{
  "url": "https://openlegislation.uk/eu-directive-1998-57/annex-II/appendix-3",
  "json_url": "https://openlegislation.uk/eu-directive-1998-57/annex-II/appendix-3.json",
  "legislation": "eudr/1998/57",
  "legislation_title": "Council Directive 98/57/EC of 20 July 1998 on the control of Ralstonia solanacearum (Smith) Yabuuchi et al.",
  "legislation_url": "https://openlegislation.uk/eu-directive-1998-57",
  "path": "annex/II/appendix/3",
  "kind": "appendix",
  "number": "3",
  "heading": null,
  "text": "A.\nCommercially available standardised control material\n(a)\nBacterial isolates\nThe following bacterial isolates are recommended for use as standard reference material either as positive controls (Table 1) or during optimisation of tests to avoid cross-reactions (Table 2). All strains are commercially available from:\n1.. National Collection of Plant Pathogenic Bacteria (NCPPB), Central Science Laboratory, York, UK\n2.. Culture Collection of the Plant Protection Service (PD), Wageningen, the Netherlands.\n3.. Collection Française de Bactéries Phytopathogènes (CFBP), INRA Station Phytobactériologie, Angers, France.\nTable 1\nSMT reference panel of isolates of R. solanacearum\nUse as standard reference strain of R. solanacearum biovar 2 (race 3).\nNCPPB code\nSMT #\nOther codes\nCountry of origin\nBiovar\nNCPPB 4153\n6\nCFBP 4582, Pr 3020, EURS11\nEgypt\n2\nNCPPB 4154\n10\nCFBP 4585, 550, EURS21\nTurkey\n2\nNCPPB 3857\n12\nCFBP 4587, Pr 1140, EURS26\nEngland\n2\nNCPPB 1584\n23\nCFBP 4598, EURS49\nCyprus\n2\nNCPPB 2505\n24\nCFBP 4599, EURS50\nSweden\n2\nNCPPB 4155\n26\nCFBP 4601, 502, EURS55\nBelgium\n2\nNCPPB 4156\n71\nPD 2762, CFBP 3857\nNetherlands\n2\nNCPPB 4157\n66\nLNPV 15.59\nFrance\n2\nNCPPB 4158\n39\nCFBP 4608, Port 448, EURS80\nPortugal\n2\nNCPPB 4160\n69\nIVIA-1632-2\nSpain\n2\nNCPPB 4161\n76\nB3B\nGermany\n2\nNCPPB 325\n41\nCFBP 2047, KEL60-1, R842\nUSA\n1\nNCPPB 3967\n42\nCFBP 4610, R285, GONg7\nCosta Rica\n1\nNCPPB 4028\n43\nCFBP 4611, R303/571, CIP310, SEQ205\nColombia\n2\nNCPPB 3985\n44\nCFBP 4612, R578, CIP312\nPeru\n2T\nNCPPB 3989\n45\nCFBP 4613, R568, CIP226\nBrazil\n2T\nNCPPB 3996\n46\nCFBP 3928, R276/355, CIP72, SEQ225\nPeru\n3\nNCPPB 3997\n47\nCFBP 4614, R280/363, CIP49, HAY0131a\nAustralia\n3\nNCPPB 4029\n48\nCFBP 4615, R297/349, CIP121, CMIb2861\nSri Lanka\n4\nNCPPB 4005\n49\nCFBP 4616, R470\nPhilippines\n4\nNCPPB 4011\n50\nCFBP 4617, R288, HEmps2\nChina\n5\nNote: Authenticity of the above strains can be guaranteed only if obtained from an authentic culture collection.\nTable 2:\nSMT reference panel of serologically- or genetically-related bacteria for use in optimisation of detection tests\nPotential cross-reacting strain in serological tests (IF and/or ELISA) with polyclonal antisera.\nStrain from which PCR product can be amplified in some laboratories of a similar size to that expected using specific primers OLI-1 and Y-2 (see Appendix 6).\nLikely to cross-react in most tests but known to occur only on banana in Indonesia.\nNCPPB code\nSMT #\nOther code\nIdentification\nNCPPB 4162\n51\nCFBP 1954\nBacillus polymyxa\nNCPPB 4163\n52\nCFBP 1538\nPseudomonas marginalis pv. marginalis\nNCPPB 4164\n—\nCFBP 2227\nBurkholderia cepacia\nNCPPB 4165\n—\nCFBP 2459\nRalstonia pickettii\nNCPPB 4166\n58\nCFBP 3567\nCSL Pr1150\nRalstonia pickettii\nNCPPB 4167\n60\nCFBP 4618\nPD 2778\nRalstonia sp.\nNCPPB 1127\n53\nCFBP 3575\nBurkholderia andropogonis\nNCPPB 353\n54\nCFBP 3572\nBurkholderia caryophylli\nNCPPB 945\n55\nCFBP 3569\nBurkholderia cepacia\nNCPPB 3708\n56\nCFBP 3574\nBurkholderia glumae\nNCPPB 3590\n57\nCFBP 3573\nBurkholderia plantarii\nNCPPB 3726\n59\nCFBP 3568\nBanana Blood Disease Bacterium\nNCPPB 4168\n61\nCFBP 4619\nIPO S339\nEnterobacter sp.\nNCPPB 4169\n62\nIPO 1695\nEnterobacter sp.\nNCPPB 4170\n63\nCFBP 4621\nIPO S306\nOchrobactrum anthropi\nNCPPB 4171\n64\nCFBP 4622\nIPO 1693\nCurtobacterium sp.\nNCPPB 4172\n65\nIPO 1696a\nPseudomonas sp.\nNCPPB 4173\n—\nPD 2318\nAureobacterium sp.\nNCPPB 4174\n81\nIVIA 1844.06\nFlavobacterium sp.