dwarf virus PDPDpearpear declinedecline PepMVPepMVPepinoPepinomosaic virusmosaic virus PFBVPFBVPelargoniumPelargonium flower break virusflower break virus PLCVPLCVPelargoniumPelargoniumleaf curl virusleaf curl virus PLPVPLPVPelargoniumPelargonium llinelline pattern viruspattern virus PLRVPLRVPotatoPotatoleafrollleafroll virusvirus

PsPs Pseudomonas Pseudomonas syringaesyringaePs.margPs.marg Pseudomonas Pseudomonas marginalismarginalisPs.viridPs.virid.. Pseudomonas Pseudomonas viridiflavaviridiflavaPsphPsph Pseudomonas Pseudomonas syringaesyringae pvpv.. phaseolicolaphaseolicolaPssPss Pseudomonas Pseudomonas syringaesyringae pvpv..syringaesyringaePstPst Pseudomonas Pseudomonas syringaesyringae pvpv.. tomatotomatoPVAPVAPotatoPotato virus virus AA PVMPVMPotatoPotato virus Mvirus M PVSPVSPotatoPotato virus Svirus S PVVPVVPotatoPotato virus virus VV PVXPVXPotatoPotato virus Xvirus X PVYPVYPotatoPotato virus Yvirus Y RsRspotatopotato brown brown rotRalstoniarotRalstonia solanacearumsolanacearumRSPaVRSPaVRupestrisRupestris stem pitting associated stem pitting associated virusvirus SLVSLVShallotShallot latent viruslatent virus TAVTAVTomatoTomato aspermyaspermycucumoviruscucumovirus TBRVTBRVTomatoTomato blacblac ring virusring virus TCSVTCSVTomatoTomato chloroticchloroticspot virusspot virus TMVTMVTobaccoTobacco mosaic virusmosaic virus ToMVToMVTomatoTomato mosaic mosaic virusvirus ToRSVToRSVTomatoTomato ringspotringspot virusvirus TRSVTRSVTobaccoTobacco ringspotringspotvirusvirus TSWVTSWVTomatoTomato spotted wilt virusspotted wilt virus TYLCVTYLCVTomatoTomato yellow leaf yellow leaf curl viruscurl virus WMVWMV--11Watermelon mosaic virus 1Watermelon mosaic virus 1 WMVWMV--22Watermelon Watermelon mosaic virus 2mosaic virus 2 XapXap XanthomonasXanthomonas arboricolaarboricola pvpv..prunipruniXccXcc XanthomonasXanthomonas campestriscampestris pvpv. . campestriscampestrisXcdXcd XanthomonasXanthomonascampestriscampestris pvpv. . dieffenbachiaedieffenbachiaeXcjXcj XanthomonasXanthomonas campestriscampestris pvpv..JuglandisJuglandisXcpXcp XanthomonasXanthomonas campestriscampestris pvpv..pelargoniipelargoniiXcphXcph XanthomonasXanthomonas campestriscampestris pvpv..phaseoliphaseoliXcvXcv XanthomonasXanthomonas campestriscampestris pvpv.. vesicatoriavesicatoriaXfXf XanthomonasXanthomonasfragariaefragariaeXyfXyfPierce'sPierce's diseaseXylelladiseaseXylella fastidiosafastidiosaXyloampXyloamp XylophylusXylophylusampelinusampelinusZYMVZYMVZucchiniZucchini yellow mosaic virusyellow mosaic virus