\n(b)\nCommercially available standardised control material\nThe following standard control material is available from the NCPPB culture collection.\nFreeze dried pellet of potato extract from 200 healthy potato tubers as negative control for all tests.\nFreeze dried pellet of potato extract from 200 healthy potato tubers containing 10 3 to 10 4 and 10 4 to 10 6 cells R. solanacearum biovar 2 (strain NCPPB 4156 = PD 2762 = CFBP 3857) as positive controls for serological and PCR tests. Since cell viability is affected during freeze-drying, these are not suitable as standard controls for isolation or bioassay tests.\nFormalin-fixed suspensions of R. solanacearum biovar 2 (strain NCPPB 4156 = PD 2762 = CFBP 3857) at 10 6 cells per ml as positive controls for serological tests.\nB.\nPreparation of positive and negative controls for the core screening tests PCR/IF and FISH\nProduce a 48 hour culture of a virulent strain of R. solanacearum race3/biovar2 (e.g. strain NCPPB 4156 = PD 2762 = CFBP 3857) on basal SMSA medium and suspend in 10 mM phosphate buffer to obtain a cell density of approximately 2 × 10 8 cfu per ml. This is usually obtained by a faintly turbid suspension equivalent to an optical density of 0,15 at 600 nm.\nRemove the heel end cores of 200 tubers taken from a white skin variety production known to be free from R. solanacearum .\nProcess the heel ends as usual and resuspend the pellet in 10 ml.\nPrepare 10 sterile 1,5 ml microvials with 900 µl of the resuspended pellet.\nTransfer 100 µl of the suspension of R. solanacearum to the first microvial. Vortex.\nEstablish decimal levels of contamination by further diluting in the next five microvials.\nThe six contaminated microvials will be used as positive controls. The four non-contaminated microvials will be used as negative controls. Label the microvials accordingly.\nPrepare aliquots of 100 µl in sterile 1,5 ml microvials thus obtaining nine replicas of each control sample. Store at -16 to -24 °C until use.\nThe presence and quantification of R. solanacearum in the control samples should be first confirmed by IF.\nFor the PCR test perform DNA extraction from positive and negative control samples with each series of test samples.\nFor IF and FISH tests perform assays on positive and negative control samples with each series of test samples.\nFor IF, FISH and PCR assays R. solanacearum must be detected in at least the 10 6 and 10 4 cells/ml of the positive controls and not in any of the negative controls.",
  "extent": "E+W+S+N.I.",
  "status": null,
  "version": "current",
  "annotations": [
    {
      "type": "F",
      "label": "F1",
      "text": "Substituted by Commission Directive 2006/63/CE of 14 July 2006 amending Annexes II to VII to Council Directive 98/57/EC on the control of Ralstonia solanacearum (Smith) Yabuuchi et al.."
    },
    {
      "type": "footnote",
      "label": "f1",
      "text": "Use as standard reference strain of R. solanacearum biovar 2 (race 3)."
    },
    {
      "type": "footnote",
      "label": "f2",
      "text": "Potential cross-reacting strain in serological tests (IF and/or ELISA) with polyclonal antisera."
    },
    {
      "type": "footnote",
      "label": "f3",
      "text": "Strain from which PCR product can be amplified in some laboratories of a similar size to that expected using specific primers OLI-1 and Y-2 (see Appendix 6)."
    },
    {
      "type": "footnote",
      "label": "f4",
      "text": "Likely to cross-react in most tests but known to occur only on banana in Indonesia."
    }
  ],
  "outstanding_effects": [],
  "provenance": {
    "source": "legislation.gov.uk",
    "source_url": "https://www.legislation.gov.uk/eudr/1998/57/annex/II/appendix/3",
    "retrieved_at": "2026-11-01T15:23:06.931886+00:00"
  },
  "licence": "Open Government Licence v3.0",
  "licence_url": "https://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/",
  "attribution": "Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives."
}